Plant Pathogenesis and Resistance: Biochemistry and Physiology of Plant-Microbe Interactions [1 ed.] 978-90-481-5750-1, 978-94-017-2687-0

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PLANT PATHOGENESIS AND RESISTANCE

Plant Pathogenesis and Resistance Biochemistry and Physiology of Plant-Microbe Interactions by

Jeng-Sheng Huang Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina, U.S.A.

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.LP. Catalogue record for this book is available from the Library of Congress.

ISBN 978-90-481-5750-1 ISBN 978-94-017-2687-0 (eBook) DOI 10.1007/978-94-017-2687-0

Printed on acid-free paper

All Rights Reserved © 2001 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 2001 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

This book is dedicated to the memory of Professor Robert N. Goodman

Contents Preface . Abbreviations

XI

XIII

SECTION I. INFECTION PROCESSES Chapter 1. Penetration of Cuticles by Plant Pathogens 1.1 Introduction, 4 1.2 Structure, chemistry, and biosynthesis of plant cuticles, 4 1.3 Enzymatic dissolution of cuticles as a means of direct penetration, 14 1.4 Mechanical force as a means of direct penetration, 28 1.5 Conclusions, 37 References, 39

1 3

SECTION II. PLANT PATHOGENESIS. Chapter 2. Degradation of Cell Walls by Plant Pathogens 2.1 Introduction, 52 2.2 Structure, chemical components, and biosynthesis of plant cell walls, 52 2.3 Degradation of cell walls by plant pathogens, 71 2.4 Cell-wall-degrading enzymes and plant pathogenesis, 101 2.5 Cell-wall-degrading enzymes and disease resistance, 106 2.6 Conclusions, 108 References, 109

49 51

Chapter 3. Bioenergetics in Plant-pathogen Interactions 3.1 Introduction, 132 3.2 An overview of energy-capture and energy-utilization processes in higher plants, 132 3.3 The energy-capture process as affected by pathogenic infection, 150 3.4 The energy-utilization process as affected by pathogenic infection, 162 3.5. Conclusions, 169 References, 169

. 131

Vlll

Chapter 4. Rhizobium-Legume Symbiosis and the Effects of Diseases on Nodulation and Nitrogen Fixation. . 175 4.1 Introduction, 176 4.2 An overview of nodulation and nitrogen fixation, 177 4.3 Effects of diseases on nodulation and nitrogen fixation, 211 4.4 Conclusions, 219 References, 220 Chapter 5. Growth Regulators and Plant Tumorigenesis .237 5.1 Introduction, 238 5.2 Crown galls caused by Agrobacterium tumefaciens, 238 5.3 Hairy roots caused by Agrobacterium rhizogenes, 261 5.4 Olive knot caused by Pseudomonas syringae pv. savastanoi, 265 5.5 Fasciation diseases caused by Rhodococcusfascians, 270 5.6 Bacterial canker of almond caused by Pseudomonas amygda/i, 271 5.7 Crown and root galls of gypsophila caused by Erwinia herbicola pv. gypsophilae, 272 5.8 Witches' broom diseases caused by Taphrina spp., 273 5.9 Galls caused by Ustilago spp., 274 5.10 Clubroot of crucifers caused by Plasmodiophora brassicae, 275 5.11 Virus-induced tumors, 276 5.12 Conclusions, 276 References, 277 Chapter 6. Phytotoxins and Plant Pathogenesis 6.1 Introduction, 292 6.2 Biochemistry and modes of action ofphytotoxins, 294 6.3 Roles of phytotoxins in plant pathogenesis, 375 6.4 Application ofphytotoxins, 378 6.5 Conclusions, 382 References, 383

. 291

SECTION III. SIGNAL TRANSDUCTION Chapter 7. Signal Transduction in Plant-Microbe Interactions 7.1 Introduction, 416 7.2 Signal molecules in host-parasite interactions, 416 7.3 Receptors and perception of signal molecules, 437 7.4 Second messengers and intracellular signal transduction, 440

.413 . 415

IX

7.5 7.6 7.7

Signal transduction in systemic acquired resistance, 457 Host responses to signals: Gene expression and regulation, 460 Conclusions, 464 References, 464

SECTION IV. DYNAMICS OF PLANT DEFENSE Chapter 8. Fortification of Plant Cell Walls as a Resistance Mechanism 8.1 Introduction, 486 8.2 Papilla Formation and Disease Resistance, 487 8.3 Lignification and Disease Resistance, 496 8.4 Suberization and Disease Resistance, 507 8.5 Cell Wall Structural Proteins and Disease Resistance, 510 8.6 Conclusions, 516 References, 516

.483 . 485

Chapter 9. Accumulation ofPhytoalexins as a Resistance Mechanism .525 9.1 Introduction, 526 9.2 Biosynthesis and metabolism ofphytoalexins, 528 9.3 Elicitation and accumulation ofphytoalexins, 575 9.4 Modes of action ofphytoalexins, 588 9.5 Phytoalexins and disease resistance, 590 9.6 Conclusions, 600 References, 601 Chapter 10. Pathogenesis-related Proteins and Disease Resistance 10.1 Introduction, 624 10.2 Characterization and biological functions of PR proteins, 625 10.3 Biosynthesis of PR proteins, 647 10.4 Roles of PR Proteins in disease resistance, 653 10.5 Conclusions, 657 References, 659

. 623

Index

.675

Preface The extensive use of molecular technology in plant pathology has generated enormous information and significantly advanced our understanding of the biochemistry and physiology of plant-microbe interactions. The objective of this book is to summarize some of these recent advancements. Each plant-microbe interaction involves a two-way molecular communication. On one hand, the microbe perceives signals from the plant, secretes chemical arsenals to establish infection courts, and produces metabolites that disrupt structural integrity, alter cellular function, and circumvent host defenses. On the other hand, the plant senses the signals from the microbe, reinforces its cell walls, and accumulates phytoalexins and pathogenesis-related proteins in an attempt to defend itself. The production of pathogenicity and virulence factors by the microbe, the elicitation of defense mechanisms by the plant, and the dynamic interactions of the two are the focal points of physiological plant pathologists. Consequently, this book is organized around the most researched areas in biochemical plant pathology: infection processes, pathogenesis, signal transduction, and resistance mechanisms. Section I deals with the infection processes. The structure and chemical composition of cuticles and the breach of these barriers by plant pathogens are examined in Chapter 1. Section II focuses on pathogenicity and virulence factors. Discussion centers on the production of cell-waH-degrading enzymes by plant pathogens (Chapter 2), the effect of pathogenic infection on plant's energy capture and utilization processes (Chapter 3), the effects of plant pathogens on nitrogen fixation (Chapter 4) and growth regulation (Chapter 5), and the production of phytotoxins and their effects on plant physiology (Chapter 6). Section m is devoted to host-parasite specificity with special emphasis on signal transduction in plant-microbe interactions. Signal molecules, their perception and transduction, as well as the plant's response to signals are discussed in Chapter 7. Section IV is concerned with the dynamics of host defense. Emphasis is placed upon the fortification of cell waHs (Chapter 8), production of phytoalexins (Chapter 9) and pathogenesis-related proteins (Chapter 10) as defense mechanisms. I wish to thank those who reviewed the manuscript: Margaret Daub, Gary Strobel, Joseph Kuc, Scott Chilton, P. E. Kolattukudy, Yasuyuki Kubo, Dean D-S Tzeng, and L. C. Van Loom. I also appreciate the valuable suggestions and needed assistance of Renee van Leeuwen and Zuzana Bernhart of the Kluwer Academic Publishers.

XII

Finally, I want to express my appreciation to my wife, Pi-Yu, for her understanding and patience during the writing of this book. Jeng-Sheng Huang

Abbreviations adenosine mono-, di-, triphosphate

AMP, ADP, ATP

barley stripe mosaic virus BSMV base pairs, ki 10 base bp,kb pairs bean pod mottle virus BPMV bean yellow mosaic virus BYMV calorie, kilocalorie cal, kcal cauliflower mosaic virus CaMV complementary DNA cDNA cucumber mosaic virus CMV cultivar cv. cyclic adenosine 3':5'monophosphate cAMP dalton, kilodalton D kD degree of polymerization Dr deoxyribonuclease DNase deoxyribonucleic acid DNA effective dosage at 50% inhibition ED50 endoplasmic reticulum ER ferredoxin Fd flavin adenine dinucleotide FAD flavin mononucleotide FMN guanosine mono, di GMP, triphosphate GDP, GTP high performance liquid chromatography HPLC hydroxyproline-rich glycoprotein HRGP isoelectric point pI lignin peroxidase LiP manganese-dependent peroxidase Michaelis constant

ORF open reading frame polyacrylamide gel electrophoresis PAGE polymerase chain reaction PCR relative molecular weight Mr ribonuclease RNase ribonucleic acid RNA messenger RNA mRNA nuclear RNA nRNA ribosomal RNA rRNA transfer RNA tRNA ribulose-I,5-bisphosphate carboxylase/oxygenase Rubisco sodium dodecyl sulfate SDS soybean mosaic virus SMV tobacco etch virus TEV tobacco mosaic virus TMV tobacco necrotic virus TNV tobacco ringspot virus TRSV ultraviolet UV uridine mono-, di, UMP, triphosphate UDP, UTP white clover mosaic WCMV ViruS

SECTION I. INFECTION PROCESSES

Chapter 1 PENETRATION OF CUTICLES BY PLANT PATHOGENS

1.1

1.2

1.3

1.4

Introduction Structure, chemistry, and biosynthesis of plant cuticles. 1.2.1 Structure 1.2.2 Chemical composition . 1.2.3 Biosynthesis of cutin monomers 1.2.3.1 Biosynthesis offatty acids in the plastids 1.2.3.2 Modification offatty acids outside the plastids 1.2.3.3 Polymerization of cutin monomers . Enzymatic dissolution of cuticles as a means of direct penetration 1.3.1 Production of cutinases by plant pathogens. 1.3.1.1 Biosynthesis of cutinases 1.3.1.2 Cutinase genes. 1.3.1.3 Molecular properties of cutinases 1.3.1.4 Catalytic properties of cutinases 1.3.2 Evidence that cutinases are involved in direct penetration of cuticles by certain plant-pathogenic fungi 1.3.2.1 Presence of cutinase at the site of penetration 1.3.2.2 Inactivation of cutinase prevents fungal penetration. 1.3.2.3 Correlation between cutinase production and virulence of the pathogen 1.3.2.4 Insertion of the cutinase gene into a wound pathogen enables it to infect an intact host Mechanical force as a means of direct penetration 1.4.1 Production of melanin by plant pathogens 1.4.1.1 Chemical and physical properties of DHN melanins. 1.4.1.2 Biosynthesis of DHN melanins. 1.4.1.3 Genetics of DHN melanin production 1.4.2 Evidence that melanin biosynthesis is a prerequisite for penetration of cuticles by appressoria of some plant-pathogenic fungi 1.4.2.1 Inhibition of melanin biosynthesis prevents fungal penetration . 1.4.2.2 Melanins mediate the build-up of a high hydrostatic pressure in the appressorium 1.4.2.3 Melanin-deficient mutants fail to penetrate nitrocellulose membranes and are nonpathogenic to intact host plants but pathogenic to wounded plants 1.4.2.4 Melanized spores with disrupted cutinase gene retain virulence

4 4 5 6 7 7 10 12 14 14 15 17 20 21 23 23 24 25 27 28 29 29 30 31 34 34 35

36 37

4 1.5

- Plant Pathogenesis and Resistance Conclusions. References .

37 39

1.1 INTRODUCTION Many fungal pathogens gain entrance into their hosts by direct penetration of the cuticle. Thus, the plant cuticle is the first barrier to be overcome by many plant-pathogenic fungi. The mechanism by which plant-pathogenic fungi penetrate the cuticle has been debated for many years. One theory states that enzymatic dissolution of the cuticle is required for fungal penetration while the other contends that the cuticle is penetrated by the mechanical force exerted by the infection structure of the penetrating fungus. Evidence supporting each theory has accumulated over the years. The objective of this chapter is to summarize and discuss recent advances relating to each of these two theories. The involvement ofthe cuticle-degrading enzymes in the direct penetration of the plant surface has been reviewed by Kolattukudy (1984, 1985), Kolattukudy and Crawford (1987), and Koller (1991). Mechanical force as a means of direct penetration has been reviewed by Bell and Wheeler (1986) and Kubo and Furusawa (1991).

1.2 STRUCTURE, CHEMISTRY, AND BIOSYNTHESIS OF PLANT CUTICLES The epidermis is made of the plant's outermost layer of cells. The outer wall of the epidermis of all aerial parts is covered by a bilayered cuticular membrane known as the cuticle. The cuticle serves as a boundary between the plant and its environment. Its physiological and biological functions are (i) to conserve water in the plant; (ii) to prevent leaching of plant components; (iii) to protect the plant from injuries due to physical abrasion, frost, and radiation; and (iv) to provide a potential barrier from attack by insects and plant pathogens. In addition, recent reports have indicated that certain cuticular components are signals in plant-microbe interactions (Kolattukudy et aI., 1995; Kerstiens, 1996). The role ofthe cuticle as a barrier to fungal invasion is supported by a direct correlation between disease resistance and cuticle thickness in several hostparasite interactions. In Solanaceae, cuticle thickness of the New Mexican-type peppers (Capsicum annuum) increases from 12 ｾｭ＠ in immature green fruit to 24 ｾｭ＠ in mature red fruit. The susceptibility of unwounded fruitto infection of

Penetration of Cuticles -

5

Phytophthora capsici decreases with increased ripening (Biles et aI., 1993). In Poaceae, the cuticle ofthe Sorghum hicolor bloom less mutant bm-22 is about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 cuticles. The reduction in cuticle deposition in bm-22 is associated with an increase in its susceptibility to Exserohilum turcicum, the leaf blight pathogen (Jenks et aI., 1994). In Brassicaceae, seven-day-old seedlings of mustard (Sinapis alba) are more resistant than those of rapeseed (Brassica napus) to Rhizoctonia solani AG2-1, the causal agent of seedling root rot. Rapeseed cuitivars do not show an obvious cuticle layer at 1 week, but a cuticle is seen through autofluorescence at 3 weeks with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular auto-fluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. The results indicate that the cuticle plays an important role in the resistance of mustard and older rapeseed plants to infection by R. solani (Yang et aI., 1992). Contradictory reports, however, are abundant in the literature. For example, no significant correlations between cuticle thickness and resistance are observed in the pathogenesis of the powdery mildew pathogen (Erysiphe cichoracearum) in Phlox (Jarosz et aI., 1982). Chemical composition, structure, and biological functions of plant cuticles have been reviewed by Kolattukudy (1980a,b; 1981, 1996), Cuiter et al. (1982), Juniper and Jeffree (1983), and Jeffree (1996). 1.2.1 Structure Structurally, the cuticle may be divided into cuticular proper and cuticular layer. The cuticular proper is the outer lamellae region that is made of cutin and wax and the cuticular layer is the inner reticulate region that consists of cutin, wax, and cellulose fibers. In addition, the cuticle bears a superficial epicuticular wax. The conventional view of the epidermis and cuticle of a leaf mesophyll cell is given in Fig. 1-1 (Jeffree, 1996). Plant cuticles can be separated from epidermis by pectinase treatment. In small bindweed (Convolvulus arvensis), Vicia major, and Philodendron spp., cuticles are separated rapidly from cell walls, frequently within 3-12 hr after the leaves are treated with pectinases. In citrus (Citrus spp.), tobacco (Nicotiana spp.), cherry (Prunus spp.), and pear (Pyrus communis), cuticles are separated from cell walls slowly, usually taking 12 hr to 3 days. Cuticles of green beans (Phaseolus vulgaris) come away as flecks, not as intact sheets. These results ind icate the existence of an uneven layer of pectinaceous materials between the cell wall and cuticle. The thickness of the cuticle varies depending on species, organs, developing stages, and environmental conditions.

6

- Plant Pathogenesis and Resistance

EWC EWF

CP

ECl

CL

ICL

SCW

Fig. I-I. The structure of the plant cuticle. The cuticle consists a bilayered cuticular membrane: the outer cuticular proper (CP) and the inner cuticular layer (CL). The CP may be amorphous or lamellate. The CL is itself a layered structure and may be divided into internal (lCL) and external cuticular layer (ECL). The epicuticular wax (EW), in crystalline (EWC) or flake (EWF) form, frequently covers the cuticular membrane. In mature cuticular membrane, secondary cell wall (SCW) may be cutinized, forming cystoliths (CYS). (Reproduced from Jeffree, 1996, in Plant Cuticles: an integrated functional approach. edited by G. Kerstiens, with permission from BIOS Scientific Publishers Ltd. Oxford).

1.2.2 Chemical Composition There are three major chemical constituents of cuticle: wax, cellulose, and cutin. The epicuticular wax, which can be extracted by dipping intact leaves in organic solvents at room temperature, contains many classes of relatively hydrophobic hydrocarbons: n-alkanes [CH3-(CH2)n-CH3' with n = 29 and 31 the most common]; primary alcohols [CH3-(CH2)n-CHzOH, with n = 26 and 28 the most common]; and fatty acids [CH3-(CH2)I1-COOH, in the range ofn = 12 to 36, even number] (Kolattukudy, 1980b). The existence of cellulose in the cuticle has been supported by electron microscopy and chemical analysis. Fibrillar materials in the cuticle have been observed under the electron microscope. These materials give a positive reaction to the periodic acid-Schiff reagent, zinc-chlor-iodide, and IKI-H 2S04 • These results indicate that the fibers are cellulose. In general, the amount of cellulose in the cuticle is small. The chemical structure of cellulose will be discussed in detail in Chapter 2. Cutin is the main structural component of plant cuticles. It is generally prepared from leaf strips by pectinase and cellulase treatment to remove pectin

Penetration of Cuticles -

7

and cellulose, followed by organic solvent treatment to remove epicuticular wax. Cutin can be depolymerized by hydrolysis with alcoholic KOH: Leaf strips

pectinase, cellulase

..

Cuticle

organic solvent

- - - - i.. ｾ＠

Cutin

alcoholic KOH

.. Cutin monomers

The monomers can be easily analyzed quantitatively and qualitatively by gas chromatography. They are mainly hydroxy and epoxy fatty acids in the C'6 and C,s families. The monomers in the C'6 family are palmitic (hexadecanoic) acid, 16-hydroxypalmitic acid, and 8,16-,9,16-, and 10, 16-dihydroxypalmitic acids. The monomers in the C,s family are mainly stearic (octadecanoic) acid, oleic acid, linoleic acid, 18-hydroxyoleic acid, 18-hydroxylinoleic acid, 9,10, 18-trihydroxystearic acid, and 18-hydroxy-9, 1O-epoxystearic acid. The composition of cutin is highly dependent upon the plant species, tissues, and developmental stage. The monomers in the C'6 family predominate in plant parts that expand rapidly. In slow-growing organs, a mixture of monomers ofC'6 and C,s families is found. The major cutin monomer of pear is 10, 16-dihydroxyhexadecanoic acid (Gerard et aI., 1993). 10, 16-Dihydroxyhexadecanoic acid and 16-hydroxy10-oxo-hexadecanoic acid are the major cutin constituents in lime fruits (Ray et aI., 1995). The main constituents (about 30%) of leaf cutin of Limonia acidissima are 9,16- and 10, 16-dihydroxyhexadecanoic acids (Das and Thakur, 1989). More than 70% of the total monomers of cucumber (Cucumis sativus) cutin is 8,16-dihydroxyhexadecanoic acid (Gerard et aI., 1994). The major monomers of wheat (Triticum aestivum) leaf cutin are octadecanoic acid; 8,16and 9,16-dihydroxyhexadecanoic acids; and 9,1 0-epoxy-18-hydroxyoctadecanoic acid (Matzke and Riederer, 1990).

1.2.3 Biosynthesis of Cutin Monomers 1.2.3.1 Biosynthesis of fatty acids in the plastids The saturated palmitic (16:0) and stearic (18:0) acids and mono-unsaturated oleic (18: I) acid are the most important fatty acids in most plant tissues. They are synthesized in plastids from acetyl-CoA via the fatty acid biosynthetic pathway. Briefly, the acyl group is esterified to the protein cofactor, acyl carrier protein (ACP), and is stepwise elongated. The extension is terminated by acylACP thioesterase which hydrolyzes the acyl-ACP and releases the free fatty acids. The free fatty acids are exported out of the plastids. They may be reesterified with coenzyme A and subjected to modification (e.g., hydroxylation, desaturation, elongation, and oxidation) and used in membrane and storage lipid synthesis and cutin and wax production. The first step in the fatty acid biosynthesis involves the formation of malonyl-CoA from acetyl-CoA and CO 2 , The reaction is catalyzed by acetylCoA carboxylase (ACCase, EC 6.4.1.2) and is ATP-dependent. Two forms of ACCase exist in higher plants: prokaryotic (multisubunit) and eukaryotic

8

- Plant Pathogenesis and Resistance

(multifunctional) (Sasaki et aI., 1995). The prokaryotic ACCase consists of several separated subunits. These subunits include nuclear-encoded ｾＵＰＭｫｄ＠ biotin carboxylase, 34- to 38-kD biotin carboxyl carrier protein (BCCP), and a plastid-encoded 65- to 80-kD carboxyltransferase. The eukaryotic ACCase has these three components arranged into a single multifunctional polypeptide. In dicots, the eukaryotic ACCase is found in the cytosol and the prokaryotic ACCase exists in the plastids. In gramineae, the eukaryotic ACCase is found in both the cytosol and plastids (Sasaki et aI., 1995). Biotin carboxylase catalyzes the attachment of CO2 to BCCP. Carboxyltransferase transfers the activated CO2 from BCCP to acetyl-CoA to form malonyl-CoA: A TP + CO 2 + BCCP

biotin carboxylase ..

COrBCCP + CHrCO-S-CoA Acetyl-CoA

CO2 -BCCP + ADP + Pi

transcarboxylase

...

-OOC-CHrCO-S-CoA + BCCP Malonyl-CoA

ACP is a 9-kD, non-enzymatic protein with phosphopantetheine as its acylbinding site. Malonyl-CoA:ACP transacylase (EC 2.3.1.39) catalyzes the transfer of the malonyl residue from coenzyme A to ACP. The enzyme has been isolated from spinach (Stapleton and Jaworski, 1984), soybean (Guerra and Ohlrogge, 1986), and Cuphea lanceolate (Briick et aI., 1994). It has a AI. of 23,500 (Briick et aI., 1994). The elongation of acyl moiety occurs when malonyl-ACP and acetyl-CoA are condensed to form ketobutyryl-ACP, a reaction catalyzed by 3-ketoacylACP synthase (EC 2.3.1.41). This four-carbon compound is subjected to reduction by 3-ketoacyl-ACP reductase (EC 1.1.1.100), dehydration by 3hydroxyacyl-ACP dehydrase, and reduction by enoyl-ACP reductase (EC 1.3.1.9) to form butyryl-ACP. Butyryl-ACP thus synthesized is ready for another elongation cycle by condensation with malonyl-ACP to form a sixcarbon ketohexanoyl-ACP. As the cycle continues, the acyl moiety ofacyl-ACP is extended stepwise by two carbons, donated by malonyl-ACP. At least four 3-ketoacyl-ACP synthases have been isolated. Synthase III is specific for short-chain acyl-ACPs as substrates and is thought to catalyze the initial elongation step in fatty acid biosynthesis (Jaworski et aI., 1989). In spinach, the synthase III has a molecular mass of 63 kD and exists as a homodimer. The purified enzyme is highly specific for acetyl-CoA and malonyl-ACP. Acetyl-butyryl-ACP and hexanoyl-ACP cannot substitute for acetyl-CoA as substrates (Clough et aI., 1992). In avocado, synthase III has a AI. of 69 kD and also exists as a homodimer (Gulliver and Slabas, 1994). Synthase III is insensitive to antibiotic cerulenin (Schuch et aI., 1994). Synthase I is highly active toward acyl-ACPs having C2 to C I4 in acyl moiety. In the developing seeds of oilseed rape (Brassica napus), the enzyme is a

Penetration of Cuticles -

9

homodimer with a M, of 86,700 (MacKintosh et aI., 1989). In barley chloroplasts, the enzyme exists in three isoforms: cx 2, cxP, and P2. The P2 isozyme is encoded by the kasI2 (ketoacyl-ACP synthase) gene (Kauppinen, 1992). Synthase II is most active with long-chain acyl-ACPs and is participating in the elongation of palmitoyl-ACP to stearoyl-ACP. A reduction in synthase II activity in sunflower mutants CAS-5 and CAS-12 results in high palmitic acid phenotype (Martinez et aI., 1999). The native synthase II from developing seeds of oilseed rape has an apparent molecular weight of87.4 kD (MacKintosh et a!., 1989). Synthase IV, detected in a Cuphea sp., has amino acid sequence similar to those of synthase II but is active toward medium chain acyl-ACPs (Dehesh et aI., 1998). The 3-ketoacyl-ACP reductase is a homotetramer (Shimakata and Stumpt, 1982a,b). A cDNA encoding the enzyme has been cloned from Cuphea lanceolata. The encoded polypeptide has 320 amino acids with 63 N-terminal, transit residues. The mature peptide has 257 residues and a molecular mass of 27 kD (Klein et aI., 1992). The 3-hydroxyacyl-ACP dehydrase has been purified from spinach leaves. It is a tetramer (Shimakata and Stumpt, 1982b). The enoyl-ACP reductase has been purified from several plants, including avocado mesocarp (Caughey and Kekwick, 1982), spinach leaves (Shimakata and Stumpt, 1982b), and rapeseed seeds (Slabas et a!., 1986). The enoy1-ACP reductase cDNA has been cloned. The ORF is predicted to code for a polypeptide of385 amino acids, including a signal peptide of73 amino acids, with a calculated molecular weight of 40,433. The mature enzyme has a calculated molecular weight of 32,890 (Kater et aI., 1991). The conversion of saturated to mono-unsaturated fatty acids is catalyzed by acyl-ACP desaturase. The most widely occurring desaturase is the 1:::. 9-18:0 desaturase, which is responsible for the introduction of a double bond at the C-9 position of stearoyl-ACP to form oleoyl-ACP (McKeon and Stumpf, 1982; Thompson et aI., 1991; Gibson, 1993; Shah et aI., 2000). Several other desaturases have been described: the I:::. 4-16:0-ACP desaturase of coriander (Coriandrum sativum) seed (Cahoon and Ohlrogge, 1994), the 1:::. 6 -16:0-ACP desaturase of blacked-eyed Susan vine (Thunbergia alata) seed (Cahoon et a!., 1994) and 1:::. 9-14:0-ACP desaturase of geranium (Pelargonium hortorum) (Schultz et aI., 1996). A reduction in desaturase activity may result in accumulation of saturated fatty acid. Sunflower (Helianthus annuus) mutants, CAS-3, CAS-4 and CAS-8, accumulate 28, 15 and 14% of stearic acid, respectively, in the seed lipids. The high stearic phenotype of these mutants is due in part to a reduced stearoyl-ACP desaturase activity (Cantisan et aI., 2000). Acyl-ACP thioesterase (EC 3.1.2.14) hydrolyzes acyl-ACPs and releases free fatty acids. Stearoyl-ACP thioesterase, an enzyme prefers stearoyl-ACP as

10 - Plant Pathogenesis and Resistance the substrate, has been found in leek (Allium porrum) (Liu and Post-Beittenmiller, 1995). Oleoyl-ACP thioesterases have been isolated from safflower (Carthamus tinctorius) (Knutzon et aI., 1992) and oilseed rape (Hellyer et aI., 1992). Genes encoding palmitoyl-ACP thioesterase have been cloned from seeds of Cuphea hookeriana (Jones et aI., 1995) and cotton (Yoder et aI., 1999). Expression of this gene in Brassica napus leads to the production of a 16:0 fatty acid-rich oil (Jones et aI., 1995). In the developing seeds of California bay (Umbellularia californica), the change in fatty acyl content from a long-chain composition to a predominance of 10:0 and 12:0 fatty acids coincides with the occurrence of lauroyl-ACP thioesterase activity (Davies, 1993; Davies et aI., 1991). A California bay medium-chain acyl-ACP cDNA under the control of the CaMV 3SS promoter has been transferred into Brassica napus. The transformed plants express lauroyl-ACP thioesterase and accumulate mediumchain fatty acids in seeds (Eccleston et aI., 1996). In the developing seeds of cocoa (Theobroma cacao), accumulation of stearate in cocoa butter, however, does not result from an increase in stearoyl-ACP thioesterase activity. Plastid preparations from two developing stages in cocoa, reflecting low (lOS days after anthesis) and high (130 days after anthesis) stearate production, show no difference in stearoyl-ACP thioesterase activity (Griffiths et aI., 1993). A schematic representation of the fatty acid biosynthesis in a plastid is given in Fig. 1-2. Free fatty acids released from acyl-ACPs can move across the plastid membrane and enter the ER where they are modified and used in cutin synthesis. 1.2.3.2 Modification of fatty acids outside the plastids Epidermis of excised leaves of Vicia jaba incorporates labeled acetate into palmitic acid, w-hydroxypalmitic acid, and 10,16-dihydroxypalmitic acid. wHydroxylation and C-I0 hydroxylation of C I6 fatty acids have been demonstrated in a cell-free microsomal fraction prepared from germinating em bryonic shoots of V.faba (Soliday and Kolattukudy, 1977, 1978). Exogenous, labeled [l-14C]palm itic acid is incorporated into 16-hydroxy- and 10, 16-dihydroxypalmitic acids (Kolattukudy et aI., 1973). Labeled 16-hydroxypalmitic acid also is incorporated without degradation into the dihydroxy acid. Thus, palmitic acid is hydroxylated first at the C-16 position followed by another hydroxylation at the C-I0 position (Kolattukudy and Walton, 1972). w-Hydroxylation is catalyzed by a cytochrome P-4S0-type hydroxylase. The enzymatic reaction requires O2 and NADPH and occurs maximally at pH 8 (Soliday and Kolattukudy, 1977). Conversion of w-hydroxypalmitic acid to 10, 16-dihydroxypalmitic acid is also catalyzed by a cytochrome P-450-type hydroxylase. The enzymatic reaction requires O 2 and NADPH and has an optimal pH of7.S and an apparentKm of SO flm for 16-hydroxypalmitic acid. w-Hydroxylation is much more sensitive to CO inhibition than C-l 0 hydroxylation, suggesting that these

Penetration of Cuticles - 11

acetyl-CoA 0 carboxylase II II --------. -O-C-CHz-C-S-CoA Malonyl-CoA

o

f

' \ ｾ｡ＱｯｮｹｬＭｃａＺ＠ .ACP transacylase

0

II

II

H3C-C-CHz-C-S-ACP

o

ft

3-Ketobutyryl-ACP

II

O-C-CHz-C-S-ACP

-ketoacyl-ACP reductase

oH I

H 3C-CH=CH-C-S-ACP Butenoyl-ACP 3-hydroxyacylｾ＠ ACP dehydrase noyl-ACP 0 reduclllSe 0

II

0 II

Malonyl-ACP

II

H3C-CH-CHz-C-S-ACP 3-Hydroxybutyryl-ACP

H 3C-CHz-CHz-C-S-ACP Butyryl-ACP

I

I

: malonyl-ACP, I 3-ketoacyl-ACP synthase I. : 3-hydroxyacyl-ACP dehydrase, I enoyl-ACP reductase

, I

12:0-ACP

lauroyl-ACPthioesterase

•

Lauric acid --------,ft-- Lauric acid

•

Palmitic acid ---H---- Palmitic acid

•

Stearic acid ----,'f----- Stearic acid

: malonyl-ACP,

,

I 3-ketoacyl-ACP synthase I, : 3-hydroxyacyl-ACP dehydrase, I enoyl-ACP reductase

16:0-ACP

paimitoyl-ACPthioesterase

I malonyl-ACP,

• 3-ketoacyl-ACP synthase II

18:0-ACP

+

stearoyl-ACP thio..terase

stearoyl-ACP desaturase

18: I-ACP

oleoyl-ACP thioesterase

PLASTID

•

Oleic acid

----t1'------- Oleic acid

CYTOSOL

Fig, 1-2_ Biosynthesis of saturated fatty acids and unsaturated oleic acid in a plastid. (Adapted from Ohlrogge, 1994; Ohlrogge and Browse, 1995),

two hydroxylations are catalyzed by two different enzymes (Soliday and Kolattukudy, 1978; Kolattukudy, 1996). In the case of the CIS monomers, a double bond is introduced at the midchain position of stearic acid to form oleic acid during fatty acid biosynthesis inside the plastids. The unsaturated fatty acid is transported out of the plastids

12 - Plant Pathogenesis and Resistance and undergoes w-hydroxylation to form 18-hydroxyoleic acid. The hydroxy acid is subsequently epoxidized to 9,1O-epoxy-18-hydroxystearic acid. The epoxide is then hydrated to form 9, I 0, 18-trihydroxy stearic acid (Kolattukudy, 1980a,b; Kolattukudy and Soliday, 1985; Kolattukudy, 1996). The biosynthesis of C I6 and CIS fatty acids is summarized in Fig. 1-3 (Kolattukudy, 1996). Acetyl-CoA + 7 Malonyl-CoA +NADPH

CHr (CH2) I4-CO-S-A CP H2 0 / " "

/

CH)-(CH 2)w COOH Palmitic acid +NADPH/O,

CHPH-{CH2)WCOOH 16-Hydroxypalmitic acid +NADPH/O,

CH 20H-{CH 2ls-CHOH-(CH2)s-COOH 10, 16-Dihydroxypalmitic acid

"

ｾｐｈ＠

ｾｮｹｬＭｃｯａＬ＠

CH)-(CH2)WCO-S-ACP _

Stearic acid

+NADPH/0 2

CH)-(CH2h-CH=CH-(CH2h-CO-S-ACP +H,O

CH)-(CH2h-CH=CH-(CH2h-COOH Oleic acid +NADPH/O,

CH 20H-(CH 2h-CH=CH-(CH 2h-COOH 18-Hydroxyoleic aicd

I

ATP CoA ,NADPH/O,

CH20H-(CH 2)rCH-CH-(CH2h-CO-SCoA _ \ /

ｾ＠

9h , IdO-Epoxty-18- 'd y roxys earlc aCI

H,O 0

CH20H-(CH2h-CHOH-CHOH-(CH2h-CO-SCoA CI6 MONOMERS

9,10,18-Trihydroxystearic acid

C 18 MONOMERS

Fig. \-3. Biosynthetic schemes for the formation ofC I6 and C IK cutin monomers. (Adapted from Koiattukudy, \996).

Alternatively, oleic acid may be first epoxidized by peroxygenase to form 9,1 O-epoxystearic acid, then hydrolyzed by epoxide hydrolase to form 9,1 O-dihydroxystearic acid, and finally w-hydroxylated catalyzed by a cytochrome P450-dependent oxidase at C-18 to form 9,10, 18-trihydroxystearic acid. It is also possible that 9, I O-epoxystearic acid is w-hydroxylated to form 9,1 0-epoxy-18hydroxystearic acid and then hydrolyzed to form 9,10, 18-trihydroxystearic acid (Fig. 1-4) (Blee and Schuber, 1993).

1.2.3.3 Polymerization of cutin monomers The C I6 and CIS monomers synthesized in the cytoplasm of the epidermal cells are transported through the wall to the outermost surface of the cell where they

Penetration of Cuticles - 13

7

Oleic acid

ｾｹｧ･ｮ｡ｳ＠

CylOChrome-P

ｾｏＢ＠

Ｂｾ＠ I 8-Hydroxyoleic acid

Ｍｾ＠

H

ｾｏｈ＠

H

ｲＢＧＴｾ＠

9, IO-Epoxystearic acid

COOH

H 9, IO-Epoxy-1 8-hydroxystearic acid

ｨｙ､ｾ＠

epoxide

ｈｏｾｃ＠

Ｚｾ＠

ｾＦＭ H 9,IO-Dihydroxystearic acid rhrome-p-450

ｈｏｾｃ＠ HilI'

HO""

OH H

9, I 0, 18-Trihydroxystearic acid

Fig. 1-4. An alternative biosynthetic pathway for the formation ofe'8 cutin monomers. (Adapted from BIte and Schuber, 1993).

are incorporated into cutin primer (Fig. 1-5). The exact mechanism involved in the polymerization process is not known. A cutin-containing, 3000 g particulate fraction from the epidermal tissue of rapidly growing Vicia faba leaves incorporated labeled hydroxy C. 6 monomers into an insoluble material. Treatment of this material with cutinase, but not other hydrolytic enzymes, released the incorporated label. These results indicate that the 3000 g fraction contains cutin synthetase. Ultrasonic treatment of an endogenous cutin preparation released a soluble enzyme that incorporates hydroxy acids into an exogenous primer. Cutin from the rapidly growing young V.faba leaves served as the best primer and the priming efficiency decreased with age of the leaf. It is possible that relatively open structure of cutin in the young leaves provides maximum free hydroxyl sites for incorporation ofthe monomer. The enzyme has been identified as hydroxylacyl CoA:cutin transacylase (Croteau and Kolattukudy, 1974).

14 - Plant Pathogenesis and Resistance

ｾｯ＠ Cutin primer

Elongated cutin

Fig. 1-5. A proposed cutin polymerization mechanism. (Adapted from Koiattukudy, 1996).

1.3 ENZYMATIC DISSOLUTION OF CUTICLES AS A MEANS OF DIRECT PENETRATION Cutinase is synthesized by many fungi and bacteria (Table 1-1). It also occurs in higher plants. For example, a polypeptide with an estimated molecular mass of 22 kD and recognized by monoclonal antibodies specific to cutinase from Fusarium solani f. sp. pisi has been found in the pollen wall of Brassica napus (Hiscock et aI., 1994). The biological function of cutinases in higher plants is not known. The following discussion will be limited to those produced by plant pathogens.

1.3.1 Production of Cutinases by Plant Pathogens Cutinase is an esterase that hydrolyzes cutin to fatty acid monomers. Cutinase activity cannot be easily measured primarily due to the structural complexity of the substrate and its insolubility in water. Current assay procedure involves the use of radioactive cutin as a substrate. Radioactive cutin can be prepared by applying 14C-labeled cutin precursors, such as acetate or palmitic acid, to the epidermal cells of rapidly expanding apple or tomato fruits. Alternatively, 3H-labeled cutin can be prepared from apple cutin exposed to 3H2 gas or grapefruit cutin treated with NaB 3H4. Hydrogen gas reduces double bonds and sodium borohydride reduces carbonyl groups of fatty acids in cutin. The activity of cutinase is assayed by measuring the radioactivity released from the cutin substrate. p-Nitrophenyl butyrate, an esterase substrate, has been used as the substrate in spectrophotometric assays of cutinase activity. The results obtained by this method indicate the presence of nonspecific esterase activity in the reaction mixture. Thus, spectrophotometry is valid only when purified cutinase is used in the assay.

Penetration of Cuticles

- 15

Table 1-1. Cutinases produced by plant pathogens. Pathogen

M,

Alternaria alternata Japanese pear pathotype Alternaria brassicicola

32,000

Botrytis cinerea

23,000 (Ae) 21,000 (B.) 18,000

Optimal pH

%CHO

Trail and Koller (1993)

6.5 8.5

40.800

Cochliobolus heterostrophus Colletotrichum capsici Colletotrichum gloeosporioides Colletotrichum lagenarium

22,000

60,000

9.0

Cryphonectria parasitica Fusarium roseum culmorum

25,000 24,300

10.0

Fusarium roseum sambucinum Fusariumsolanif. sp. pisi

24,800

Helminthosporium sativum Magnaporthe grisea Monilinia fructicola

6.5

24,000

22,000 (I) (II) 26,300

16.0

10.0 10.0

9.0

8.5

Streptomyces scabies

17,000 18,500 26,000

Ulocladium consortiale

25,100

Venturia inaequalis

22,000

Rhizoctonia solani

6.0 6.0

24,000 18,600 20,800 24,000

Phytophthora capsid

Reference Tanabe et al. (1988a)

4.3 5. I 4.5

3.5 4.0 6.0

5.4

Van der VlugtBermans et al. (1997) Gindro and Pezet (1999) Trail and Koller (1990) Ettinger et al. (1987) Dickman et al. (1982) Bonnen and Hammerschmidt (1989a) Varley et al. (1992) Soliday and Kolattukudy (1976) Lin and Kolattukudy (I 980a) Purdy and Kolattukudy (1975a) Lin and Kolattukudy (1980a) Sweigard et al. (1992a) Wang et al. (2000) Munoz and Bailey (1998) Trail and Koller (\ 990) Lin and Kolattukudy (\ 980a) Lin and Kolattukudy (1980a) Koller and Parker (1989)

1.3.1.1 Biosynthesis of cutinases Cutinase activities have been detected in culture filtrates of more than 20 species of plant- pathogenic fungi (Koller, 1991). The enzyme has been isolated and purified to homogeneity from culture filtrates of more than a dozen plantpathogenic fungi (Table 1-1). F. solani f. sp. pisi produces small quantities of cutinase in media containing glucose as a carbon source. Significant amounts of cutinase are produced only

16 - Plant Pathogenesis and Resistance when glucose is depleted and cutin monomers or hydrolysates are added to the media (Woloshuk and Kolattukudy, 1986). Thus, expression of the cutinase gene is induced by cuticular components and is catabolite-repressed by glucose. In vitro translation ofpoly(AfmRNA isolated from induced cultures produced a peptide that was immunologically cross-reacted with anti-cutinase IgG. The peptide, with a ｾ＠ = 25.5 kD, was 2.1 kD larger than the mature cutinase. Cutinase added to the translation mixture effectively competed with this in vitro translated peptide for the antibody. These results indicate that the 25.5 kD peptide is synthesized as a precursor of cutinase. The peptide is subsequently subjected to post-translational proteolysis and glycosylation. Proteolysis removes a 3 kD-leader sequence. Glycosylation introduces monosaccharides at the hydroxyl groups of serine, threonine, ｾＭｨｹ､ｲｯｸｰ･ｮｬ｡ｩＬ＠ and ｾﾭ hydroxytyrosine before excretion (Flurkey and Kolattukudy, 1981). Transcriptional activation of cutinase genes has been studied using isolated nuclei (Podila et aI., 1988). When nuclei isolated from uninduced F. so/ani f. sp. pisi cultures are incubated with [32 p]UTP, little label is incorporated into cutinase transcripts as determined by hybridization with a cutinase cDNA probe. When both a cutin monomer and a soluble fraction prepared from the fungal extract are incubated with [32 p]UTP, an increase in the incorporation of label into cutinase transcripts is observed. Neither the cutin monomer nor the soluble extract alone is effective. Heating the soluble extract eliminates the transcription-stimulating activity. The active factor in the extract has been identified as a protein with a size of 100 kD. Activation of the cutinase gene by the cutin monomer and the protein factor is specific, as the transcription of other genes has been found unchanged by the addition of these two components. Novobiocin, an antibiotic that inhibits the initiation of transcription, severely inhibits the transcription of cutinase. These results indicate that the cutin monomer and protein factor stimulate initiation of transcription of the cutinase gene (Podila et aI., 1988). Among the cutin monomers, w-hydroxy fatty acids with one or more midchain hydroxyl groups, such as 10, 16-dihydroxypalmitic acid and 9,10,18trihydroxystearic acid, are essential for transcription activation. w-Hydroxy fatty acids that have a hydroxyl group at the nonreducing ends, but lack midchain hydroxyl groups, are not effective activators. Removal of the mid-chain hydroxyl group decreases its activation effect (Podila et aI., 1988). Ricinoleic acid [CH3(CH2)5CHOHCH2CH=CH(CHz)7COOH], which has a mid-chain hydroxyl group but lacks a w-hydroxyl group, is not active. Thus, cutinase gene transcription activation requires the unique structural elements found in the cutin monomers. The protein factor that activates transcription of the cutinase gene in isolated nuclei exhibits binding to a 32P-labeled 360-bp segment ofthe 5'-flanking region of the cutinase gene. A similarly sized DNA fragment from the 3'-flanking

Penetration of Cuticles - 17 region or other sources does not show any binding activity. These results strongly indicate that the protein factor is a DNA-binding transcription factor. The transcription factor aggregates in the absence of high ionic strength, and a nuclear extract from F. solani f. sp. pisi successfully substitutes for the protein factor in the cutinase transcription assays. These results indicate that the protein is of nuclear origin. Electrophoresis of the 360-bp segment of the 5'flanking region of cutinase gene after incubation with either this nuclear extract or the protein factor from the supernatant shows the same gel retardation band. These results indicate that the protein factor is a nuclear protein that binds to the upstream region of the cutinase gene and serves as a transcription-activating factor. The role of cutin monomer in transcription activation of the cutinase gene remains unclear. Incubation of isolated nuclei with protein factor and monomer in the presence of [32p]_ATP shows phosphorylation of a ｾＵＰＭｫｄ＠ protein, whereas neither protein factor nor monomer alone is adequate to achieve maximum phosphorylation of the protein. Cutinase gene transcription is severely inhibited when isolated nuclei are preincubated with the protein factor, cutin monomer, and protein kinase inhibitor H-7 before the addition of transcription components including [32 p]UTP. The results indicate that phosphorylation is required for transcription. A schematic representation of cutinase production by a fungal spore as proposed by Kolattukudy et al. (1989a) is given in Fig. 1-6.

Fig. 1-6. Induction of cutinase biosynthesis in a fungal spore. (Reproduced from Kolattukudy et aI., 1989a, with permission from NRC Research Press, National Research Council of Canada).

1.3.1.2 Cutinase genes Cutinase genes have been cloned from a number of plant-pathogenic fungi, including F. solani f. sp. pisi (Soliday et aI., 1989), Botrytis cinerea (Van der Vlugt-Bergmans et aI., 1997), Colletotrichum capsici, C. gloeosporioides (Ettinger et aI., 1987), Magnaporthe grisea (Sweigard et aI., 1992a), and

18 - Plant Pathogenesis and Resistance Alternaria brassicicola (Yao and Koller, 1994). A genomic library ofF. solani f. sp. pisi (=Nectria haematococca) isolate T-8 has been constructed in lambda Charon 35 vector. Using a labeled cutinase cDNA as a probe, the library has been screened for the cutinase gene. The clones that contained the cutinase gene have been isolated and a 2,818-bp DNA fragment sequenced. A 690-nucleotide open reading frame (ORF) was identified within the fragment (Soliday et aI., 1989). The polypeptide deduced from this ORF has a molecular weight of about 25.5 kD, which is similar to the in vitro translated procutinase (Flurkey and Kolattukudy, 1981). The 940 nucleotides in the 5'-flanking region preceding the ATG initiation codon have also been sequenced. The classical TATAA box commonly found in eukaryotic genes has not been found, but a T AAATAT sequence does exist at the position -111. There is a 260-bp nontranslated sequence atthe 3 '-flanking region that contains no polyadenylation site (Soliday et aI., 1989). Cutinase genes from C. capsici and C. gloeosporioides have also been cloned and sequenced. These two genes, similar to the one from F. solani f. sp. pisi, lack a TAT A box but do have aTAAATAT box at -177 and -160 from the translational start sites, respectively (Ettinger et aI., 1987). The residues involved in the catalytic triad and disulfide cross-linking of cutinases are strongly conserved, yet only 43% of the residues are conserved among the three enzymes (Ettinger et aI., 1987). A gene from M grisea has been cloned using a cDNA clone of the C. gloeosporioides cutinase gene as a heterologous probe. A 2-kb DNA segment containing the gene (CUT1) has been sequenced. The predicted polypeptide has 228 amino acids and a molecular weight of24,276 D. The 5'-flanking region of CUT1 contains the sequence TATAA starting at -117. No classical AATAAA is found at the 3'-flanking region. The CUT1 gene contains two introns, 115 and 147 bp in length. The CUT1 gene product shows significant similarity in amino acid sequence when compared with cutinases from C. capsici (69%), C. gloeosporioides (74%), and F. solani f. sp. pisi (68%) (Sweigard et aI., I 992a,b). Alternaria brassicicola produces two cutinase isozymes in the presence of cutin monomers. Cutinase Ac has the pH optimum of 6.5 and a molecular mass of23 kD. Cutinase B. has the pH optimum of 8.5 and a molecular mass of21 kD (Trail and Koller, 1993). A cDNA library of the fungus has been constructed from poly(AtRNA isolated from mycelia incubated with cutin monomers and transfected into E. coli. Cutinase-specific cDNA clones have been identified by Southern analysis of plasmid DNA using a mixture of two heterologous cutinase cDNA and one cutinase gene as probes. The largest 984bp insert found among positive clones contains the entire cutinase coding region composed of209 amino acids. Southern analysis of genomic DNA ofA. brassicicola yields a similar result. The structural gene of cutinase (CUTAB1)

Penetration of Cuticles - 19 is contained within a 1545-bp genomic DNA fragment. Nucleotide sequences of the cDNA and the gene are identical, with the exception of one intron of 56 bp (Yao and Koller, 1994). The CUTABl gene has been disrupted and CUTAB 1- mutants obtained. During saprophytic growth with cutin as the sole carbon source, CUTAB 1mutants excrete no cutinase Ac and possibly no cutinase Ba. These results indicate that CUTABl encodes one cutinase and that isozymes Ac and Ba evolved from posttranscriptionl or posttranslational modification. Two serine hydro lases with molecular weights of 31 and 19 kD, however, are induced by cutin. The mixture of these hydrolases exhibits cutinase activity. The same hydrolases are also expressed by a wild-type strain during the early stages of host infection. The CUT AB 1- mutants of A. brassicicola remain pathogenic on both leaf and stem tissues of Brassica oleracea, without affecting the direct penetration of the pathogens (Yao and Koller, 1995). Plant cutin monomers trigger the expression of the fungal cutinase gene. The 5'-flanking region of the gene from F. solani f. sp. pisi has been tested for promoter activity. In these experiments, plasm ids contained the 5'-flanking region or its deletion of the cutinase gene from F. solani f. sp. pisi was fused with a promoterless hygromycin-resistance gene. These plasm ids were used to transform F. solani f. sp. pisi, and hygromycin-resistant transform ants were assessed by Southern blot analysis using labeled probes for the hygromycinresistance gene and the putative promoter. The results indicate that promoter activity resides at the 5'-flanking segment of the cutinase gene and that a 360-bp segment immediately upstream to the cutinase initiation codon is sufficient to generate .hygromycin-resistant transformants (Soliday et aI., 1989). Since hygromycin-resistant transformants cannot be used to quantitatively measure promoter activity and inducibility by the cutin monomers, a different approach has been employed. A plasmid has been constructed to contain a constitutive promoter from Cochliobolus heterostrophus to drive the hygromycin resistance gene. In this same plasmid, the promoter sequence of the cutinase gene from F. solani f. sp. pisi has been fused at the 5'-end of the coding region for chloramphenicol acetyltransferase (CAT) gene. The plasmid has been introduced into the protoplasts of F. solani f. sp. pisi by electroporation and hygromycin-resistant transform ants have been selected. In these transformants, CA T activity is inducible by cutin monomers, and this induction is repressed by glucose. The 5'-flanking region of the cutinase gene is progressively shortened, and the transformants obtained with such constructs have been tested for CAT induction by cutin monomers and for glucose repression. Results from this approach indicate that the inducible promoter activity resides in the -225 to -360 region of the cutinase gene (Bajar et aI., 1991). A more detailed analysis of the promoter region has identified the existence of four regulation elements. First, a silencer, between -249 and -287 upstream

20 - Plant Pathogenesis and Resistance from the ATG start codon, keeps the constitutive transcription low and reduces inducibility. Second, an activator element between -360 and -310 functions as an antagonist to the silencer. Third, an element necessary for constitutive transcription is located within 141 bp immediately to the 5' of the ORF. Finally, a GC-rich palindrome at -170 serves as the cutinase-transcription-factor-l binding site (Kamper et aI., 1994). The cutinase transcription factor has been identified as a 49-kD nuclear protein (Bajar et aI., 1991). The promoter activity has also been found in the 5'-flanking regions of the cutinase gene in C. gloeosporioides and C. capsid (Ettinger et aI., 1987). The expression of cutinase genes in plant-fungus interactions has been reviewed by Kolattukudy (1992) and Kolattukudy et al. (1989a,b; 1995).

1.3.1.3 Molecular properties of cutinases Cutinases are extracellular enzymes. They are glycoproteins consisting of3 to 16% carbohydrates. All fungal cutinases so far examined have similar amino acid compositions. They contain one to three methionines, one to five histidines, one to four tryptophans, and two to five cysteines per molecule (Kolattukudy, 1985). Most of them have a molecular weight of about 25 kD. Exceptions, however, do exist. Cutinases from Colletotrichum lagenarium have a M. of 60 kD (Bonnen and Hammerschmidt, 1989a), and those from Rhizoctonia solani have M. = 17 to 18.5 kD (Trail and Koller, 1990). Despite great similarities in amino acid compositions, immunological heterogeneity exists among cutinases from different fungi. Ouchterlony double diffusion experiments have shown that cutinases from Fusarium roseum sambudnum, Ulocladium consortiale, Streptomyces scabies, and Helminthosporium sativum did not cross-react with the antibody prepared against F. solani f. sp. pisi cutinase I. Furthermore, the enzymatic activity of cutinase from these fungi was either not inhibited or inhibited only slightly by the antibody (Lin and Kolattukudy, 1980a). Two cutinase isozymes, I and II, have been isolated from the extracellular fluid of Fusarium solani f. sp. pisi grown on a cutin medium. The molecular weight of cutinases I and II is 22,000. The optimal pH for the enzyme is 10 (Purdy and Kolattukudy, 1975a,b). Cutinases I and II contain 4.3 and 5.1% carbohydrates, respectively. Treatment of cutinase with alkaline NaWH4 breaks carbohydrates from the protein and generates labeled sugars and amino acid residues at the sites of detachment. Hydrolysis of the labeled protein followed by chromatographic analyses of the products shows that serine, threonine, phydroxyphenylalanine, and p-hydroxytyrosine account for nearly all of the 3H contained in the protein. Labeled sugars are mannose, arabinose, N-acetylglucosamine and glucuronic acid (Lin and Kolattukudy, 1980b). The cutinase from F. solani f. sp. pisi releases free amino acids when treated with carboxypeptidase. The N-terminal amino group of cutinase I does not react

Penetration of Cuticles - 21 with phenylisothiocyanate. These results indicate thatthe cutinase has a free Cterminus and that the N-terminus is not free. Furthermore, N-gulonyl-glycine has been isolated from the protease digest of the labeled protein. It appears that the N-terminus has glycine in amide linkage with glucuronic acid (Lin and Kolattukudy, 1980b). Cutinases from F. roseum culmorum, F. roseum sambucinum, Ulocladium consortiale, Streptomyces scabies, and Helminthosporium sativum are similar to those produced by F. so/ani f. sp. pisi in molecular weight and carbohydrate content. Treatment of cutinases with alkaline NaB3H4 reveals that the amino acids involved in O-glycosidic linkages are serine, threonine, and ｾＭｨｹ､ｲｯｸﾭ phenylalanine in U. consortiale; serine and ｾＭｨｹ､ｲｯｸｰ･ｮｬ｡ｩ＠ in F. roseum culmorum; and serine in F. roseum sambucinum. The O-glycosidically attached sugars for the three cutinases are man nose, an unidentified neutral sugar, glucosamine, and glucuronic acid. Cutinases from H sativum and S. scabies have no O-glycosidically linked sugars and give no labeled amino acids. The carbohydrates of these two cutinases are probably attached by alkali-stable asparaginyl linkages (Lin and Kolattukudy, 1980b).

1.3.1.4 Catalytic properties of cutinases All of the fungal cutinases that have been purified to homogeneity show similar catalytic properties. All hydrolyze cutin at maximal rates at pH 9-10. Cutinase hydrolyzes cutin initially to oligomers and further hydrolyzes to monomers. This enzyme shows specificity for primary alcohol esters. The structurefunction relationship of cutinase has been reviewed (Longhi and Cambillau, 1999). All fungal cutinases are severely inhibited by reagents reacting with a catalytic triad containing an active serine commonly found in serine proteases (e.g., bovine a-chymotrypsin, bovine y-chymotrypsin, bovine chymotrypsinogen, bovine trypsin, and bovine trypsinogen). Chemical modification of the serine hydroxyl group with diisopropyl fluorophosphate [DIFP, F-P-{OCH (CH 3 )2}2=O] inhibits the enzyme activity. Treatments of the carboxyl group with carbodiimide (cyanamide, H2NC=N) and of the imidazole group of histidine with diethyl pyrocarbonate (C2HS-OOC-O-COO-C2HS) also inhibits the enzyme activity. Thus, the presence of the catalytic triad in cutinases is established. The mem bers of the catalytic triad in the cutinase from F. solani f. sp. pisi have been identified as Asp 99, Ser 136 , and His 204 by Kolattukudy (1987). The identification is based on the amino acid sequence deduced from the nucleotide sequence of cloned cDNA for cutinase and direct amino acid sequencing of tryptized peptide. Identification of histidine is simple since there is only one molecule in the enzyme. The active serine is identified by treating the enzyme with [3H]-DIFP, followed by proteolysis and amino acid sequencing of the

22 - Plant Pathogenesis and Resistance tryptic peptide containing the modified residue (Soliday and Kolattukudy, 1983). The active carboxyl group was similarly identified by first treating the enzyme with carbodiimide and [14C]-glycine ethyl ester. The protein was subsequently hydrolyzed, the labeled peptide isolated, and the amino acid sequence determined (Soliday et aI., 1984). The three-dimensional structure of a recombinant cutinase from F. so/ani f. sp. pisi and expressed in E. coli has been studied. The results reveal that the active site of the mature cutinase (with a 16-amino acid signal peptide removed) is composed of the triad Ser 120, His 188, and Asp l75. The reassignment of the triad aspartic acid at position 175, but not at 83 (99 in procutinase), is that ASpl75 is conserved among the cutinase sequences. Furthermore, the triad amino acids in Iipases commonly occur in the sequence order of SerH / ｾ＠

OH

0-' , 0

OH

coo·

L

0..,

ｾｈ＠

ｾ＠

\

+

6H OH

pOH OH 00

0

coo·

Pecta!!.::

Fig. 2 .. 7. Enzymatic degradation of pectic substances. (Adapted from Fogarty and Ward, 1974).

Erwinia chrysanthemi E. chrysanthemi strains B374 and 3937 each has five genes: petA, pe/B, petC,

pelD, and petE, encoding PeIA, PeIB, PeIC, PeID, and PeIE, respectively (Hugouvieux-Cotte-Pattat et aI., 1992). PelA is an acidic protein with an pI between 4.0 to 5.0; PeIB and PelC are neutral with pIs between 7.0 to 8.5; and PelD and PelE are alkaline with pI values of 9.0 to 10. These five genes are arranged into two clusters with pelB and pelC on a 2.5-kb DNA fragment while pelA, pelD, and pelE are on a 5.0-kb DNA fragment. Not every strain of E. chrysanthemi contains five pel genes. Strain EC 16 has petA, pe/B, pelC, and

74

- Plant Pathogenesis and Resistance

pelE (Barras et aI., 1987), and strain CUCPB 1237 has pelB and pelC (Kotoujansky et aI., 1987). Table 2-1. Pectate Iyases (endo-Pel, EC 4.2.2.2; exo-Pel, EC 4.2.2.9) produced by plant pathogens. Pathogen

Type

Molecular Optimal pI mass, kD pH

Reference

Botrytis cinerea

endo

31

7.5

Cladosporium cucumerinum Colletotrichum gloeosporioides Colletotrichum gloeosporioides f. sp. malvae Erwinia carotovora subsp. atroseptica strain SCRI 1043 Erwinia carotovora subsp. atroseptica strain SR8 Erwinia carotovora subsp. carotovora Erwinia carotovora subsp. carotovora strain EC Erwinia carotovora subsp. carotovora strain SCRI 193 Erwinia carotovora subsp. carotovora strain SCC3193 Erwinia carotovora subsp. carotovora strain 71 Erwiniachrysanthemistrain B374 Erwinia chrysanthemi strain ECI6

endo

9.7

endo

27 39 33.2 32.8 39 (Pell) 39 (PeI2) 31

8.4 8.3 10.3 10.0 8.5-9.0 9.2

Movahedi and Heale (1990) Robertsen (1990) Wattad et al. (1997) Shih et al. (2000) McMillan et al. (1992) Allen et al. (1987)

exo

76

8.5

Kegoya et al. (1984)

endo

8.5 8.3

endo

44 (PelA) 44 (PelB) 42 (PeIC)

endo

35 (PelB)

9.5

endo

35

7.96

endo endo

endo

43 (PeID) 44 (PeIA) 39 (PeIB) 39 (PelC) 76 25 (race 0) 37 (race 5) 23.4

endo

25

endo endo

41 41

10.0

endo

42

9.7

endo

36-41

endo

41

endo endo

Erwinia chrysanthemi strain EC 16 exo Fusarium oxysporum f. sp. ciceri endo Fusarium oxysporum f. sp. lycopersici 42-87 Fusarium solani f. sp. pisi Pseudomonas fluorescens CY091 Pseudomonas marginalis strain N6301 Pseudomonas viridiflava strain SF-312 Xanthomonas campestris pv. campestris Xanthomonas campestris pv. malvacearum B414

8.1

9.4 9.4 10.3

9.0 >10 8.6 4.2-4.6 8.9-9.5 8.8 8.8-9.5 9.0 7.5-8.0 8.6 9.5 9.0 10.5 9.0 7.4

Lei et al. (1987, 1988) Hinton et al. (1989) Heikinheimo et al. (1995) Liu et al. (I 994a) Van Gijsegem (1989) Barras et al. (1987)

Brooks et al. (1990) Artes and Tena (1990) Huertas-Gonzalez et al. (1999) Crawford and Kolattukudy (1987) Liao (1991) Nikaidou et al. (1993) Liao et al. (1988) Dow et al. (1987)

9.7

Liao et al. (1996)

Degradation of Cell Walls -

75

In strain 3937, the pelA gene has an ORF of 1179 bp encoding a polypeptide of 41,555 Da (Hugouvieux-Cotte-Pattat et a!., 1992). Mutations in the pelA, pe/D, and pe/E genes have resulted in a significant reduction of virulence on Saintpaulia ionantha. Mutations in the pel genes in the other cluster do not alter bacterial virulence on this plant (Boccaraet a!., 1988). On pea, the PelA mutant has reduced virulence, and a mutant deleted of all the genes except pelA (peIBCDE) was as virulent as the wild-type strain. These results indicate that PelA is essential for virulence on pea (Beaulieu et a!., 1993). Recent results indicate that strain 3937 has a second set of pectate lyases. A pectate lyase gene has been cloned from a genomic library of a derivative deleted of the five major genes. The gene, pelL, has an ORF of 1275 bp, encoding a polypeptide of 425 amino acids, including a 25 amino acid signal sequence. No homology has been detected between pelL and other bacterial pel genes. Transcription ofpelL is induced by pectic catabolic products, suppressed by catabolic repression and affected by growth phase. The PelL· mutant has reduced virulence on potato tubers and Saintpaulia ionantha plants, indicating the importance of this enzyme in soft-rot disease (Lojkowska et a!., 1995). In strain B374, the pelA, pe/D, and pelE genes, in lengths of approximately 1.2, 1.3, and 1.4 kb, respectively, are clustered in the order ofpe/D-peIE-pelA. Each gene constitutes an independent transcript unit (Reverchon et a!., 1986). The pelB and pelE genes from strain EC 16 have been cloned and expressed in E. coli. The pelB gene encodes a protein of375 amino acids with a molecular weight of 40,213. The mature PelB has 353 amino acids and a molecular weight of37,922 by leaving a signal peptide of22 amino acids at N-terminal. The pelE gene encodes a protein with 385 amino acids and a molecular weight of41,115. Again, the mature PelE is composed of 355 amino acids with a molecular weight of 38,037 by leaving a 30-amino acid peptide at N-terminal. The isoelectric points ofPelB and PelE are 8.8 and 9.9, respectively. E. coli cells contain ingpelE macerate potato tuber tissue as efficiently as EC 16 cells. E. coli cells containingpelB are less effective. Thus, there is a difference in macerating abilities between pelB and pelE products (Keen and Tamaki, 1986). In strain CUCPB1237, PelB and PeIC, encoded by pelB and pelC, have molecular masses of 30 and 33 kD and pIs of 7.6 and 8.1, respectively. Either Pel contributes significantly to bacterial virulence toward maceration of potato tubers. TLC of reaction products and viscometric assays reveals little difference between these two isozymes (Schoedel and Collmer, 1986).

Erwinia carotovora subsp. carotovora E. carotovora subsp. carotovora also produces multiple pectate lyase isozymes. Strain 71 produces Pell to PelS with pIs of >10.0,9.7, 9.2, 8.0, and 6.6, respectively (Willis et a!., 1987). A 2.2-kb DNA fragment containing the pell gene has aI, 122-bp ORF that can encode a pre-Pell of 374 amino acid

76

- Plant Pathogenesis and Resistance

residues. A signal peptide of 22-amino acid residues is present within the Nterminal region of the protein. The mature Pel I is predicted to have a molecular mass of 38.5 kD and an pI of 10.0 (Chatterjee et aI., 1995a). There is tight linkage between pel3, the gene encoding Pe13, and pehl, a gene encoding an endopolygalacturonase. A 3,500-bp chromosomal segment that contains the ORFs for Pel3 and Peh I has been cloned. The 1,041-bp pe13 ORF and the 1,206-bp peh-l ORF are separated by a 579-bp sequence. These genes are transcribed from their own promoters, and their expression in vivo is activated by plant signals. The pel3 gene is predicted to encode a pre-Pel3 of347 amino acid residues having a M;. of 37, 179 and an pI of 8.08. The first 21 amino acid sequence at the N-terminal end is the signal peptide. There is no significant homology between pe13 gene and the pel genes of E. chrysanthemi (Liu, I 994a). Strain SCC 3193 produces four Pel isozymes in culture media. The nucleotide sequence of pelB contains a 1,040-bp ORF for a predicted 347amino acid polypeptide with a M;. of 37,482. There is a 22-amino acid signal leader at the N-terminus. The predicted molecular mass of the processed PelB is 35,051 (Heikinheimo et aI., 1995). The predicted amino acid sequence of PelB has 93% amino acid homology to that of Pel3 of strain 71, indicating a common origin of these two Pels (Heikinheimo et aI., 1995). Strain SCRI193 has four pel genes encoding four different Pel isozymes. The pIs for the two major Pels are 9.1 and 9.3 (Plastow et aI., 1986). Strain ER(AMS6082) has one pel gene that has been cloned. In strain ECI4, a 3.4-kb DNA fragment has been transformed into E. coli. Three proteins with pectate lyase activity have been produced by the transformants. Two of the Pels have pI of9.5 and can macerate potato tuber slices (Roberts et aI., 1986). Erwinia carotovora subsp. atroseptica The pelA and pelB genes from E. carotovora subsp. atroseptica strain EC have been cloned and sequenced (Lei et aI., 1987, 1988). The PelA and PelB have been purified from E. coli harboring recombinant plasm ids containing the pelA and petB genes. The pH optima are 8.5 for PelA and 8.25 for PelB. The purified PelA or PelB alone can macerate potato slices. The N-terminal and C-terminal peptide sequences of the purified PelA and PelB have been determined and compared with the polypeptide sequences deduced from the DNA sequences of pelA andpelB. An ORF encoding a protein of374 amino acids has been found on each nucleotide sequence. Both pelA and pelB code for the same signal peptides of22 amino acids with the exception of2 amino acids. Both PelA and PelB start at amino acid 23 of the predicted sequence withoutthe 22-amino-acid leader peptide. Thus, the matured PelA and PelB deduced from the DNA sequences contain 352 amino acids and have a calculated molecular weight of about 3 8,000. They have an apparent molecular weight of 44,000 as determined

Degradation of Cell Walls -

77

by SDS-PAGE, identical to some of the Pels produced by strain EC. Nucleotide sequences of pelA and pelB are 82% homologous. The deduced polypeptide sequences of PelA and PelB are 88% homologous (Lei et aI., 1988). Due to the similarity between PelA and PelB, it is possible that pelA and pelB are duplicates. PelB of E. carotovora subsp. atroseptica strain EC and PelB of E. chrysanthemi strain EC 16 contain 352 and 353 amino acids, respectively. The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical (Lei et aI., 1987). It is probable that pelB in these two bacteria are diverged from a common ancestor. Strain ECI4 has one pel gene encoding an endo-Pel with aM,0f31 ,000 and pI of 9.2 (Allen et aI., 1987).

Xanthomonads Xanthomonas campestris pv. campestris produces three Pels. The gene for the isozyme I has been constructed in the plasmid pIJ3051 and cloned in the nonpectolyticX campestris pathovars translucens and vesicatoria. The isolates containing pIJ3051 produce a Pel isozyme with a molecular mass of 33 kD, similar to one of the three isozymes produced by the wild type. Mutants lacking isozyme I, induced by Tn5 insertion into the wild type, are as virulent as the wild type in pathogenicity tests. These results indicate that Pel isozyme I is not necessary for pathogenicity (Dow et aI., 1989). Pseudomonads Certain strains of Pseudomonas jluorescens, P. marginalis, and P. viridiflava are postharvest pathogens causing soft rot of fruits and vegetables in storage and at markets. Although Pem, Peh, and Pnl have been detected in some strains, Pel is considered the principal enzyme responsible fortissue maceration. Unlike erwinias which produce multiple Pel isozymes, pseudomonads produce only one or two Pels. The pel gene from P. jluorescens strain CY091 encodes an alkaline Pel with an pI of 10.0 and a M. of 41,000 (Liao, 1991; Liao et aI., 1997). P. marginalis also produces Pels. Strain N630 I contains an 1140-bp ORF encoding a Pel of 380 amino acids, including a 29-amino acid signal peptide (Nikaidou et aI., 1993). Strain MAFF 03-01173 produces a Pel of 43 kD (Hayashi et aI., 1997). P. viridiflava strain SJ074 produces a Pel with a Mr of 42,000 and an pI of9.7. The pel gene has been cloned. The mutant MEl, which has the pel gene interrupted by Tn5, produces 70- to 100-fold less Pel than the wild type and fails to cause tissue maceration in plants (Liao et aI., 1992). Plant Pathogenic Fungi Several plant pathogenic fungi produce pectate Iyases. A pectate lyase produced by F. solani f. sp. pisi (=Nectria haematococca, mating population VI) is essential for host infection (Crawford and Kolattukudy, 1987). The gene,

78

- Plant Pathogenesis and Resistance

designated pelA , encoding the Pel has been isolated from this fungus. A probe has been synthesized by PCR with oligonucleotide primers based on the known amino acid sequences of two regions of the mature protein and first-strand cDNA as templates. Both cDNA and the gene has been isolated and sequenced. This lyase sequence shows Iitt Ie homology to those of other pectolytic enzymes. The pelA gene mRNA is detected only when F. solani f. sp. pisi is grown with pectin, and there is no detectable transcript accumulation when the fungus is grown with glucose as the sole carbon source. The levels of transcription decrease rapidly prior to maximal enzyme accumulation, indicating a mechanism of self catabol ite repression (Gonzalez-Candelas and Kolattukudy, 1992). A pectate lyase from F. oxysporum f. sp.lycopersici has been purified and characterized (Di Pietro and Roncero, 1996c). PH, the gene encoding the enzyme has also been cloned (Huertas-Gonzalez et aI., 1999). It encodes a 240amino acid polypeptide with an N-glycosylation site and a 15-amino acid Nterm inal signal peptide. The deduced protein has aM. of23.4 kD and a pI of7.4 (Huertas-Gonzalez et aI., 1999). Colletotrichum gloeosporioides f.sp. malvae is a plant pathogenic fungus used in control of the annual weed mallow (Malva pusilla). The fungus produces Peh, Pnl and Pel to macerate host tissues. Two full length pel genes have been cloned from this fungus (Shih et aI., 2000). B. Pectin Iyases (Pnl, EC 4.2.2.10) Pectin Iyases (Pnls) split a-l,4-bonds between methylgalacturonides in the pectin molecule in a lytic fashion, resulting in the formation of an unsaturated bond between C-4 and C-5 of a uronide in the reaction product (Fig. 2-7). Many soft-rot erwinias produce Pnls when the cultures are treated with DNA-damaging agents, such as mitomycin C, nalidixic acid, or UV light (Table 2-2). The Pnl production is frequently accompanied by cell lysis and bacteriocin production (l1oh et aI., 1980). The Pnl from E. chrysanthemi EC 183 has aM,. of34,500, an optimum pH of8.3, an pI of9.45 (Tsuyumu et aI., 1985). The Pnl produced by E. carotovora subsp. atroseptica strain SCRI 1043 has a M. of 31,000, an optimum pH of 8.0, and an pI of 9.4 (Godfrey et aI., 1994). E. carotovora subsp. carotovora strain 71 produces a 32.1- kD Pnl with an pI of 9.92. The gene pnlA, which encodes the enzyme Pnl, is located on a 3.4 kb EcoRI DNA fragment and is contained in an ORF of 870 bases. The sequence of the first 20 amino acid residues of the enzyme purified from the culture filtrate of the pathogen agrees completely with the predicted amino acid sequence of the N -term inal segment of the 0 RF. Th is indicates that the enzyme is not subjected to processing by a signal peptidase (Chatterjee et aI., 1991). The Pnl from Pseudomonas marginalis N630 1 has a M. of 34,000 and an optimum pH of 8.0 (Nikaidou et aI., 1995). The pnl gene that encodes the Pnl

Degradation of Cell Walls -

79

has been cloned and sequenced (Nikaidou et aI., 1992). Deletion clones of pnl have been constructed and expressed in E. coli. Pnl activities have been detected only in E. coli with recA +, indicating the requirement of recA for Pnl expression (Nikaidou et aI., 1994). Production ofPnl from fungal pathogens has also been reported (Table 2-2). Using oligonucleotide primers designed from the amino acid sequence ofPnID from Aspergillus niger, three different 220-bp fragments have been cloned from Glomerella cingulata. One of the 220-bp PCR products has been used as a probe to isolate a Pnl-encoding gene from a A genomic DNA library prepared from G. cingulata. This gene encodes a putative, 380-amino acid peptide (Templeton et aL, 1994). Table 2-2. Pectin Iyases (endo-Pnl, EC 4.2.2.10) produced by plant pathogens. Pathogen

Colletotrichum gloeosporioides Colletotrichum lindemuthianum race y (ATCC 56987) Erwinia carotovora subsp. atroseptica strain SCRI 1043 Erwinia carotovora subsp. carotovora strain 71 Erwinia carotovora subsp. carotovora strains Er, Ar, Ar13,6083 Erwinia chrysanthemi strain ECI83 Pseudomonas marginalis N6301 Rhizoctonia solani strain 82 Rhizoctonia solani AG2-2

pI

Reference

9.4

Templetonetal. (1994) Wijesundera et al. (1989) Godfrey et al. (1994)

Molecular mass, kD

Optimal pH

23 (I) 28 (II) 31

9.4 8.2 9.7 8.0

32.1

9.9

28

8.0

9.6

Itoh et al. (1982)

34.5

8.3

9.5

Tsuyumu et al. (1985)

34 45.8 35

8.0 8.4 8.0

8.1 10.1

Nikaidou et al. (1995) Marcus et al. (1986) Bugbee (1990)

ｃｨ｡ｴ･ｾｩ＠

et al. (1991)

C. Polygalacturonases (endo-Peh, EC 3.2.1.15; exo-Peh, EC 3.2.1.82) Polygalacturonases attack (X-l,4 glycosidic bonds of pectic substances by a hydrolytic mechanism. Thus, they are pectate hydroJases (Pehs) (Fig. 2-7). Pehs may attack their substrates randomly or in a terminal manner and are designated as endo-Pehs (EC 3.2.1.15) and exo-Pehs (EC 3.2.1.82), respectively. The activity ofPeh may be measured by loss of viscosity and increase in reducing groups. These two assays can be used in combination to distinguish between endo and exo enzymes. Endo enzymes will normally induce a 50% viscosity loss of high molecular weight substances with only 0.5-3% hydrolysis, whereas 20% hydrolysis or more may be required for an exo enzyme to induce a similar viscosity loss. These two techniques, however, do not permit determination of whether the enzyme is a hydrolase or a lyase. Lyase activity is determined by

80

- Plant Pathogenesis and Resistance

UV absorption or the TBA method described previously (see Section 2.3 .1.1.A). Generally the pH optima for Pehs are acidic. The activity of Peh is often reduced by Ca+ 2, presumably due to the formation of insoluble calcium pectate. Pehs are produced by numerous plant pathogens (Table 2-3). Endo-Pehencoding genes, peh, have been cloned from strains 71 and SCRI193 of E. carotovora subsp. carotovora (Plastow et aI., 1986; Liu et aI., 1994a). In strain 71, the peh gene was found linked to a pel gene. The coding region of pehl is 1206 bp, which is sufficient to encode a pro-Peh I of 402 amino acid residues with a molecular weight of 42.6 kD. The 26 amino acid residues at the Nterminal are the signal peptide (Liu et aI., I 994a). Strain EC of the subspecies atroseptica produced Peh with an apparent molecular mass of 43 kD. The enzyme macerated carrot, potato, and turnip slices. When the peh gene was cloned in E. coli, it produced Peh similar in molecular weight to the one produced by the bacterium. Pseudomonas solanacearum strain A W contains a pglA gene that encodes a 52-kD endo-Peh. The gene, contained on a 1.8-kb DNA fragment, translated a pre-Peh with a N-terminal signal sequence of21 amino acids. The mature Peh has a M, of 52,000 and an pI of 9.0. An inactivated pglA gene has been used to mutate the chromosomal pglA gene of P. solanacearum by marker exchange mutagenesis. The resulting mutant was deficient in production of the 52 kD Peh and took twice as long to wilt and kill tomato plants as compared to the wild type in bioassay experiments. Complementation in trans with the pglA gene restored virulence to near wild-type levels (Schell et aI., 1988). The signal sequence has been shown to direct the export of the Peh across of the inner membrane to periplasm (Huang and Schell, 1990). Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. It secretes a Peh with an pI of 4.5 into culture media. Other biovars produce neither root decay of grape seedlings nor extracellular Peh in culture (McGurie et aI., 1991). Biovar 3 strain CG49 contains a Peh-encoding gene pehA that is located on a 2.5-kb HindIII-SalI fragment. When cloned in E. coli, the bacterial cells harboring the pehA gene produce a Peh in culture with the same pI as the enzyme produced by strain CG49 (Rodriguez-Palanzuela et aI., 1991 ). Sclerotinia sclerotiorum is known to secrete a set of enzymes that macerate plant tissues (Riou et aI., 1991, 1992). The gene pgl, which encodes an endogalacturonase (Peh 1) has been cloned and sequenced. The coding region consists of a non interrupted 1143-bp ORF. The nucleotide sequence encodes a polypeptide of380 amino acids with a calculated M, of37,849 (Reymond et aI., 1994). The amino acid sequence is comparable to other fungal Peh sequences (Caprari et aI., 1993; Scott-Craig et aI., 1990) and shows a high level of homology (41.5 to 59.8%) (Reymond et aI., 1994).

Degradation of Cell Walls -

81

Table 2-3. Polygalacturonase (endo-Peh, EC 3.2.1.15; exo-Peh, EC 3.2.1.82) produced by plant pathogens. Pathogen

Type

Molecular Optimal pI mass, kD pH

Reference

Alternaria alternata Alternaria alternata rough lemon pathotype Aspergillus niger Botrytis cinerea isolate 347

exo endo

43 60

Nozaki et al. (1997) Isshiki et al. (1997)

endo endo

4.0-5.0 4.0-5.0 4.0-5.0 4.0-5.0 5.0 5.0

5.0

8.8 5.1 8.8 4.9 4.9 3.5

Cervone et al. (1986) Johnston and Williamson (1992)

Cladosporium cucumerinum Cochliobolus carbonum race 1

endo endo exo

33.5 36 (I) 36 (II) 65 70 38 32 48

Colletotrichum gloeosporioides

endo endo endo

62 (I) 68 (II) 33

5.1 5.1 5.0

4.95 5.00 9.4

endo

39

5.4

9.4

Cryphonectria parasitica Ep 155

endo exo endo

36 90 42

4.5-5.0

5.7 6.5 8.0

Diplodia natalensis

endo

5.0 5.0

Erwinia carotovora subsp. atroseptica strain SCRI 1043 Erwinia carotovora subsp. carotovora strain SCRI 193 Erwinia carotovora subsp. carotovora strain 71 Erwinia chrysanthemi strain CUCPB1237 Fusarium moniliforme Fusarium oxysporum f. sp. ciceri race 0

endo

64 (I) 45 (II) 39

Stanley and Brown (1994) Gao and Shain (1994) Barmore et al. (1984)

10.3

endo

43

4.0

9.3

McMillan et al. (1992) Plastow et al. (1986)

endo

40

5.5

9.94

Liu et al. (1994a)

exo

67

6.0

8.3

endo endo

39.5 44 (I) 44 (II) 76 35.5 74 (II) 63 (III)

Collmer et al. (1982b) Cervone et al. (1986) Artes and Tena (1990)

exo

Colletotrichum lindemuthianum race y (ATCC 56987) Colletotrichum lindemuthianum races ｾ＠ and y Colletotrichum musae

Fusarium oxysporum f. sp. lycopersici isolate 42-87 Fusarium oxysporum f. sp. melonis

exo endo exo

exo

58

4.5 4.5 4.5 5.0

5.0

9.1

6.7 8.0 6.4 4.2 6.2 4.5 7.0 6.4

Robertsen (1987) Walton and Cervone (1990); Scott-Craig et al. (1998) Pursky et al. (1989) Wijesundera et al. (1989) Keon et al. (1990)

Di Pietro and Romcero (1996a, b, 1998); Maceira et al. (1997) Martinez et al. (1991)

82

- Plant Pathogenesis and Resistance

Table 2-3. (Continued) Pathogen

Type

Molecular Optimal pi mass, kD pH

Reference

Fusarium oxysporum f. sp. radicis-lycopercisi Fusarium solani Gaeumannomyces graminis var. tritici Geotrichum candidum Leptosphaeria maculans Phomopsis cucurbitae Pseudomonas cepacia

exo

68

5.6

endo endo

38 42

6.0 4.5-5.5

9.5 8.1

Vazquez et al. (1993) Zhang et al. (I 999b ) Dori et al. (1992)

endo endo endo endo

38 42 54 46

4.2

7.8

5.0 4.5

4.2 8.1

Rhizoctonia solani

endo

Rhizopus stolonifer

endo

34 (I) 37 (II) 32

4.8 5.4 4.8

6.8 7.4 8.0

Sclerotinia sclerotiorum

endo exo

30 68

4.8 5.0

Venturia inaequalis

exo

90.5

5.5

Verticillium albo-atrum

endo

37

4.6-5.0

8.6

Barash et al. (1984) Sexton et al. (2000) Zhang et al. (1999a) Gonzalez et al. (1997) Marcus et al. (1986) Lee and West (l981a) Marciano et al. (1982); Riou et al. (1992) Valsangiacomo and Gessler (1992) Huang and Mahoney (1999)

E. chrysanthemi does produce exo-Peh (Table 2-3). The exo-Peh produced by strain CUCPBI237 has an pI of 8.3 and a M. of 67,000 (Collmer et aI., 1982b). The pehX gene encoding the exo-Peh has been isolated from a derivative of strain EC 16. The cloned pehX gene is expressed highly in E. coli, but most of the enzyme is localized in the periplasm. The nucleotide sequence of pehX reveals the presence of an N-terminal signal peptide and an ORF encoding a preprotein of 64,608 daltons (He and Collmer, 1990). D. Pectin methylesterase (Pem, EC 3.1.1.11) Pem removes the methoxyl groups on C-6 ofpolymethylgalacturonate to form polygalacturonate (Fig. 2-7). These enzymes are hydro lases and are widely distributed among higher plants (Rillo et aI., 1992; Bordenave and Goldberg, 1993; Nighojkar et aI., 1994; Richard et aI., 1994; Mareck et aI., 1995; McMillan and Perombelon, 1995). They are involved mainly in fruit maturation (Tieman et aI., 1992). Many plant-pathogenic bacteria and fungi also produce Pem (Table 2-4). In general, Pem of microbial origin, particularly those from fungi, have a lower optimum pH for activity than those from higher plants. The activity ofPem can be measured by determ ining the amount of acid or methanol produced.

Degradation of Cell Walls -

83

Table 2-4. Pectin methylesterase (Pem, EC 3.1.1.11) produced by plant pathogens. Pathogen

Molecular mass, kD

Aspergillus oryzae KBN616 38.5 Botrytis cinerea 42 (I, II) Erwinia chrysanthemi strain B374 37 (Pem) Erwinia chrysanthemi strain 3937 37 (PernA) 45 (PemB) Phytophthora infestans 45 (I) 35 (II) Rhizoctonia solani 26 Uromyces viciae-fabae 33.5 (A) 40.0 (B)

Optimal pH

pi

5.0 7.0-7.4 5-9

Kitamoto et al. (1999) Reignault et al. (1994) 9.6-9.9 Plastow (1988) Laurent et al. (1993) Shevchik et al. (1996) 9.6 Forster and Rasched (1985) Marcus et al. (1986) 6.2 Frittrang et al. (1992) 8.4 5.7

7.5 7.0 6-8 7.7 6.0 5.5-7.5

Reference

E. chrysanthemi strain 3937 produces two pectin methylesterases, PernA and PemB (Kotoujansky et aI., 1985, 1987). The gene pem has been cloned and mutagenized by mini-Mu transposable elements. The Pem' derivative of the strain 3937 is noninvasive when inoculated onto Saintpaulia plants (Boccara and Chatain, 1989). E. Rhamnogalacturonanases RG-I is the backbone of pectic substances. It consists of 1,4-linked a-Dgalacturonan chain interrupted at intervals by single 1,2-linked a-L-rhamnopyranosyl residues. The hydroxyl group at the C-4 of the Rha residues is the site where side chains arabinan, arabinogalactan, and rhamnogalacturonan II attach (see Section 2.2.2.I.A). The region with side chains attached, known as the hairy region, is not degraded by pectate lyase. Recently, enzymes with specificity toward the hairy region of pectin have been isolated from Aspergillus aculeatus and Botrytis cinerea. These fungi produce rhamnogalacturonan a-L-rhamnopyranohydrolase (RG-hydrolase), which hydrolyzes RG-I fragments and releases Rha from the nonreducing end (Mutter et aI., 1994; Fu et aI., 200 I). The cDNA clones encoding RG-hydrolase have been isolated from A. aculeatus. One clone contains a 1320-bp ORF (rhgA) for a predicted 440-residue polypeptide of 45,943 D. There is an IS-residue signal peptide at the N-terminus. This mature RG-hydrolase has a predicted molecular weight of 44,162 (Kofod et aI., 1994; Suykerbuyk et aI., 1995). The other clone contains a 1584-bp ORF (rhgB) encoding a 527-residue polypeptide with a calculated molecular weight of 56,187. There is a signal sequence of 19 amino acids at the N-terminus. The molecular weight of the mature, second RG-hydrolase is 54,200 (Kofod et aI., 1994). A. aculeatus also produces rhamnogalacturonan aL-rhamnopyranosyl-(1-4)-a-D-galactopyranosyl uronide lyase (RG-Iyase). The enzyme cleaves the linkage between Rha and GaIA in the backbone, leaving an

84

- Plant Pathogenesis and Resistance

unsaturated galacturonic acid at the nonreducing end and an Rha atthe reducing end of the product. It has an optimum pH of 6.0 and pI of 5.1 (Mutter et aI., 1996). F. a-L-Arabinofuranosidase (EC 3.2.1.55) and endo-l ,5-a-L-Arabinanase (EC 3.2.1.99) a-L-Arabinofuranosidase activity has been detected in fruits of Japanese pear (Pyrus serotina var. culta cv. Hosui). The enzyme has a M, of 42,000 (Tateishi et aI., 1996). Aspergillus niger produces both exo- and endo-enzymes that hydrolyze arabinan. The exo-enzymes, arabinofuranosidases A and B, act on pnitrophenyl-a-L-arabinofuranoside and 1,5-a-L-arabinofuranose oligomers and release arabinose as the sole product. They have molecular masses of 128 and 60 kD, respectively (Rom bouts et aI., 1988). The endo-arabinanase hydrolyzes 1,5-a-L-arabinan and produce di- and tri-saccharides. The enzyme has a Ai, of 35,000 to 42,500 (Dunkel and Amado, 1994; Rombouts et aI., 1988). A similar endo-arabinanase with a M, of 45,000 has also been isolated from A. aculeatus (Beldman et aI., 1993). Cochliobolus carbonum produces an a-arabinofuranosidase when grown on maize cell walls. The enzyme has a molecular mass of63 kD, a pH optimum of 3.5-4.0 and a temperature optimum of 50 C. The enzyme releases both aarabinose and larger oligosaccharides, suggesting it acts as an a-arabinosidase and an arabinanase (Ransom and Walton, 1997).

2.3.1.2 Enzymes that degrade hemicelluloses A. Xylanase (EC 3.2.1.8) Degradation of the (1 ｾＴ＠ )-P-I inked xylans has been reviewed (Thomson, 1993). Xylans are hydrolyzed by endo-xylanase (EC 3.2.1.8) to oIigomers. Exoxylanase or p-xylosidase (EC 3.2.1.37) is required to convert the oligomers to xylose. Endo-xylanase has been isolated from culture media of several plant pathogens (Table 2-5). Cochliobolus cm'bonum secreted three endo-xylanases into a culture medium containing xylan or isolated maize cell walls as a carbon source. These isozymes have Ai, of22,000 to 24,000 with pIs >9.3 (Holden and Walton, 1992). The genes, XYLl, XYL2, and XYL3, have been cloned (Apel et aI., 1993; Apel-Birkhold and Walton, 1996). Recent results indicate that C. carbonum also secretes an exo-xylanase. The cDNA encoding the enzyme has been cloned. The predicted protein has 328 amino acid residues with a M, of 36,700 with four N-glycosylation sites (Wegener et aI., 1999). Magnaporthe grisea strain ken60- I 9 produces two xylanases, XYN33 and XYN22. The genes encoding these two enzymes, xyn33 and xyn22, occur as single copies in the haploid genome of the fungus. The xyn22 gene encodes a polypeptide of 233 amino acids. The first 39 amino acids appear to be a

Degradation of Cell Walls -

85

hydrophobic signal peptide that is cleaved from the secreted enzyme. The predicted pI and molecular mass of the mature polypeptide are9.71 and 21,300, respectively. The xyn33 gene encodes a putative polypeptide of 331 amino acids. The first 28 amino acids constitute a signal peptide. The calculated pI and molecular mass are 9.95 and 32,800, respectively. Thus, the amino acid sequences of XYN33 and XYN22 are not homologous (Wu et aI., 1995). Table 2-5. P-I ,4-xylanase (endo type, EC 3.2.1.8) produced by plant pathogens. Pathogen

Molecular mass, kD

Optimal pH

Aspergillus oryzae variant D5 Bipo[aris sorokiniana strain H83

24 30

3.6 5.5

Claviceps purpurea

21.5 (cpxyll) 33.8 (cpxyI2) 21 (XYLI) 5.0 22 (XYL2) 22 (XYLJ) 42 5.5

Cochliobolus carbonum

Erwinia chrysanthemi strain SRI20A Fusarium oxysporum f. sp. melonis Fusarium oxysporum f. sp. pisi Gaeumannomyces graminis var. avenae isolate Gg 178 Helminthosporium turcicum Macrophomina phaseolina Magnaporthe grisea strain ken60-19

80

pi

9.5 8.9 7.0 9.1

8.8

5.0

22 26.5 5.0-5.5 28 5.0-5.5 22.5 5.5-6.5 22 14 33 (XYN33) 22 (XYN22)

10.5 10.5 7.4

9.9 9.7

Reference Golubev et al. (1993) Peltonen and Karjalainen (1994) Giesbert et al. (1998) Apel et al. (1993); Apel-Birkhold and Walton (1996) Braun and Rodrigues (1993) Alconada and Martinez (1994) Dean et al. (1989) Southerton et al. (1993) Degefu et al. (1995) Dean et al. (1989) Wu et al. (1995)

One of the common side chains attached to the xylan backbone is 4-0methyl-D-glucopyranosyl uronic acid. The uronic acid is attached to C-2 of the xylosyl residue in a (1 ｾＲＩ＠ linkage (see Section 2.2.2.1.8). The white-rot fungus Phanerochaete chrysosporium produces an extracellular a-( 4-0-methyl)-Dglucuronidase that hydrolyzes a-linked 4-0-methyl-D-glucuronosyl residues. The enzyme has a M. of 112 kD, pI of 4.6 and optimum pH of 3.5 (Castanares et aI., 1995). B. Glucanase ｾＭＱ＠ ,3-glucan is a minor component of plant tissue, but it is important in plant disease resistance because it occurs primarily in cell wall appositions and papillae in the form of callose in response to fungal penetration (see Chapter 8).

86

- Plant Pathogenesis and Resistance

Many plant pathogen produce (Table 2-6).

ｾＭｬ＠

,3-glucanase, which degrades the glucan

Table 2-6. P-I ,3-glucanases (endo type, EC 3.2.1.39; exo type, EC 3.2.1.58) produced by plant pathogens. Pathogen

Type

Molecular Optimal mass, kD pH

pI

Reference

Botrytis cinerea strain Lu 1458 MaB

exo

endo

4.9 5.2 3.6 3.4 9.0

Stahmann et al. (1993)

Claviceps purpurea strain T5a

? (GluI) 75 (GluIl) 84 (GluIII) ? (GluIV) 90 4.5

Cochliobolus carbonum

exo

63

6.3

6.0

Brockmann et al. (1992) Van Hoof et al. (1991)

,3-glucanase. The Claviceps purpurea strain T5a produces an ･ｮ､ｯＭｾｬ＠ enzyme is a glycoprotein with aM. of 90,000 and an pI of9.0 (Brockmann et aI., 1992). The ｾＭＱ＠ ,3-glucanase produced by Cochliobolus carbonum acts on laminarin in an exolytic manner. The enzyme has a M. of 63,000 and an pI of 6.3 (Van Hoof et aI., 1991). Based on partial amino acid sequences of this enzyme, two oligonucleotides have been synthesized and used as PCR primers to amplifY a 1.I-kb fragment of corresponding genomic DNA. The PCR product has been used to isolate the genomic copy of the gene exgJ. Partial sequencing of the genomic DNA confirms that the PCR product corresponds to exgJ. A strain of the fungus specifically mutated in the exgJ gene has been constructed. ,3-glucanase activity of the mutant is reduced by 98%, but it is still The ･ｸｯＭｾｬ＠ pathogenic to maize (Schaeffer et aI., 1994; N ikolskaya et aI., 1998). C. Galactanase An exo-l ＬＳＭｾｧ｡ｬ｣ｴｮｳ･＠ with high activity on ｾＭＱ＠ ,3-linked galactan backbone of type II arabinogalactan has been purified from a culture supernatant of Aspergillus niger. This enzyme is able to bypass the branching points of ｾＭＱ＠ ,6linkage. It has a molecular mass of 66 kD (Pellerin and Brillouet, 1994). A. niger also produces an enzyme that hydrolyzes ｾＭＱ＠ ,4-D-galactan. The ･ｮ､ｯＭｾ 1,4-D-galactanase (EC 3.2.1.89) has aM,. of32,000 and an optimum pH 00.5 (Yamaguchi et aI., 1995).

2.3.1.3 Enzymes that degrade cellulose The complete degradation of native cellulose to glucose requires three enzymes: endo-p-I ,4-glucanase (EG, or cellulase, CEL, EC 3.2.1.4), cellobiohydrolase

Degradation of Cell Walls -

87

(CBH, or exo-glucanase, EC 3.2.1.91), and p-glucosidase (BG, EC 3.2.1.21). Carboxymethyl cellulose (CMC) is the substrate of choice in cellulolytic activity assay as cellulose in its native form is water insoluble. Cellulolytic activity can be determined by measuring the reduction in viscosity of CMC solution and identifying the reaction products in reaction mixtures. EG first hydrolyzes amorphous regions of the cellulose fibrils. The nonreducing ends thus generated can then be attacked by CBH, releasing cellobiose. The action ofCBH then proceeds into the crystalline region. BG hydrolyzes cellobiose to glucose:

·&

b"9.0 ＢｾＩＮ＠ "0·vb 9 ｾＱＩＺ［ＬＢ＠ P9"O'\J b"VOPO1r--/\vQ I

I

t

CelioblOhydrolase

Crystalline regIOn

I

I

Crystallme region

t:ndo-Glucanase

Annrphous n:gK>Il

The biological degradation of cellulose has been reviewed (Beguin and Aubert, 1994). Several plant pathogens produce EG, CBH, and/or BG (Table 2-7). E. chrysanthemi strains 3665 and 3937 produce two cellulases, EGZ and EGY. The genes encoding these two enzymes, celZ and celY, respectively, have been cloned from both strains of the bacterium. Strain 3665, however, produces a 10fold higher level of cellulase activity than strain 3937. The difference in activity is possibly due to differences in the regulation of gene expression (Boyer et aI., 1987b). In strain 3937, EGZ is a major endoglucanase and EGY is a minor one. They account for 95% and 5% ofthe total activity, respectively (Boyer et aI., 1987a, 1987b). Sequence analysis of celZ and celY reveal no similarity in predicted amino acid sequences between these two enzymes. In contrast, a high level of identity, both at the nucleotide and the predicted amino acid levels, is found between celYand an EG-encoding gene from Cellulomonas uda, a grampositive bacterium taxonomically distant from E. chrysanthemi. These results indicate a possible interspecies transfer of the celY gene (Guiseppi et aI., 1991).

88 - Plant Pathogenesis and Resistance In E. chrysanthemi, EGZ consists of an N-terminal catalytic domain, a linker region, and a C-terminal cellulose-binding domain (CBD). Recent results indicate that mutation of a single tryptophan residue (Try-43) in the CBD prevents the binding between EGZ and cellulose (Chapon et aI., 2000). Table 2-7. Cellulose-degrading enzymes (endo-p-I,4-glucanase, CEL or EG, EC 3.2.1.4; cellobiohydrolase, CBH. or exo-glucanase. EC 3.2.1.91; P-I,4-glucosidase. BG, EC 3.2.1.21) produced by plant pathogens. Pathogen

Type

Clavibacter michiganensis subsp. michiganensis NCPPB382 Clavibacter michiganensis subsp. sepedonicus Cochliobolus carbonum Coniophora puteana strain EMPA62 Cryphonectria parasitica

CELA

78

CEL

28

CELl CBHI CBHIJ CBH

46.4 52 50

CELS CELV

Erwinia carotovora subsp. carotovora strain SCC3193 Erwinia carotovora subsp. carotovora SCRI 193 Erwinia chrysanthemi strain 3937

EGY EGZ Erwinia chrysanthemi strain 3665 EGY EGZ Fusarium oxysporum strain F3 EG BG Fusarium oxysporum f. sp. BG melonis Globodera rostochiensis EG-I EG-2 Heterodera glycines EG-I EG-2 Phanerochaete chrysosporium CBH Pseudomonas solanacearum strain AW Sclerotinia sclerotiorum

Xanthomonas campestris pv. campestris

Molecular Optimal mass. kD pH

pI

Jahr et al. (2000) 6.0

-5.0 -5.0

3.6 3.6

27

6.8

5.5

50

7.0

4.5

5.5 6.2-6.5 5.5 7.0 4.5 5.0-6.0 5.5

8.8 4.3 8.2 4.5 6.4 3.8 5.0

35 43 35 45 41.7 110 66

Reference

49.7 42 49.8 34.7

Baer and Gudmestad (1995) Sposato et al. (1995) Schmidhalter and Canevascini (1993) Wang and Nuss (1995) Saarilahti et al. (1990) Cooper and Salmond (1993) Guiseppi et al. (1988, 1991) Boyer et al. (1987a) Christakopoulos et al. (1995) Alconada and Martinez (1996) Smant et al. (1998) Smant et al. (1998)

EGL

43

7.5

Wymelenberg et al. (1993) Schell (1987)

EGI EG2 BG EG

48 34 240 53

6.2 3.7

Waksman (1988, 1991)

6.0

Gough et al. (1988)

Pseudomonas solanacearum strain A W produces a43-kD EG. This enzyme reduces rapidly viscosity of 0.5% CMC and releases cellobiose but no glucose

Degradation of Cell Walls -

89

(Schell, 1987). The gene encoding the enzyme, egl, is located on a 2.7-kbXhoISaIl ON A fragment and has been cloned and expressed in E. coli (Roberts et al., 1988). The enzyme is synthesized as a proprotein with a 45-residue leader sequence preceding the N-terminus of the mature EG. The sequence is cleaved during export to the extracellular environment (Huang et aL, 1989). Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial ring rot of potato. The bacterium produces a 28-kD EG (Baer and Gudmestad, 1995). Fusarium oxysporum strain F3 produces a BG of 110 kD with maximal activity at pH 5.0 - 6.0. The Km values are 0.093 and 1.07 mM when pnitrophenyl P-D-glucopyranoside and cellobiose are used as substrates, respectively (Christakopoulos et aL, 1994). Coniophora puteana strain EMPA 62, a brown-rot fungus, produces CBH I and CBH II in culture media. Both enzymes are glycoproteins and ｨ｡ｶ･ｾＮ＠ of 52,000 and 50,000 and pI of3.6 and 3.55, respectively. They act on cellulose, liberating cellobiose. They are inactive on cellobiose but hydrolyze cellotriose to cellobiose and glucose (Schmidhalter and Canevascini, 1993). Phanerochaete chrysosporium, a white-rot fungus, produces several CBHs and BGs (Covert et aL, 1992; Lymar et al., 1995; Wymelenberg et aL, 1993). The gene cbhl-4, encoding the dominant isozyme CBHl, has been identified and sequenced (Wymelenberg et aL, 1993). Among the 96-,98-, and 114-kD BGs produced by the fungus, the 114-kD enzyme is able to bind to cellulose and retain its catalytic activity against p-nitrophenyl glucoside. It is possible that 96- and 98-kD proteins are derived from the 114-kD protein and that BG from P. chrysosporium may consist oftwo domains, one for cellulose binding and the other for p-glucoside catalysis (Lymar et aL, 1995). Two cyst nematodes, Globodera rostochiensis and Heterodera glycines, synthesize EG in the esophageal glands. Two cDNA from each nematode species have been isolated and characterized. The ORFs of G. rostochiensis are 1414 and 1179 bp, encoding EG of 49.7 and 42 kD. The ORFs of H glycines consists of 1428 and 957 bp, encoding EG of 49.8 and 34.7 kD (Smant et aL, 1998; Van et aL, 1998).

2.3.1.4 Enzymes that degrade proteins Many plant-pathogenic bacteria produce proteases (Table 2-8). The gene encoding extra-cellular protease,prtl, has been cloned in plasmid pSKlfrom E. carotovora subsp. carotovora EC 14. The pSK 1 contains an ORF of 1041 bp that encodes a polypeptide of347 amino acids with a calculated molecular mass of 38,826 Da. E. coli transformed with the plam id produces Prt 1 intracellularly with a ｾ＠ of38,000 and an pI of 4.8. The prtl promoter is located between 173 and 1173 bp upstream of the ORF (Ky6sti6 et al., 1991).

90

- Plant Pathogenesis and Resistance

E. chrysanthemi strain B374 produces metalloproteases A, B, and C with molecular masses of 50, 53, and 55 kD, respectively. The genes encoding for proteases Band C have been sequenced. The predicted amino acids of the two proteases do not have typical sequences of signal peptides at their N-termini. They do possess a secretion signal located within the last 40 C-terminal amino acids that is analogous to a-hemolysin, a protein with a C-terminal secretion signal that is secreted by some strains of E. coli (Delepelaire and Wandersman, 1990). The proteases are secreted into the external medium as inactive precursors (zymogens Band C) where they are activated by divalent cations. Protease B shows a high degree of homology with the secreted 50-kD metalloprotease of Serratia marcescens, which also lacks a signal peptide (Delepelaire and Wandersman, 1989). Table 2-8. Proteases (Prt) produced by plant pathogens. Pathogen

Molecular mass, kD

Cochliobolus carbonum

25 30 38 38

Erwinia carotovora subsp. carotovora strain EC 14 Erwinia carotovora subsp. carotovora strain SCC3193 Erwinia chrysanthemi

Erwinia chrysanthemi strain EC 16 Pseudomonas tolaasii Xylellafastidiosa strain P

(Alpla) (Alplb) (Alp2) (Prt1)

Optimal pH

Reference Murphy and Walton (1996)

4.8

Kyostio et al. (1991)

51 (Prtw)

Marits et al. (1999)

50 (PrtA) 55 (PrtC) 52 (PrtG)

Wandersman (1989, 1990); Ghigo and Wandersman (1992a, 1992b) Dahler et al. (1990) Baral et al. (1995) Fry et al. (1994)

51(PrtC) 50 54 (Prtl) 50 (Prt2)

5.25 7.0 9.0 9.0

A 14-kb BamHI-EcoRI DNA fragment cloned from E. chrysanthemi strain EC 16 contained three prt genes encoding metalloproteases and one gene encoding a metalloprotease inhibitor. The prt genes are separated from the inhibition gene by a -4-kb region. One of the protease genes,prtC, has been sequenced and has high homology to a metalloprotease (prtB) gene of E. chrysanthemi strain B374. The ORF of the prtC gene encodes a protein of 478 amino acids with a calculated M. of 51, I 00 and an pI of5.25. Marker exchange mutants of E. chrysanthemi EC 16 defective in production of one or all of the extracellular proteases are not impaired in virulence on plant tissues (Dahler et aI., 1990).

Degradation of Cell Walls -

91

Xylellafastidiosa is a xylem-limited bacterium that causes Pierce's disease of grapevines and symptoms of marginal leaf necrosis on several other hosts. The bacterium produces proteases PI and P2 with molecular masses of 54 and 50 kD, respectively, on the defined medium PD3 amended with gelatin. The role of these proteases in plant pathogenesis, however, has not been elucidated (Fry et al., 1994). 2.3.1.5 Enzymes that degrade Iignins Enzymatic degradation oflignin involves three extracellular enzymes. They are laccase (EC 1.10.3.2), lignin peroxidase (LiP, EC 1.11.1.7), and manganesedependent peroxidase (MnP, EC 1.I1. I. 7). Several white-rot fungi (e.g., Phanerochaete chrysosporium, Phlebia radiata, Dichomitus sequalens, Rigidoporus lignosus, Ceriporiopsis subvermispora, and Junghuhnia separabilima) produce one or more of these enzymes and are efficient lignin degraders in nature (Hatakka, 1994) (Table 2-9). The lignin-modifying enzymes from the white-rot fungi have been reviewed (Tien, 1987; Hatakka, 1994; Thurston, 1994; Tuor et al., 1995). LiP and MnP are heme-containing glycoproteins that require hydrogen peroxide as an oxidant. LiP is capable of cleaving the Ca-C p bond in P-O-4 and P-I type linkages in a variety of lignin model compounds and synthetic lignin (Tien and Kirk, 1983). P-O-4 linkage is a bond between the p-carbon of the propyl side chain of one monolignol and the oxygen of the C-4 of the adjacent phenol ring. This is the predominant linkage in lignin structure and accounts for 60% of the interunit linkages in spruce lignin (Alder, 1977). The P-l type of linkage is the bond between p-carbon of the propyl side chain of one monolignol and the C-l of the phenol ring.

P-0-4 linkage

ｾＭｬ＠

linkage

MnP oxidizes Mn(H) to Mn(III), which then oxidizes phenol rings to phenoxy radicals. These radicals lead to decomposition oflignin. Laccase is a

92

- Plant Pathogenesis and Resistance

copper-containing oxidase that uti I izes molecu lar oxygen as an oxidant and also oxidizes phenol rings to phenoxy radicals. Table 2-9. Lignin-degrading enzymes (lignin peroxidase, LiP, EC 1.I1.1. 7; manganese peroxidase, MnP, EC 1.11.1.7; laccase, EC 1.10.3.2) produced by wood-rotting fungi. Pathogen

Type

Molecular Optimal pI mass, kD pH

Bjerkandera adusta MnP Bjerkandera sp. strain BOS55 MnP liP

46.5 43 40-42

Ceriporiopsis subvermispora

MnP Laccase

52.5

Ganoderma lucidum Junghuhnia separabilima

Laccase Laccase I Laccase 2 Laccase 3 LiPH2 LiPH3 LiPH4 MnP

40,66 62 60 58 47 44 43 50

MnP Laccase LiPI LiP2 LiP3 LiP4 LiP5 LiPI LiP2 LiP3 MnP Laccase MnPI MnP2

43 64 40 44 38 43 44,46 42 45 44 49 53 42 42

Nematoloma frowardii Panus tigrinus strain 8/18 Phlebia ochraceofulva strain 75144

Phlebia radiata strain 79 (A TCC 64658)

Rigidoporus /ignosus

Reference

Yoshida et al. (J 996) 3.9 3.9 Mester and Field 2.5-3.0 (J 998); ten Have et al. (J 998) 4.1-4.6 Lobos et al. (1994) 3.5-3.7 Sethuraman et al. (1998) 3.0-5.1 D'Souza et al. (1999) Vares et al. (1992) 3.6 3.4 3.5 3.5 3.4 3.4 3.2 Hofrichter et al. (1999) 2.95 Maltseva et al. (1991) 3.00 Vares et al. (J 993)

3.0-4.5

4.0 6.0 4.5 4.5

4.1 3.9 3.2 3.8 3.8 3.5 3.7

Niku-Paavola et al. (1988); Karhunen et al. (1990) Gallianoetal. (1991)

Panus tigrinus synthesizes MnP and laccase but not LiP. MnP has a M, of 43,000 and pI of2.95. Laccase has aM,of64,000 and pI of3.0 (Maltseva et aI., 1991 ). Ceriporiopsis subvermispora also produces MnP and laccase (Lobos et aI., 1994). The genes encoding liP have been cloned from P. chrysosporium, Trametes versicolor, and Phlebia radiata (Tien and Tu, 1987; Walther et aI., 1988; Saloheimo et aI., 1989; Jonsson and Nyman, 1992). One of the liP genes from

Degradation of Cell Walls -

93

P. chrysosporium has an ORF of 1122 bp encoding a 28-amino acid signal peptide and a mature protein of345 amino acids with a calculated M,0f37,072. The protein is glycosylated (Tien and Tu, 1987). The liP gene from T versicolor encodes a protein of 346 amino acid residues, preceded by a 26residue signal peptide. The calculated M, for the mature liP is 37,000 (Jonsson and Nyman, 1992). There is a 65% homology between these two LiP (Jonsson and Nyman, 1992). The genes encoding MnP have been cloned from P. chrysosporium (Pease et aI., 1989).

2.3.2 Biosynthesis of Cell-Wall Degrading Enzymes In most instances, cell-wall-degrading enzymes are produced inductively rather than constitutively, e. g., the enzymes are produced only, or much more abundantly, when specific inducers are in the medium. There are two types of inducers: catabolic products of the enzymes and DNA-damaging agents. The first type of inducers are the monomers and/or oligomers released from the cell wall polysaccharides by the basal level of cell-wall-degrading enzymes. These catabolic products are taken up by the pathogen and serve as signals for enzyme induction (autocatalytic induction). When the concentration of the catabolic products is high, synthesis of these enzymes is repressed (self-catabolic repression). The production of these enzymes is also subjected to repression by glucose and other sugars and derepression by certain compounds. Production of Pels and Pehs is generally induced by catabolic products. The second type of inducers are DNA-damaging agents, such as UV light, mitomycin C, and nalidixic acid. The production ofPnls is induced by the second-type inducers. In addition to catabolic induction, catabolic repression, and DNA-damaging agents, production of Pels, Pehs, and Pnls is further subjected to a number of global regulatory mechanisms. These include growth-phase inhibition (Gold et aI., 1992). 2.3.2.1 Pee tate Iyases The rate of Pel synthesis by erwinias in cultures increases in the presence of isolated plant cell walls, polygalacturonates, or oligogalacturonates. Induction by high-molecular-weight polysaccharides or insoluble pectic polymers begins with an extracellular digestion phase in which oligogalacturonates, particularly the dimers, are released by the basal level of pectic enzymes. The digalacturonates are taken up by the bacteria and catabolized intracellularly through the pectin-degradative pathway. The unsaturated dimers are first split by the oligogalacturonide lyase (Ogl, encoded by the ogl gene) to form two molecules of5-keto-4-deoxyuronate (DKI). This metabolite is isomerized to 2,5-diketo-3deoxyglconate (DKII) by DKI ketol isomerase (encoded by kduI) and then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by KDG dehydrogenase (encoded by kduD). Saturated oligomers are first catabolized by Ogl to form

94

- Plant Pathogenesis and Resistance

one molecule of galacturonic acid and one molecule ofDKI. Galacturonic acid is subsequently catabolized by the enzymes encoded by the operon xuaCBA to form KDG (Fig. 2-8). KDG is phosphorylated at C-6 to form 2-keto-3-deoxy-6phospho-D-gluconate (KDPG) by KDG kinase (encoded by kdgK) and is cleaved by KDPG aldolase (encoded by kdgA) to yield pyruvate and glyceraldehyde-3-phosphate (Fig. 2-8). The genes involved in intracellular catabolism of plant pectin have been located in E. chrysanthemi strain 3937 (HugouvieuxCotte-Pattat and Robert-Baudouy, 1992). Pectin

pectin methyl esterase

pectin lyase (pili)

(pem)

Polygalacturonate

Unsaturated oligomethylgalacturonate pectin methylesterase

polygalacturonase (peh)

(pelll)

Saturated oligogaIacturonate (GiIU)n

Unsaturated oligogalacturonate (uGiIU)n

oligogalacturonate lyase

External medium Bacterial cytoplasm

J""lyase...,._m

(og/)

(og/)

!

Galacturonate glucuronate keto-

5-Keto-4-deoxyuronate (DKI)

Isomerase

JＢＧｾｭ＠

(uxaC)

!

Isomerase (kduf)

Tagaturonate atronate oxidoreductase

2.5-Diketo-3-deoxygluconate (DKII)

(uxoB)

AI""",,, ｾＮＧ＠

ｾＭＢＧ＠

hydrolyase (uxoA)

dehydrogenase (kduJ)

2-Keto-3-deoxygluconate (KDG)

+ketodeoxygluconate kinase

(kdgK)

2-Keto-3-deoxy-6-phosphogluconate (KDGP) ｾ＠

ketodeoxyphosphogluconate aldolase (kdgA)

Pyruvate + 3-Phosphoglyceraldehyde

Fig. 2-8. Pectin degradative pathway. (Adapted from Hugouvieux-Cotte-Pattat et ai., 1994).

Pel synthesis by mutants of E. chrysanthemi EC 16 deficient in Ogl has been induced by DKI but not by isolated plant cell walls, polygalacturonate, or

Degradation of Cell Walls -

95

digalacturonate. On the other hand, Pel synthesis by the mutant with a kduD gene interrupted by Tn5 transposon insertion, which is devoid of KDG dehydrogenase and therefore accumulates DKII, is hyperinduced by pectic metabolites (Chatterjee et aL, 1985). These results indicate that catabolic products of digalacturonate, DKI, DKII, KDG, and KDGP are the actual inducers of PL synthesis. The kdgK gene of E. chrysanthemi strain 3937 has been cloned. It has an ORF of 981 bp, corresponding to a protein of 329 amino acids with a M.: of 36,377. Expression of kdgK is controlled by the regulatory gene kdgR. Tn5 transposon mutagenesis at the kdgR gene results in the constitutive expression of genes for the pectin-degrading pathway and PelD and PeIE. This indicates that the product of the kdgR gene is a negative regulator of PL synthesis. Specific binding of the KdgR protein to the operators of pel genes (pelABCE), pectin catabolism genes (kdgK, kdgT, ogl, kdu!-kduF), and pectinase secretion gene (outT) has been demonstrated in vitro (Hugouvieux-Cotte-Pattat et aL, 1994; Nasser et aL, 1994). Thus, the KdgR protein regulates pectinolysis, pectinase synthesis, and pectinase secretion (Hugouvieux-Cotte-Pattat et aL, 1996). The production of Pel, Peh, Cel, and Prt in E. carotovora subsp. carotovora appears to be mediated by common regulatory steps. For example, the production of these cell-wall-degrading enzymes is concomitantly activated when the bacteria of strains 71 and SCRI 193 are grown in media supplemented with celery extract (Liu et aL, 1993; Murata et aL, 1991). Recent results indicate that rex (regulation of exoenzymes), rsm (repressor of secondary metabolites), and aep (activator of extracellular protein production) genes are regulators in extracellular enzyme production. Certain strains of E. carotovora subsp. carotovora are able to produce carbapenem (I carbapen-2-em 3 carboxylic acid), a p-Iactam antibiotic. Transposon and chemical mutagenized Car" mutants are also Rex' (regulation of exoenzymes). They fail to produce the antibiotic and are defective in synthesis of pectolytic, cellulolytic, and proteolytic exoenzymes and are avirulent. These mutants are also defective in the production of N-3(oxohexanoyl)-L-homoserine lactone (HSL):

ｾｎＨ｜ｏ＠

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Addition of exogenous HSL to these mutants restores their abilities to synthesize the antibiotic, exoenzymes, and virulence in planta. These mutants can also be genetically complemented by the lux! (bioluminescence) gene from Vibrio fischeri. HSL is synthesized by a 22-kD protein encoded by the lux!

96

- Plant Pathogenesis and Resistance

gene, possibly using S-adenosyl methionine and 3-oxohexanoic acid (an intermediate in fatty acid metabolism) as substrates (Jones etal., 1993). Similar results have also been obtained by Pirhonen et al. (1993). Thus, the production of exoenzymes in E. earotovora subsp. earotovora is regulated by the lux autoinducer. AC5070, a RsmA mutant derived from strain 71, overproduces extracellular enzymes. The overproduction is due to the stimulation of transcription as basal levels of pelJ, pel3, a:nd pehl mRNAs are higher in this mutant than in the RsmA + parent, AC5047. The transcription is stimulated in the absence ofHSL, a starvation/cell-density-sensing signal required for extracellular enzyme production in Rsm+. Thus, RsmA is a global repressor of extracellular enzyme production (Chatterjee et aI., 1995b). Strain 71 also harbors a gene, RsmB, that positively regulates extracellular enzyme production (Liu et aI., 1998). Strain 71 produces higher levels of Pel, Peh, Cel, and Prt in a medium containing celery extract than in a medium containing pectate. Transposon mutagenesis results in the formation of pleiotropic mutants that are deficient in exoenzyme production and attenuated in their ability to macerate plant tissues. A cosmid in the strain 71 gene library, pAKC264, is able to restore exoenzyme production and tissue maceration in these mutants but does not affect the levels of peri plasmic enzymes (e.g., cyclic phosphodiesterase and p-Iactamase). This cosmid carries no pel, peh, eel, or prl genes. The gene is designated as aepA (Murata et aI., 1991). Subsequent study has identified an activator locus designated as aepH* in a mutant of strain 71, AC5034, that overproduces pectolytic enzymes and proteases. The nucleotide sequence of the aepH* DNA segment reveals an ORF of 141 bp that could encode a small (5.45- kD), highly basic (pI 11.7) protein of 47 amino acid residues. Analyses of deletions and MudI insertions indicate that the activator function requires a 508-bp DNA fragment that contains this ORF. The wild-type locus aepJr is localized within a DNA segment upstream of aepA. An AepR mutant obtained by exchanging aepH+ with aepH·::MudI is deficient in Pel, Peh, Cel, and Prt production. Production of these exoenzymes is restored upon the introduction of a plasmid carrying aepJr or aepJt (Murata et aI., 1994). Thus, aep appears to be another regulator of extracellular enzyme production. 2.3.2.2 Pectin Iyases Pnl production is regulated differently from Pel and Peh biosynthesis. It is induced in E. ehrysanthemi and E. earotovora by mitomycin C, bleomycin, nalidixic acid, UV light, and other DNA-damaging agents but not by pectic substances (Chatterjee et aI., 1991; Itoh et aI., 1980, 1982; Tsuyumu and Chatterjee, 1984). Induction ofPnl production is associated with activated cell lysis (Lss) and carotovoricin (Ctv) synthesis in E. earotovora and the temperate phage in E. ehrysanthemi. It is possible that the Pnl genes in erwinias are

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97

located within or near a temperate or defective phage (bacteriocin) (Itoh et ai., 1980). RecA protein is encoded by the recA gene. It is required in DNA repair and recombination. A functional recA gene has been shown to be involved in Pnl production in E. carotovora subsp. carotovora strain 71 (Zink et aI., 1985) and in P. marginalis N6301(Nikaidou et aI., 1994). The recA+ gene has been cloned from strain 71 and inactivated by Tn5 insertion. A mutant that contains the recA' has been constructed. Production of Pnl and Ctv by the mutant is not induced by mitomycin C, but the RecN phenotype is restored in the mutant with the recA DNA. Clones with the promoter region ofpnl from P. marginalis deleted have been transformed into E. coli strains 600 (recA+) and DHSa (recA). Pectin lyase activities have been detected only in the recA+, but not in the recA-, strain of E. coli. The recA from P. marginalis complements the recAstrain of E. coli to form RecA+ phenotype (N ikaidou et ai., 1994). These results indicate that RecA is required in Pnl production. Recently, Reck mutants that are not restored by the recA DNA have been obtained from strain 71, indicating that recA gene may not be involved in Pnl production in E. carotovora (McEvoy, 1990, 1992). Addition of the cosmid pAKC280, however, restores inducibility ofPnl, Ctv, and Lss by mitomycin C. The genes responsible for restoration have been located and designated as rdg (regulator of damage-inducible genes) (Liu et aI., 1994b). In the rdg locus, two regulatory genes, rdgA and rdgB. have been identified. RdgA, a polypeptide of 244 amino acid residues with aM; of26,78S and pI ofS.09, has high homology with the repressors of lambdoid phages. RdgB is a small polypeptide of 117 amino acid residues with a Mr of 13,551 and pI of 9.56. It has significant homology with the transcriptional activator of the Mu phage. By itself, rdgB can activate Pnl production in E. coli. Transcription ofrdgB occurs only after the bacteria are treated with mitomycin C. A substantial level ofrdgA mRNA, however, is detected in the absence of the antibiotic. Thus, these two closely linked rdgA and rdgB genes are expressed differently in strain 71 (Liu et aI., 1994b).

2.3.2.3 Polygalacturonases Colletotrichum gloeosporioides produces two endo-Peh isozymes of 62 and 68 kD based on SDS-PAGE. Purified Peh macerated avocado and cucumber tissues. The enzyme activity might be regulated by epicatechin, which is abundant in the peel of unripe, resistant avocado fruit and less abundant in ripe, susceptible fruit (Prusky et aI., 1989). 2.3.2.4 Hemicellulases Information related to regulation of hemicellulase production by plantpathogenic bacteria is limited. E. chrysanthemi produces xylanase and is

98

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responsible for cornstalk rot (Braun and Kelman, 1987). E. earotovora subsp. earotovora produces arabinanses and xylanases (Kaji and Shimokawa, 1984).

2.3.2.5 Cellulases E. ehrysanthemi and E. earotovora subsp. earotovora produce cellulase extracellularIy. The cellulase purified from culture supernatants of E. ehrysanthemi

strains 3665 and 3937 has a molecular mass of 45 kD, an pI of 4.3, and an optimum pH for activity between 6.2 and 7.5. The eel genes have been cloned from E. ehrysanthemi strains 3665 (Barras et aI., 1984; Boyer et aI., 1987a, 1987b), 3937 (Boyer et aI., 1987b; Kotoujansky et aI., 1987), and B374 (Van Gijsegem et aI., 1985). The endoglucanases encoded by eelYand eelZ are immunologically distinct. Both genes are expressed constitutively and are subjected to catabolic repression (Boyer et aI., 1984; Chambost, 1986). The eelVl gene encoding cellulase CelVlhas been cloned from E. earotovora subsp. earotovora SCC3193. The gene contains an ORF of 1511 bp and codes for an exported protein of504 amino acids. The predicted amino acid sequence of CelVI is highly similar to that of CelV of strain SCRII93 but significantly different from the CelS ofthe strain SCC3193. Gene fusions to the laeZ reporter have been used to characterize the regulation ofCelVI and CeIS. Both genes are coordinately induced in a growth-phase-dependent manner and are catabolite repressed. Expression of eelVI, but not eelS, is stimulated by plant extracts. CelV I is the major cellulase produced by strain SCC3193 since the eelVl gene is expressed at a much higher level under all conditions tested. Inactivation of the eelVl gene by maker exchange significantly reduces virulence of the mutants (Mae et aI., 1995).

2.3.3 Secretion of Synthesized Enzymes Cell-wall-degrading enzymes must be exported from the cytoplasm across the membranes and secreted into the external milieu to be functional. Mepanipyrim [N-( 4-methyl-6-prop-I-ynyl pyrimidin-2-yl) aniline] is a fungicide that affects the intracellular transport process of secretory proteins, leading to their intracellular accumulation. Botrytis cinerea treated with mepanipyrim fails to secrete cell wall-degrading enzymes and has reduced infectivity (Miura et aI., 1994). Mutants of virulent pathogens that synthesize Pels but fail to secrete them to the milieu are avirulent (Andro et aI., 1984; Wattad et aI., 1995). Colletotrichum magna (=Glomerella magna) wild-type strain and C. gloeosporioides isolate Cg-14 colonize mesocarp tissue of avocado fruits equally well. C. magna is also able to decay the peri carp, although the decay is significantly delayed compared to that caused by C. gloeosporioides. A nonpathogenic mutant strain (path-I) that fails to cause disease symptoms in either the pericarp or the mesocarp has been obtained from C. magna. Isoelectrofocusing reveals the presence of Pel and Peh at pIs of 7.9 and 4.8,

Degradation of Cell Walls -

99

respectively, in the culture filtrates of the wild type of C. magna as well as isolate Cg-14, but not in the path-I ofC. magna. Western blots detect Pel in avocado fruit tissue infected with Cg-14 and the wild-type strain of C. magna. DNA of the three strains hybridize to a pel probe from C. gloeosporioides indicating that no deletion in the pel gene is evident in strain path-I. Similar levels of pel mRNA expression are found for the three strains. Pel antibodies cross-react with lysate of path-l but not Cg-14. These results indicate that the path-l accumulates Pel but fails to secrete it into the inoculated tissue. The malfunction in extracellular secretion of Pel results in the differential pathogenicity of path-l strain on avocado (Wattad et aI., 1995). The envelope of gram-negative bacteria typically consists of inner membrane, periplasm, and outer membrane. "Secretion" is the term to describe the transfer of a synthesized protein from cytoplasm to periplasm and from periplasm to the extracellular medium. The term "export" is used to describe the movement of a protein from cytoplasm, across the inner membrane, to the periplasm (Salmond, 1994). The mechanisms of protein secretion have been intensively investigated. Two distinct secretion pathways are involved in the secretion of cell-walldegrading enzymes: the sec-dependent, two-step general secretory pathway; and the sec-independent, one-step secretory pathway.

2.3.3.1 sec-Dependent, two-step general secretory pathway In this pathway, an N-terminal signal sequence of the presecretory protein is first recognized by the sec gene product located on the inner membrane. The protein is processed by signal peptidase and exported across the cytoplasmic membrane into the periplasm. The exact form of exported proteins in the periplasm is not known. The protein is subsequently transferred to the extracellular medium by various Out proteins (Pugsley, 1993). An Ouf mutant, obtained by chemical mutagenesis of E. carotovora subsp. carotovora, synthesizes Pel, Cel, and Prt but secretes only Prt. The mutant exports Pel and Cel to the periplasm but not to the milieu. Cosmid clones carrying the wild-type DNA with the ability to restore the Out+ phenotype of the mutant have been identified. The complementing DNA resides in a 12.7 kb DNA fragment with 13 ORFs corresponding to 13 out genes (outCDEFGHIJKLMNO) (Reeves et aI., 1993). Some minor differences do exist in other Erwinia spp. For example, E. chrysanthemi does not have the equivalent of the outN gene but has outBST genes, which are absent in E. carotovora. Most Out proteins are not localized in the outer membrane but span across the inner membrane and lie within the periplasm. The functions of these Out proteins are not fully understood. OutO is a signal peptidase (Reeves et aI., 1994). OutO is located in outer membrane

100 - Plant Pathogenesis and Resistance and may form a pore, gate, or channel through which the exported proteins are released into the milieu (Lindberg and Collmer, 1992; Reeves et aI., 1993). Pel, Peh, and Cel synthesized by erwinias and Prt synthesized by xanthomonads are secreted via this pathway (He et aI., 1991 a, 1991 b).

8

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8

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8 The excretion of cell-wall-degrading enzymes has also been investigated in P. solanacearum. P-l,4-glucanase is initially synthesized by the bacterium as

a higher molecular-mass precursor with a 45-residue leader sequence. During the first step of export across the inner membrane, this precursor is cleaved with a signal peptidase at residue -26 of the leader sequence concomitantly with fatty acylation resulting in the formation of a transient lipoprotein intermediate. The remaining residues of the lipid-modified leader sequence is subsequently removed during the second step of export (Huang et aI., 1989). The export of the 52-kD endo-Peh, the product ofpglA, by P. solanacearum is slightly different from that of P-l ,4-glucanase. The endo-Peh is initially synthesized with a 21-amino acid residue, N-terminal leader sequence. The signal sequence is cleaved in the first step of export. The second step of export is possibly directed by a sequence from the C-terminus of the mature endo-Peh. Endo-Peh, lacking the last 13 C-terminal residues, accumulates in the periplasm of the bacterium and is not exported across the outer membrane (Huang and Schell, 1990). P. viridiflava produces an extracellular Pel that is the pathogenicity factor of the bacterium. A Tn5-induced mutant that produces no extracellular Pel, but high levels of Pel activity in sonicated cell extracts, fails to macerate plant tissue (Liao et aI., 1988). In E. chrysanthemi, genes coding for the enzymes involved in pectin degradation and secretion are negatively regulated by the kdgR repressor (Condemine and Robert-Baudouy, 1995).

Degradation of Cell Walls - 101

2.3.3.2 sec-Independent, one-step secretory pathway The proteins secreted through this pathway lack the homology of the N-terminal signal sequence that is recognizable by Sec. They do possess a glycine-rich repeated motif close to their C-termini (Wandersman, 1992). This signal sequence is not cleaved in the translocation process. E. chrysanthemi proteases are secreted via this pathway. The proteins transported through this pathway do not stop in the periplasm. They are transported through a channel or pore formed between the inner and outer membranes. The secretion ofPrtA synthesized by E. chrysanthemi B374 requires three accessory proteins, PrtDEF. These proteins are associated with membranes. The PrtD is an ATP-binding cassette motif and is probably an inner membrane A TPase that hydrolyzes ATP to provide energy for the secretion process. PrtE is located in the inner membrane, and PrtF is located in the outer membrane (Ghigo and Wandersman, 1992a). Milieu Outer membrane Periplasm

Inner membrane

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ADP ATP

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ATP

88 8 PrtG is another protease secreted by Erwinia chrysanthemi through the secindependent secretory pathway. Deletion mapping of a short, secretion competent C-terminal peptide reveals that the last 29 residues ofPrtG is needed to promote the secretion. A low but significant level of secretion can be promoted by the last 15 residues. The last four C-terminal sequence of Dxxx is a conserved motifin all constructs that are secreted through the E. chrysanthemi transporter. The x's in the motif are hydrophobic residues (Ghigo and Wandersman, 1994).

2.4 CELL-WALL-DEGRADING ENZYMES AND PLANT PATHOGENESIS The ability of an organism to produce cell-wall-degrading enzymes in vitro is no proof of involvement in plant disease. In order for a cell-wall-degrading enzyme to be important in plant pathogenesis, the enzyme should meet several criteria: (i) it should be detected at the infection sites and in infected tissues; (ii)

102 - Plant Pathogenesis and Resistance the enzyme should reproduce disease symptoms; and (iii) enzyme production should positively correlate with virulence of the producing pathogen. Testing the importance of pectic enzymes in pathogenesis is complicated by the fact that pectic enzymes in many tissues are produced by higher plants. The involvement of cell-wall-degrading enzymes in plant pathogenesis has been reviewed (Barras et aL, 1994; Collmer, 1987; Collmer et aL, 1982a; Collmer et aL, 1989; Kotoujansky, 1987).

2.4.1 Presence of Cell-Wall-Degrading Enzymes at Infection Sites and in Infected Tissues Participation of cell-wall-degrading enzymes in the infection process has been demonstrated through electron microscopy using gold-tagging lectin with specific binding affinity for a particular carbohydrate molecule. The lectin from gonads of the sea mollusk (Aplysia depi/ans) binds specifically to polygalacturonic acid. Incubation of thin sections of bean leaf with gold-complexed Aplysia lectin resulted in heavy labeling of primary walls and middle lamella matrices at the junction between adjacent cells but not in the cytoplasm, subcellular organelles, or vacuoles. In contrast, gold particles were scattered on shredded host cell walls closely neighboring the invaded Colletotrichum lindemuthianum cells, indicating a reduction in polygalacturonic acid in infected tissues (Benhamou et aL, 1991). Similar techniques have also been used to demonstrate pectin and cellulose degradation in tobacco roots infected by Phytophthora parasitica var. nicotianae. The epidermis is colonized 24 hr after inoculation and the cortex 96 hr after inoculation. The pathogen enters the roots by intercellular growth through dissolution of middle lamellae or by direct penetration of tangential and radial primary walls. The strong alteration of middle lamella matrices without major deformation of penetrated primary cell walls supports the idea that tobacco root invasion by P. parasitica var. nicotianae is achieved primarily enzymatically rather than mechanically. Colonization of the root tissues is associated with marked alterations of pectin and cellulose in host cell walls and middle lamella matrices. Using gold-complexed Aplysia lectin as a probe, pectin molecules have not been detected in primary walls and middle lamellae in areas near the invading hyphae. Significant reduction in electron density is found in these areas when gold-complexed exoglucanase (specific to cellulosic P-l,4-glucans) is used as a probe. These observations indicate that both pectin and cellulose are degraded by pectinases and cellulase secreted by the pathogen (Benhamou and Cote, 1992). A polygalacturonase-inhibiting protein (PGIP) isolated from bean cell walls has strong affinity toward endo-Peh produced by C. lindemuthianum. Using gold particle-tagging PGIP as a probe, endo-Peh produced by the fungus in infected bean tissues can be located. Incubation of sections from actively

Degradation of Cell Walls - 103 growing colonies of C. lindemuthianum with gold-complexed PGIP results in the deposition of few gold particles over the outermost fungal cell walls. When the sections from inoculated bean leaf tissue are incubated with the goldcomplexed PGIP, there are large amounts of gold particles over walls of invading hyphae as well as host cells closely neighboring fungal cells (Benhamou et aI., 1991). These observations demonstrate the participation of endo-Peh in the infection process.

2.4.2 Reproduction of Disease Symptoms with Purified Cell-WallDegrading Enzymes The macerating ability of several cell-wall-degrading enzymes has been tested on plant tissues. Maceration of potato tuber tissue by E. chrysanthemi EC 16 derivatives with various pel deletions has been studied. An isolate with pelE deleted - but still producing PeIA, PelB, and PelC - has 47% maceration ability as compared to the wild type. Among the Pel isozymes, with the exception of PeIA, all induce maceration, electrolyte leakage, and cell death. Mutant UMI 005, derived from EC 16, is deficient in PeIA, PelB, PeIC, and PelE and produces less than 0.1 % of the extracelluar Pel activity of the wild type. The specific growth rate ofUM 1005 is unchanged related to the wi ld type in a minimal medium with polygalacturonic acid as the sole carbon source. Virulence is reduced 79 to 98% in potato tuber maceration tests, but the mutant is still able to cause significant maceration in potato, carrot, and pepper tissues. These observations indicate that Pel is not necessary for pectate utilization and tissue maceration by E. chrysanthemi (Ried and Collmer, 1987). A purified Pel from Pseudomonas viridiflava, devoid of Pnl, Peh, and Prt activities, readily macerates plant tissues. Tn5-induced pet mutants fail to induce soft rot in tested plants. These results indicate that Pel is the pathogenicity factor of P. viridiflava (Liao et aI., 1988). Verticil!ium albo-atrum produces an array of cell-wall-degrading enzymes. Of these Peh and Pel are capable of causing wilt symptoms in tomato cuttings. The abilities of three pectate lyase-deficient mutants, obtained by chemical mutagenesis, to infect and cause symptoms in tomato plants have been compared to the wild type. Symptoms are absent, less severe, or delayed in plants infected with these mutants. With the exception of mutant C23 (a secretory mutant that accumulates Pel intracellularly), mutants 34i (defective in galacturonide utilization) and 24d (produces reduced levels of Pel isozymes) are able to colonize plants to levels comparable to that of the wild type. These results imply that Pel is a virulence factor but not a pathogenicity factor (Durrands and Cooper, 1988).

104 - Plant Pathogenesis and Resistance

2.4.3 Correlation of Enzyme Production with Virulence of the Pathogens Several reports provide circumstantial evidence that cell-wall-degrading enzymes are virulent factors in plant pathogenesis, particularly in soft-rot diseases. For example, CelV is the major P-l ,4-endo-glucanase secreted by E. carotovora subsp. carotovora seRI 193. A cellulase-deficient mutant, GS7000, was obtained by ethylmethylsulfonate mutagenesis. This mutant has a growth rate equal to the wild type and produces wild-type levels of other extracellular enzymes. Inoculation of this mutant to potato tubers fails to cause maceration, indicating that Cel plays an important role in soft rot (Walker et aI., 1994). In addition to soft-rot diseases, correlation between cell-wall-degrading enzymes and crown gall and necrotrophic diseases have also been reported. Agrobacterium tumefaciens biovars 1 and 2 cause crown gall on various plants. No strain ofbiovar 1 or 2 has pectolytic activity. Biovar 3 causes both crown gall and root decay of grape, and all strains produce a single Peh with an optimum pH of 4.5 and pIs ranging from 4.8 to 5.2. Lesions on grape seedling roots inoculated with a strain of biovar 3 yield Peh activity with an pI sim i1ar to that of the enzyme produced by the bacterium in culture. These results support the hypothesis that the Peh produced by A. tumefaciens biovar 3 has a role in the decay of grape roots (McGuire et aI., 1991). CBH, a cellulose-degrading enzyme, is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. A hypovirulent strain of the pathogen, resulted from hypovirus infection, fails to secrete detectable CBH activity. These results indicate that CBH is important in chestnut blight pathogenesis (Wang and Nuss, 1995). Cell-wall-degrading enzymes also contribute to the aggressiveness of fungal pathogens. When grown in liquid culture with carrot cell walls as a carbon source, four isolates of Mycocentrospora acerina secrete Pem, Peh, Pel, and P-I,4-glucanase. All activities are positively correlated with isolate aggressiveness. Time-course experiments show that aggressive isolates produce Peh and Pem more rapidly than hypoaggressive ones. The rate at which carrot cell walls are solubilized and depolymerized is also related to isolate aggressiveness, suggesting a strong contribution ofcell-wall-degrading enzymes in M acerina virulence (Le Cam et aI., 1994). Aspergillus jlavus requires a wound or natural opening to invade cotton bolls. After initial penetration, pectolytic enzymes are important for this fungus to successfully colonize the infected tissues. Sixteen isolates of A.jlavus with known ability to spread between cotton locules and rot them have been assayed for pectinase production. Aggressiveness of isolates during the infection process is correlated with their ability to secrete pectinases on sterilized cottonseed, on pectin-containing liquid media, and in living host tissues. Four isolates with reduced ability to spread through boll tissues lack a major endoPeh activity that is always present in highly aggressive isolates. Thus, endo-Peh

Degradation of Cell Walls - 105 produced by A ..flavus during host infection contributes to fungal aggressiveness (Cleveland and Cotty, 1991). For many plant pathogens, cell-wall-degrading enzymes are not pathogenicity factors. Consequently, the ability of a pathogen to produce cell-walldegrading enzymes has no bearing on its pathogenicity. The role ofEGZ and EGY in E. chrysanthemi pathogenicity on Saintpaulia ionantha has been assessed by mutagenizing cloned celZ and celYand recombining them with the chromosomal alleles. Strains with an omega interposon in celZ, a deletion in celY, or a double eel mutant are as virulent as the wild-type strain. In the strain with a deletion in celY, a delay in the appearance of symptoms occurs and then maceration progresses as in plants infected with the wild-type strain, indicating that E. chrysanthemi endo-glucanases playa minor role in soft-rot disease development (Boccara et a\., 1994). A gene (PGNl) encoding extracellular endo-Peh has been isolated from Cochliobolus carbonum race 1. A probe was synthesized by PCR using oligonucleotides based on the endo-Peh amino acid sequence. Genomic and cDNA copies of the gene have been isolated and sequenced. The corresponding mRNA is present in the fungus grown on pectin, but not on sucrose, as the carbon source. The single copy of PGNI in C. carbonum is disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Peh activity in one transformant is 10-35% of the wild-type activity based on viscometric and reducing sugar assays. End-product analysis indicates that the residual activity in the mutant is due to an exo-Peh. Pathogenicity on maize of the mutant lacking endo-Peh activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction, endo-Peh is not required (Scott-Craig et a\., 1990). Twenty-two percent of 522 strains of Xanthomonas campestris pv. vesicatoria have been found to be pectolytic by secreting a single pectate lyase with pI of 8.8. Mutants deficient in synthesis of the pectate lyase have been selected after nitrosoguanidine treatment. The mutants are not altered in their ability to evoke disease symptoms, to grow in plants, or to induce the HR in nonhost plants. Thus, the pectate lyase gene appears to play no essential role in pathogenicity (Beaulieu et a\., 1991), The ability of E. chrysanthemi to cause soft-rot diseases in plants has been linked to its production of endo-Pel. Mutant UMlO05, mutated from E. chrysanthemi strain EC 16, contains deletions in the pel genes that encode the endo-Pel but still macerates plant tissues. Screening the gene library for pectolytic activity resulted in the identification of a clone (pPNL5) that contains the gene for exo-Pel. The gene is located on a 3.3-kb EcoRV fragment that contains an ORF for a 79.5 kD polypeptide. Exo-Pel purified from E. coli DH5a(pPNL5) has an apparent molecular mass of 76 kD with pI 8.6. The enzyme can utilize pectate and pectin as substrates. An endoPel'exoPel' mutant of EC 16 has been constructed that exhibits reduced growth on pectate but

106 - Plant Pathogenesis and Resistance retains pathogenicity on chrysanthemum equivalent to that ofUM1005. These results indicate that exo-Pel does not contribute to the residual macerating activity ofUMI005 (Brooks et aI., 1990). The importance of endo-p-l ,4-glucanase (EGL) in wilt disease has been investigated in Pseudomonas solanacearum. A 2. 7-kbXhoI-SaII DNA fragment containing the egl gene has been cloned and mutagenized with Tn5 and used to specifically mutate the chromosomal egl gene of P. solanacearum by sitedirected mutagenesis. The resultant mutant is identical to the wild-type strain in production of extracellular polysaccharides, extracelluar Peh, and several other extracellular proteins but produces at least 200-fold less EGL. This mutant strain is significantly less virulent on tomato than the wild-type strain in bioassay experiments. Virulence of the EGL-deficient strain is restored to near wild-type level by complementation with the cloned egl gene, indicating that the egl gene is important, but not absolutely required, for pathogenesis (Roberts et aI., 1988). In E. chrysanthemi strain 3937, EGL plays a minor role in soft-rot disease development in Saintpaulia ionantha. The cloned cel genes (celZ and celY) have been mutagenized and recombined with the chromosomal alleles. Strains with an Q interposon in celZ, a deletion in celY, or a double cel mutant are as virulent as the wild-type strain (Boccara et aI., 1994).

2.5 CELL-WALL-DEGRADING ENZYMES AND DISEASE RESISTANCE Cell-wall-degrading enzymes play an important role during plant pathogenesis by facilitating fungal and bacterial colonization through theirdegradative effect on plant cell walls. Many plants are able to synthesize protein or glycoprotein inhibitors that inhibit cell-wall-degrading enzymes (Table 2-10). For example, mature green fruits of pear (Pyrus communis) produce a 43-kD glycoprotein that inhibits endo-Peh produced by Botrytis cinerea (Stotz et aI., 1993). In Phaseolus vulgaris, there is an early and rapid accumulation of polygalacturonase inhibitory protein (PGIP) mRNA in an isogenic line that exhibits hypersensitive resistance to race y of Colletotrichum lindemuthianum (N uss et aI., 1996). Cell-wall-degrading enzymes themselves and their degradation products have been implicated as signal molecules that elicit host defense responses by eliciting phytoalexin accumulation (Esquem!-Tugaye etal., 2000). For example, Peh from Rhizopus stolonifer elicits casbene synthesis activity in castor bean seedlings, resulting in the accumulation of cas bene, a castor bean phytoalexin (Bruce and West, 1982; lin and West, 1984; Lee and West, 1981b). Pel from Erwinia carotovora elicits accumulation of glyceollin, a soybean phytoalexin, in soybean cotyledons (Davis et aI., 1984). Similarly, endo-Peh (Peh-II and PehVI) purified from Sclerotinia sclerotiorum are el icitors of glyceollin I synthesis

Degradation of Cell Walls - 107 in soybean hypocotyls (Favaron et aL, 1988). The endo-Peh from Fusarium moniliforme in suspension-cultured carrot cells induces both the accumulation of6-methoxymellein, a carrot phytoalexin, and the activityof6-hydroxymellein O-methyltransferase, the enzyme that specifically methylates the C-6-hydroxyl group in 6-hydroxymellein to give 6-methoxymellein (Marinelli et aL, 1994). Oligosaccharides of different sizes are released from intact Citrus limon seedlings treated with endo-Peh obtained from Alternaria alternata. The treatment also increases PAL activity and the formation of a phytoalexin (Roco et aL, 1993). Table 2-10. Polygalacturonase inhibitory proteins (PGIPs). Plant

Chemical nature

Enzyme inhibited

Reference

Bean (Phaseolus vulgaris)

46-kD glycoprotein

Lafitte et al. (1984)

Bark of Chinese chestnut

protein

Stalk tissue of leek (Allium porrum)

39-42 kD protein

Rind of Valencia orange (Citrus sinensis) Fruit of pear (Pyrus communis) cv. Bartlett Immature fruit of raspberry

54-kD protein

endo-Peh from Colletotrichum lindemuthianum endo-Peh from Colletotrichum lindemuthianum, Cryphonectria parasitica endo-Peh from Scierotinia scierotiorum, Botrytis aciada endo-Peh from Diplodia natalensis endo-Peh from Botrytis cinerea endo-Peh from Botrytis cinerea. Aspergillus niger endo-Peh from Botrytiscinerea

43-kD glycoprotein 38.5-kD protein

Fruit of tomato 35-41kD (Lycopersicon esculentum) glycoprotein

Gao and Shain (1995)

Favaron et al. (1997)

Barmore and Nguyen (1985) Stotz et al. (1993) Johnston et al. (1993) Stotz et al. (1994)

Cell-wall-degrading enzymes and their products may elicit a resistant response by accumulating PR proteins. The transcript for p-I,3-glucanase rapidly accumulates in tobacco plants inoculated with E. coli harboring genes for Pel and Peh, but not cellulase, from E. carotovora subsp. carotovora (PaIva et aL, 1993). The endo-Peh from Colletotrichum lindemuthianum race p increases P-I ,3-glucanase activity earlier in resistant than in susceptible, nearisogenic lines of Phaseolus vulgaris. Elicitation of defense is abolished by the addition of exo-Peh to the bioassay. It is possible that pectic fragments of a critical size are required for endo-Peh-mediated defense (Lafitte et aL, 1993).

108 - Plant Pathogenesis and Resistance A pectic polysaccharide, possibly released by tomato Peh activity, has been identified as a proteinase inhibitor-inducing factor (Bishop et aI., 1981). This polysaccharide also serves as a phytoalexin elicitor (Walker-Simmons et aI., 1983). Cell-wall-degrading enzymes and their degradation products may reinforce cell wall structure by lignification. The endo-Peh from Cladosporium cucumerinum elicits lignification in cucumber hypocotyls at a concentration of about 70 ng/mL. The enzyme has an optimum pH between 5.0 and 5.5 and a molecular weight of about 38 kD (Robertsen, 1987). Cell-wall-degrading enzymes and their degradation products may regulate HRGP accumulation in plant cell walls. Treatment of bean cell walls with Peh purified from cultures of Colletotrichum lindemuthianum race p released oligogalacturonides and pectic fragments. Small oligogalacturonides with a DP of2-3 elicit a 40-70% hydroxyproline increase within 48 hr at 450 nmol/bean cutting. High-molecular-weight pectic fragments, on the other hand, suppress hydroxyproline deposition by 30-40%. Northern-blot analysis of RNA shows that changes in the transcription intensity occur within 12 hr after the treatment. These results indicate that the change in HRGP in plant cell walls may affect a plant's defense and development (Boudart et aI., 1995).

2.6 CONCLUSIONS Pathogenicity of an organism is determined by several factors, including the chemical weapons the organism possesses, the defense mechanisms that exist in the host plant, and environmental conditions. Cell-wall-degrading enzymes may be regarded as one of the most powerful weapons possessed by certain plant pathogens, particularly for those causing soft rot and wilt. Many plant cell walls possess polygalacturonase inhibitory proteins that inactivate the cell-wall degrading enzymes and thus reduce their detrimental effects on host cell structure and function (Table 2-10). For example, a protein, with an apparent molecular weight of 54 kD, inhibits Peh secreted by Diplodianatalensis. Under acidic conditions, the inhibitor forms a complex with the Peh. The complex is dissociated at pH 8.0, and the Peh restored its activity when assayed at pH 5.0 (Barmore and Nguyen, 1985). Cell-wall-degrading enzymes may be of little or no importance in diseases caused by leaf spot pathogens. For example, disruption of a gene (PGNI) encoding extracellular endo-Peh in Cochliobolus carbonum race I makes the pathogen a Peh-negative mutant. Pathogenicity on maize of this mutant is qualitatively indistinguishable from the wild-type strain. The results indicate that Peh is not required for pathogenicity in foliar diseases (Scott-Craig et aI., 1990).

Degradation of Cell Walls - 109

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Chapter 3 BIOENERGETICS IN PLANT-PATHOGEN INTERACTIONS

3.1 3.2

3.3

3.4

3.5.

Introduction An overview of energy-capture and energy-utilization processes in higher plants 3.2.1 The energy capture process. 3.2.1.1 The photosynthetic apparatus 3.2.1.2 The light reactions . 3.2.1.3 The dark reactions . A. The C 1 pathway B. The C4 pathways 3.2.2 The energy-utilization process 3.2.2.1 Mitochondrion. 3.2.2.2 Glycolysis . 3.2.2.3 TCA cycle. 3.2.2.4 Electron transfer chain and oxidative phosphorylation 3.2.2.5 Alternative electron transfer system The energy-capture process as affected by pathogenic infection. 3.3.1 Photosynthesis of diseased plants 3.3.2 Factors affecting pathological photosynthesis 3.3.2.1 Reduction in CO 2 diffusion into diseased leaves 3.3.2.2 Degeneration of chloroplasts 3.3.2.3 Alteration in photochemical reactions 3.3.2.4 Alteration in photosynthetic carbon assimilation 3.3.2.5 Alteration in translocation of photosynthetic products 3.3.3 Photosynthesis and disease resistance The energy-utilization process as affected by pathogenic infection 3.4.1 Respiration of diseased plants 3.4.2 Factors affecting pathological respiration 3.4.2.1 Energy utilization by pathogens 3.4.2.2 Uncoupling of oxidative phosphorylation and increased participation of cyanide-resistant respiration 3.4.2.3 Stimulation of biosynthetic processes 3.4.2.4 Change in hexose catabolic pathways 3.4.2.5 Dysfunction of mitochondria Conclusions References .

132 132 132 132 138 139 139 141 141 143 144 146 147 150 150 150 152 152 153 156 158 160 162 162 162 164 164 164 166 167 168 169 169

132 - Plant Pathogenesis and Resistance

3.1 INTRODUCTION The objective of this chapter is to understand how disease affects energycapture and energy-utilization processes in plants. Since energy capture and utilization take place mainly in chloroplasts and mitochondria, respectively, certain concepts of photosynthesis and respiration and their metabolic regulation will be briefly reviewed. In higher plants, energy for metabolic machinery comes from the sun's energy. The sun's energy is first converted into chemical energy in the form of ATP which in turn is used to fix carbon dioxide into organic compounds. The process, photosynthesis, takes place in chloroplasts. The organic compounds are converted to carbohydrates and are translocated to various parts of the plant. The energy stored in carbohydrates is released by the respiration process in mitochondria for metabolism, growth, and reproduction. Thus, photosynthesis in chloroplasts and respiration in mitochondria govern bioenergetics in higher plants. Fig. 3-1 summarizes the relationship between energy capture and energy utilization in higher plants. Monographs that detail the structure and function of the chloroplast and mitochondrion are available (Hall and Rao, 1994; Hoober, 1984; Lawlor, 1993; Tzagoloff, 1982). Biochemistry and physiology of these two subcellular organelles also have been extensively reviewed (Avron, 1981; Robinson and Walker, 1981). Plant pathogens have numerous ways to affect directly and indirectly structure and function of chloroplasts and mitochondria. Consequently, bioenergetics are altered. For bioenergetics in host-parasite interactions, readers are referred to Kosuge and Kimpel (1981) and Hutcheson and Buchanan (1983).

3.2 AN OVERVIEW OF ENERGY-CAPTURE AND ENERGYUTILIZATION PROCESSES IN HIGHER PLANTS 3.2.1 The Energy-Capture Process The energy in the plant metabolic machinery comes from the electromagnetic energy of sun I ight. The electromagnetic energy is first converted to biological useful chemical energy in the form of A TP via a process known as photophosphorylation. A TP in turn is used in reduction of carbon dioxide into organic compounds. These assimilates are first converted to starch in the chloroplasts and later transported to various sites for metabolic activity, growth, and reproduction. 3.2.1.1 The photosynthetic apparatus The process of photosynthesis in higher plants takes place in chloroplasts. They are lens-shaped subcellular organelles, 1-3 x 5-7 /-un, typically 50 to 200 per cell. Fig. 3-2 illustrates the ultrastructure of a mature chloroplast in a mesophyll cell of a tobacco leaf. The chloroplast is bounded by a double-membrane envelope. Within the envelope are thylakoid membranes and the stroma. The

Bioenergetics - 133 thylakoid membrane may be stacked or unstacked and are called granal thylakoids and stromal thylakoids, respectively. It is made up of lipids and proteins and is the site of light reaction of photosynthesis. Electromagnetic energy of sunlight is trapped by lipoid pigment molecules and converted into chemical energy in the form of A TP and reducing power in the form of NADPH. The stroma contains enzymes essential for dark reaction of photosynthesis. A TP and NADPH are used to fix CO 2 into carbohydrates and other organic compounds in a series of enzymatic reactions. Light ATP

+

ADP NADPH

Photophosphorylation

Photoassimilation

NADP

1 --------------------------------l----------Photoassimilates

ｾＭ

ｾｓｩｯＭ

I

Glycolysis

ｾ＠

ｾ＠

ATP

+

Metabolism, Growth, Reproduction

Electron transfer system

ｾ＠

+

I

Acetyl-CoA

•

(:) cycle

ADP+Pi

Fig. 3-1. Relationship between energy capture and energy utilization.

The major lipid components of the thylakoid membrane are chlorophylls, carotenoids, and galactolipids. The most important lipid is chlorophyll a. It is found in all photosynthetic plant cells. It consists of a cyclic tetrapyrrole porphyrin and a long, isoprenoid alcohol phytol. The first compound committed to the synthesis of the cyclic tetrapyrrole porphyrin portion of chlorophyll is 5aminolevulinic acid. The carbon skeleton of 5-aminolevulinic acid is derived from glutamic acid. Two molecules of 5-aminolevulinic acid are condensed to form porphobilinogen, a reaction catalyzed by 5-aminolevulinic acid dehydratase. Four porphobilinogen molecules are subsequently converted to the tetrapyrrole hydroxymethylbilane (preuroporphyrinogen). The reaction is

134 - Plant Pathogenesis and Resistance catalyzed by porphobilinogen deaminase (l-hydroxymethylbilane synthase). The cyclization ofhydroxy-methylbilane is carried out by uroporphyrinogen III synthase. The cyclic tetrapyrrole is then subjected to decarboxylation and oxidation reactions to form protoporphyrinogen IX. During these reactions, four acetic acid side chains are shortened to methyl groups, two propinoic acid side chains are trimmed into vinyl groups and two double-bonds are added to the molecule. By a series of modifications, including insertion of Mg2+, methylation, and cyclization to form ring V, proto-porphyrinogen IX is converted to protochlorophyllide. The reduction of ring IV converts protochlorophyllide to chlorophyll ide.

Fig. 3-2. Electron micrograph of a mature chloroplast in the parenchyma cell ofa apple stem. The chloroplast is bounded by a double membrane. It contains the stroma, stacks of thylakoid membranes (grana) and intergranal lamella, and starch granules (X 36,500). (Reproduced from Huang and Goodman, 1976, with permission from the American Phytopathological Society).

The tail portion of chlorophyll, phytol, is derived from geranylgeranyl pyrophosphate which is synthesized via the acetate-polymevalonate pathway (see Chapter 9). The esterification between phytol and chlorophyll ide is catalyzed by chlorophyll synthetase. Chlorophyll b has the same structure as chlorophyll a with the exception that CH3 group in ring II of chlorophyll a is replaced with a CHO group. The biosynthesis of chlorophyll has been reviewed in detail by Von Wettstein et al. (1995) and is summarized in Fig. 3-3.

Bioenergetics - l35

COOli

:I1l9' CI6:2112,9, CI6:3112,4,9' in addition to a series of C 18 to C20 (w-l )hydroxylated fatty acids. A deletion of nodE resulted in the absence of C 16:2 and C 16:3 fatty acids, which were replaced with CI8:llllI' The proportion of (w-l )hydroxylated fatty acids, remained unchanged, however. A nodF deletion resulted in the same alterations in the N-acyl composition. These results indicate that both nodF and nodE are required for the synthesis of the C 16 polyunsaturated fatty acids (Demont et aI., 1993). R. leguminosarum bv. viciae nodulates plants of the genera Vicia, Pisum, and Lens. The closely related R. leguminosarum bv. trifolii nodulates only plants of the genus Trifolium. The nodE genes of these two biovars have been sequenced. The proteins encoded by these genes have a predicted molecular mass of 42 kD and share a 78% homology (Spaink et aI., 1989). The difference in host range is determined by 44 non-conserved amino acid residues in the central domain of these two NodE proteins. The wild-type strain RBL5560 of the biovar viciae produces Nod factors containing CI8:4112,4,6,1I or C18:llllI and nodulates Vicia sativa. Strain RBL601, which has the pSym interrupted by Tn5 at nodE, produces only Nod factors containing C18:llllI and fails to nodulate vetch. Thus, the NodE protein is involved in biosynthesis of CI8:4112,4,6,1I fatty acid (Spaink et aI., 1991). In biovar trifolii, NodE synthesizes C 18 :2 and C 18:3 fatty acyl chains. Thus, the host range of the biovars viciae and trifolii may be determined by the degree of hydrophobicity of the polyunsaturated fatty acyl moieties of their Nod factors (Bloemberg et aI., 1995).

Acetylation at C-6 of the nonreducing residue - The role of nodL All Nod factors of R. leguminosarum bv. viciae and S. meliloti contain an 0acetyl group at the C-6 of the terminal, non-reducing glucosamine residue. The acetylation is catalyzed by acetyl transferase. The nodL gene from R. leguminosarum encodes a protein of 190 am ino acids with a Ai. of 20,105 (Surin and Downie, 1988). There is a significant homology between the protein encoded by nodL and the proteins encoded by the lacA and cysE genes of E. coli. The lacA gene from the lactose operon encodes thiogalactoside acetyl transferase and cysE encodes serine acetyl transferase, an enzyme involved in the biosynthesis of cysteine. The strongest homology occurs in the region between amino acid residues 139 and 169 of all three proteins. These findings suggest that nodL encodes an acetyl transferase (Downie, 1989).

Rhizobium-Legume Symbiosis - 193

S. meliloti strain 2011 produces O-acetylated Nod factors. The nodL mutants produce the same Nod factors without acetylation (Ardourel et aI., 1994). Similarly, R.leguminosarum bv. viciae strain RBL5560 (a wild-type containing Sym plasmid) produces O-acetylated Nod factor and strain 5793 (a nodLmutant containing nodL::Tn5phoA) produces the same Nod factor without 0acetylation (Spaink et aI., 1991). These results suggest that O-acetylation is specified by the nodL gene. N-methylation of the non-reducing residue - The role of nodS In A. caulinodans strain ORS571 nodABCSUIJ forms a single operon. Mutants with Tn5 insertions in the genes nodS, nodU and nodJ were delayed in the nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences ofORS571, B.japonicum and Rhizobium sp. strain NGR234 contain a consensus with similarity to the methyltransferase that utilizes S-adenosylmethionine (SAM) as the substrate (G6ttfert et aI., 1990b; Lewin et aI., 1990). A naringenin-induced nodS-dependent protein of approximately 25-kD reacted with labeled SAM, suggesting the presence of a SAM-binding site in the NodS protein. In vivo application of L-[Me- 3H]methionine, a precursor of SAM, resulted in the formation of methylated Nod factors by the wild-type strain but not the nodS mutants. These results suggest that the NodS protein is a SAMutilizing methyltransferase (Gee len et aI., 1993). It is tempting to assume that the molecular site of N-methylation is at the nonreducing glucosamine residue, as the Nod factors of A. caulinodans and Rhizobium sp. strain NGR234 are Nmethylated atthe nonreducing ends. The Nod factors produced by B.japonicum are notN-methylated although this bacterium harbors a nodS gene. It is possible that the expression of this gene is too low to be functional in B.japonicum. Fucosylation at C-6 of the reducing residue - The role of nodZ One of the common modifications found in Nod factors of B.japonicum is the presence of a 2-0-methylfucose (or fucose or other substituted fucose) group at the reducing residue (Carlson et aI., 1993; Sanjuan et aI., 1992). The addition of the methylfucose to the reducing N-acetylglucosamine residue requires the presence of the nodZ gene. Mutations in nodZ result in defective nodulation of siratro (Macroptilium atropurpureum) and altered nodulation ability on a few varieties of soybean (Stacey et aI., 1994). The nodZ gene encodes a protein of323 amino acids with a predicted M, of 36,600. Using the internal 895-bp of the nodZ as a probe, positive hybridization was found in all B.japonicum strains, other Bradyrhizobium spp., S.fredii and Rhizobium sp. strain NGR234. These results agree with the reports that these rhizobia produce fucosylated Nod factors (see Table 4-4). Unlike other nod genes, the expression of nodZ is not regulated by the NodD protein (Stacey et aI., 1994).

194 - Plant Pathogenesis and Resistance

Glycosylation at C-J of the reducing residue - The role ofnolO The nolMNO genes are components of a 9-kb nodYABCSUIJnolMNO operon in B.japonicum USDA 110. The nolO and nolNO mutants produced fucosylated Nod factors that are normally produced by the wild-type bacteria and several other modified compounds that are not produced by the wild-type. These modified compounds included lipooligosaccharides that are not fucosylated but linked glycosidically to glycerol at the reducing end. These mutants had reduced nodulation efficiency (Luka et aI., 1993). The presence of glycerol has been found also in Nod factors of B. elkanii USDA61 (Carlson et aI., 1993). Sulfation at C-6 of the reducing residue - The role of nodPQ , and nodH In E. coli, cysN and cysD genes encode subunits of ATP sulfurylase (EC 2.7.7.4), the enzyme which catalyzes the formation of adenosine 5'phosphosulfate (APS) from A TP and cysC encodes APS kinase (EC 2.7.1.25), the enzyme which phosphorylates APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The sulfuryl moiety of PAPS is reduced to sulfite and then sulfide before being used in cysteine biosynthesis. The nodP and nodQ genes of S. meliloti encode ATP sulfurylase and APS kinase and are homologous to the cysNDC genes in E. coli. The products of nodPQ form a sulfate activation complex that requires GTP for activity (Schwedock et aI., 1994). The nodPQ and nodH genes in R. tropici CIAT899 have also been characterized (Folch-Mallol et aI., 1996). The ORF of nodP encodes a protein of 300 amino acids with a calculated ｾ＠ of 34,756, while the ORF of nodQ encodes a protein of 633 residues with a deduced ｾ＠ of 69,712. They are respectively 75% and 74% homology with the nucleotide sequences of S. meliloti (Cervantes et aI., 1989; Folch-Mallol et aI., 1996; Schwedock and Long, 1989). The ORF of nodH encodes a polypeptide of 250 amino acids with a predicted ｾ＠ of 28,492. It has a 70% nucleotide sequence homology with the nodH of S. meliloti (Debe lie and Sharma, 1986; Folch-Mallol et aI., 1996). In addition to nodPQ, in vitro experiments have shown that NodH encoded by nodH of S. meliloti has sulfotransferase activity (Schultze et aI., 1995). The nodH mutants are unable to produce sulfated Nod factors and do not nodulate alfalfa (Medicago sativa). Instead, these mutants gain the ability to induce nodules on vetch (Vicia sativa subsp. nigra). The nodP or nodQ mutants produce a mixture of sulfated and nonsulfated molecules (Roche et aI., 1991) and are able to form nodules on both Medicago and Vicia (Schwedock and Long, 1992). Thus, Nod factor sulfation is an important factor in determining host specificity.

sot.

Rhizobium-Legume Symbiosis

-

195

Table 4-3. The biological functions of the nodulation gene products. Gene

nodA

Product (kD) Function 22-25

N-acyltransferase

Rhizobia Sm Sf

nodB

node

23-25

44-47

Chitooligosaccharide N-deacetylase

N-acetylglucosaminyltransferase

Rg Rlt Sm Ac Sf Rlt Ac Sf Sm Rio

nodD,

35

nodD}

36

nodDJ

37

nodE

42

I3-Ketoacyl synthase

nodF

10

Acyl carrier protein

nodG nodH

27 29

Ribitol dehydrogenase Sulfotransferase

nodI

34

ATP-binding transport protein Secretion of Iipochitin oligosaccharides

nodJ

28

nodL

20

Hydrophobic protein; secretion of Iipochitin oligosaccharides Acetyl transferase

Transcriptional activator of inducible nod genes

B·.I B·.I Sm Bj Sm Sm

Rit Sm Rlv Sm Sm Sm Rt Rlv Rio Rlt Re Rlv Rlt Re Rlv Re Sm

Reference Atkinson et al. (1994) Rohrig et al. (1994) Krishnan and Pueppke (1991a) Riisiinen et al. (1991) Spaink et al. (1994) John et al. (1993) Krishnan and Pueppke (199Ia) Spaink et al. (1994) Geremia et al. (1994) Krishnan and Pueppke (199Ia) Atkinson and Long (1992) Collins-Emerson et al. (1990) Debelle et al. (1992) Gottfert et al. (1992) Gottfert et aI. (1986) Gottfert et al. (1992) Gottfert et al. (1986) Gottfert et al. (1986) Kondorosi et al. (1991a) Rushing et al. (1991) Spaink et al. (1989) Debelle and Sharma (1986) Shearmen et al. (1986) Platt et al. (1990) Debelle and Sharma (1986) Schultze et al. (1995) Roche et al. (1991) Folch-Mallol et al. (1996) Evans and Downie (1986) Young et al. (1990) Spaink et al. (1995) Cardenas et al. (1996) Evans and Downie (1986) Spaink et al. (1995) Cardenas et al. (1996) Downie (1989); B10emberg et al. (1994) Corvera et al. (1999) Baev and Kondorosi (1992)

196 - Plant Pathogenesis and Resistance Table 4-3. (continued) Gene

Product (kD) Function

nodM

66

Glucosamine synthetase

nodN

18

Unknown

nodO

30

nodP

35

Interacting with plant plasma membrane to form ion channels Determinant of host range

nodQ

71

A TP-sulfurylase GTP-binding protein

nodR nodS

14 25

nodT

51-58

nodU nodV nodW nodX

99 25 41

nodY nodZ nolA nole

16 35 27 44

nolE naiF nolG nolH noll nolK

12 34 31 24 49

nolL

42

nolM nolN nolO nolP naiR

6 14 40 II 13

Rhizobia Sm Rlt Rlv Rlv Sm Rlv Sm Rt Sm

Rt Unknown S-adenosylmethionine methylAc transferase Outer-membrane transport protein Rlt Rlv 6-0-carbamoy Itransferase NGR234 Ac B'J Sensor protein Transcriptional regulatory protein Bj O-acetyltransferase catalyzes Rlv O-acetylation of the C-6 of the reducing sugar Unknown Fucosy!transferase 8j DNA-binding regulatory protein Bj 8'J Unknown

Reference Baev et al. (1991) Lewis-Henderson and Djordjevic (1991) Surin and Downie (1988) Surin and Downie (1988) Baev et al. (1991) De Maagd (1989) Sutton et al. (1993) Schwedock and Long (1989) Folch-Mallol et al. (1996) Schwedock and Long (1989); Cervantes et al. (1989); Faucher et al. (1989) Folch-Mallol et al. (1996) Geelen et al. (1993) Rivilla et al. (1995) Surin et al. (1990) Jabbouri et al. (1995) D'Haeze et al. (1999) Gottfert et al. (I 990a) Gottfert et al. (1990a) Davis et al. (1988); Firmin et al. (I 993)

Stacey et al. (1994) Sadowsky et al. (1991) Krishnan and Pueppke (1991 b) Protein in the periplasmic space Rip Davis and Johnston (1990) Unknown Sm Baev et al. (1991) Unknown Baev et al. (1991) Sm Unknown Baev et al. (1991) Sm Unknown Baev et al. (1991) Sm GDP-4-keto-6-deoxy-mannose Mergaert et al. (1996) Ac 3,5-epimerase/4-reductase Sf Lamrabet et al. (1999) O-acetyltransferase catalyzes 4-0- Rio Scott et al. (1996) acetylation of the fucose residue Corvera et al. (1999) Re B'J Unknown Luka et al. (1993) 8'J Unknown Luka et al. (1993) 3-0-Carbamoyl transferase NGR234 Jabbouri et al. (1998) Unknown Davis and Johnston (1990) Rip Repressor of the nod regulon Sm Kondorosi et al. (1991 b)

Rhizobium-Legume Symbiosis - 197 Table 4-3. (continued) Gene

noeC noeE noel noeJ noeL

Product (kD) Function

Rhizobia

Arabinosylation Fucose sulphation 2-0-Methyltransferase GDP-Mannose pyrophosphorylase GDP-Mannose 4,6-dehydratase

Ac NGR234 NGR234 NGR234 NGR234

Reference Mergaert et al. (1996) Hanin et al. (1997) Freiberg et al. (1997) Freiberg et al. (1997) Freiberg et al. (1997)

Ac=Azorhizobium caulinodans; Bj=Bradyrhizobium japonicum; Re=R. etli; Rg=Rhizobium galegae; Rlp=R. leguminosarum biovar phaseoli; Rlt=R. leguminosarum biovar trifolii; Rlv=R. leguminosarum biovar viciae; Rlo=R. loti; Rt=R. tropici; NGR234=Rhizobium sp. strain 234; Sf=Sinorhizobiumji"edii; Sm=s. meliloti

Acetylation at C-6 of the reducing end - The role of nodX Strain TOM is one of the R. leguminosarum biovar viciae strains that is able to nodulate Afghanistan peas. Most R. leguminosarum biovar viciae strains produce nod factors consisting of four or five P-l,4-N-acetylglucosamine residues in which the non-reducing end carries an O-acetyl group and a C 18:4 or C 18: 1 N-acyl fatty acid. One of the Nod factors synthesized by strain TOM has an additional O-acetyl group at C-6 of the reducing end. This acetylation is NodX-dependent. Thus, NodX mediates O-acetylation of a Nod factor. The amino acid sequence of the NodX protein has no similarity to NodL, which acetylates C-6 of the non-reducing glucosamine residue (Firmin et a!., 1993). The other nod genes There are two genes that encode glucosam ine synthase in R. legumin osarum bv. viciae: glmS on the chromosome and nodM on the pSym. The enzyme forms glucosamine by replacing a hydroxyl group with an amino group on the C-2 of glucose. Glucosamine is the precursor of N-acetylglucosamine, the building block of Nod factors. The nodM gene is expressed when the rhizobia are in the rhizosphere and within the infection threads. When the bacteria are released from the infection threads to form bacteroids, nod genes, including nodM, are no longer expressed. Normal development of the bacteroid requires the expression of glmS. glmS mutants develop nodules containing bacteroids that are abnormally shaped, highly vacuolated, and rapidly senescent. These nodules have reduced levels of symbiotic nitrogen fixation (Marie et a!., 1994). In R. leguminosarum biovar viciae pRL 111, the nodO gene encodes a protein of284 amino acids with aM. of30,002. The NodO protein is secreted into the culture medium but it does not have the N-terminal signal sequence commonly found in proteins secreted by gram-negative bacteria. The NodO protein has limited homology with hemolysin of E. coli which is also a secreted protein without an N-terminal signal sequence (De Maagd et a!., 1989). Recent results have indicated that NodO forms Ca2+-regulated ion channels in an artificial

198 - Plant Pathogenesis and Resistance membrane. It is possible that the biological function of the NodO protein is to initiate a specific ion flux across the plasma membrane and to facilitate the entrance of Nod factors into root hairs (Sutton et aI., 1994).

4.2.2.4 Exporting the Nod factors In R. ieguminosarum bv. viciae pRL 1JI, nodIJ are located 2-kb downstream of nodABC and are in the nodABCIJ operon whereas nodT is located 168 nucleotide downstream of nodMN (Surin et aI., 1990). The nodI and nod! mutants delay nodulation. The ORFs corresponding to the nodI and nodJ genes encoded proteins with predicted M, of 34,301 and 27,683, respectively. The predicted amino acid sequence of NodI is homologous to hisP and malK gene products. HisP is involved in the active transport of histidine and MalK is involved in transport of maltose. Both HisP and MalK proteins have high affinity toward ATP. The sequence of the predicted nod! product suggests that the protein is hydrophobic and may be an integral membrane protein as well. Thus, the products of nodIJ genes may be involved in active transport. The substrates being transported have been determined to be lipochitin oligosaccharides (Spaink et aI., 1995; Cardenas et aI., 1996). The kinetics of lipochitin oligosaccharides secretion by R. etli wild-type strain and derivatives carrying disrupted nodI and nodJ genes has been studied on bean. Lipochitin oligosaccharides were detected in the culture media of the wild-type strain as early as 1 hr after nod gene induction. In contrast, nodI and nod! mutants secreted less Iipochitin oligosaccharides and accumulated the oligosaccharides intracellularly. These mutants have a delayed nodulation phenotype and a reduction in the number of nodules formed in bean roots (Cardenas et aI., 1996). The ORF corresponding to nodT is predicted to code a protein of 483 amino acids with aM, of51,471. Mutation ofnodThas no effect on nodulation phenotype (Surin et aI., 1990). In R. leguminosarum bv. trifolii, nodT is located immediately downstream of nod! and is in the nodABCIJT operon. The gene is predicted to code a protein of 468 amino acids with a M, of 50,345. The predicted protein has a sequence of ser-gly-cys at positions 16-18, a typical cleavage site for outermembrane proteins that have lipid on the cysteine (Wu and Tokunaga, 1986). Thus, the nodT product has an N-terminal signal peptide of 17 amino acids (Surin et aI., 1990). The presence of the signal peptide can be assayed using gene fusions with a phoA construct lacking an N-terminal signal peptide. The PhoA activity is expressed by cells only when PhoA is translocated across the inner membrane. Cells of E. coli containing a protein fusion of NodT and PhoA produced alkaline phosphatase activity, indicating the N-terminus of NodT could trans locate PhoA across the inner membrane. Cellular fractionation suggested that the NodT: :PhoA fusion is targeted to the outer membrane. The amino acid

Rhizobium-Legume Symbiosis - 199 sequence ofNodT is similar to those ofTolC from E. coli (Wandersman and Delepelaire, 1990), CyaE from Bordetella pertussis (Glaser et aI., 1988), PrtF from Erwinia chrysanthemi (Delepelaire and Wandersman, 1991) and AprF from Pseudomonas aeruginosa (Duong et al., 1992). All of these proteins are located in the outer membranes and are involved in protein secretion. TolC is necessary for the secretion of hemolysin and colicin V, CyaE is involved in secretion of cyclolysin toxin, PrtF trans locates metalloproteases PrtB and PrtC, and AprF is implicated in secretion of alkaline protease and a lipase. Thus the function ofNodT may be in the secretion of Nod factors (Rivilla et a!., 1995).

4.2.2.5 Naming the Nod factors The functions of the nod genes required for the biosynthesis and transport of Nod factors have been reviewed (Carlson et aI., 1994). In general, nodABC encode the proteins for the synthesis of a basic N-acetylglucosamine oligomer. The DP of the oligosaccharide may be 3 (III), 4 (IV) or 5 (V). The N-acetyl group at the nonreducing end of the oligosaccharide is attached with a fatty acid with different degrees of unsaturation. The N-acetyl group may be N-methylated (NMe). The C-3 and C-4 in B. elkanii USDA61 and Rhizobium sp. strain NGR234 may have one or two carbamoyl groups. The C-6 on the nonreducing residue may be substituted with an acetyl (Ac) or carbamoyl (Carb) group. The C-6 on the reducing end may be substituted with sulfate (S), 2-0-methylfucose (MeFuc), or 2-0-methyl 3-0-acetyl fucose (MeAcFuc) group. C-l on the reducing end may be substituted with glycerol in B. elkanii USDA61 and mannose (Man) in R. tropici CIAT899 (Folch-Mallol et aI., 1996). Thus, NodRm-IV(S,C I62 ) denotes the Nod factor produced by S. meliloti which has four N-acetylglucosamine units with a sulfate substitute at the C-6 of the reducing end. The fatty acid attached to the N-acyl group has 16 carbons with 2 unsaturated double bonds. NodBj-V(Ac,C I8 : I ,MeFuc) refers to the Nod factor of B. japonicum which consists of five N-acetylglucosamines with an acetyl group at the nonreducing end. The fatty acid has 18 carbons and 1 unsaturated double bond. The reducing end is substituted with a 2-0-methylfucose:

ｾｏ＠

OSO,H

C/H,

CH,OH

CH,OH

CH,OH

0=(,'

o=c'"

0=(''''

0=("

\

\

\

\

ｈｾ＠

CH

CH,

CH, CH, . .

\ (CH,); I

HC

ＨｃｾＲＩＵ＠

'CH NodRm-IV(S,C I6 :2 ) \

CH,

200 - Plant Pathogenesis and Resistance

ｈＳｃｏｾＺ＠ ｾｯ｜＠

PCOCH 3 ｾＮＧ＠

ｏｾ｜＠

ｏｾ｜＠ ｏｾ｜＠

O=C'

ｏｾ｜＠

O=C'

(tH,),

O=C'

'cH 3

ｲｏｾｌＳ＠

p OH

ｾ＠

O=C'

'CH3

O=C' 'cH3

'CH3

He'

\CH I

(CH,),

'cH 3

NodBj-V(Ac,Cls:I,MeFuc)

The Nod factors may be subjected to further modifications. Nod factors produced by Rhizobium sp. strain NGR234 carry a variety of substitutions. For example, one or both C-3 and C-4 on the reducing end may have a carbamoyl (NH 2CO') group (Price et aI., 1992). The following is a hypothetical pathway of Nod factor biosynthesis (Fisher and Long, 1992): Monosaccharides • NodM (glucosamine synthase)

Glucosamine I

: Node (N-acetylglucosaminyltransferase)

•

NodL (acetyitransferase)

"OCOC H3

NodA (N-acyltransferase)

I

ｾｏ｜＠

NodS (chitin oligosaccharide deacetylase)

NodH (sulfolransferase) OS 03H , ....0---:--- NodPQ (ATP sulfurylase, APS kinase) C H2

ｾ＠

CHz

OH

0

,

a,

O=C

O=C

\

Fatty acid

n

\

CH 3

t

NodE ＨｾＭｫ･ｴｯ｡｣ｹｬ＠ synthase) NodF (acyl carrier protein)

The structures of Nod factors produced by selected rhizobia are given in Table 4-4.

4.2.3 Rhizobial Genes Involved in Nitrogen Fixation The expression of nitrogen fixation (nifandfix) genes is independent of plant inducers. Many free-living prokaryotes are able to fix nitrogen. In fact, our understanding of biochemistry and molecular biology of nitrogen fixation is

Rhizobium-Legume Symbiosis - 201 Table 4-4. Structures of Nod factors produced by rhizobia.

Non-reducing End

Species and strain RI A. caulinodans ORS571 B. e/kanii USDA61 B. japonicum USDAIIO B. japonicum USDAI35

Me Me, H H

Reducin End

R2

R)

C18:0 C18:1 C18:1

H, Carb Ac, H2, H,Carb H H H2

ｃＱＸｾＹ＠

R4

H2

H

C16:0 H2 C I6:IM

H

C18:1 ｃｉＸＺＱｾ＠

Rs

R6

n

Reference

H, Ara Fuc, MeFuc MeFuc

H

2,3

Mergaert et al. (1993) Carlson et al. (1993)

H, 2,3 Glycerol H 3

Carlson et al. (1993) Carlson et al. (1993)

H, Ac

MeFuc H

3

H2

H

H2

Ac

Fuc, H MeFuc H H

1,2,3 Bec-Ferte et al. (1994) 2,3 Spaink et al. (1991)

H2

Ac

S

H

H, H S MeFuc H AcMeFuc MeSFuc

ｃｉＸＺＱｾＹ＠

S. fredii USDA257 R. /eguminosarum by. viciae RBL5560 S. meliloti 1021 R. tropici CFN299 Rhizobium sp. NGR234

H

C ＱＸａｾＲＮＴＶ＠ H ｃｉＶＺＲｾＮＹ＠

ｃｉＶＺＳｾＲＮＴＹ＠

Me

C18:1

H2

Me

C16:0 C18:1

H,Carb H Carb 2

H

2,3

Schultze et al. (1992)

3

Poupot et al. (1993)

3

Price et al. (1992, 1996)

derived mainly from studies of nif and fix genes in Klebsiella pneumoniae, Azotobacter vinelandii and several other free-living bacteria. Significant differences in nitrogen fixation between the free-living and symbiotic bacteria do exist. For a review of genetics of nitrogen fixation, see Ligon (1990), Elmerich (1991) and Fischer (1994),

4.2.3.1 Roles ofnifandflXgene products in the biosynthesis of nitrogenase Nitrogenase is a complex enzyme that consists of two protein components: dinitrogenase (EC 1.18.6,1, also known as component I or MoFe protein) and dinitrogenase reductase (also known as component II or Fe protein), Dinitrogenase consists of two dissimilar polypeptides with a conformation of (X2P2 and a molecular mass of 200 to 250 kD. The (X and P subunits are

202 - Plant Pathogenesis and Resistance encoded by the nifD and nifK genes, respectively. The enzyme also contains four [4Fe-4S] clusters and two iron-molybdenum cofactors (FeMo-co), a proposed site ofN 2 reduction. In K. pneumoniae, synthesis ofFeMo-co requires the participation of the niJB. nifH, nifQ. niJN. nifE and nifV genes (Hoover et aI., 1987a,b). The nifV gene encodes homocitrate synthase which synthesizes homocitric acid [(R)2-hydroxy-l ,2,4-butane-tricarboxylic acid] (Hoover et aI., 1987a,b). Homocitrate may function in the uptake, processing or targeting of metal ions for FeMo-co synthesis because it forms complexes with Fe3+ and MoO/+ (Hoover et aI., I 987a,b). The product of nifQ is also required for the uptake of molybdenum (Joerger and Bishop, 1988). The niJNE genes encode a protein containing equimolar amounts of the niJN and nifE gene products with a molecular mass of about 200 kD. The molecular mass is 49 kD for the niJN product and 50.2 kD for the nifE product. Thus, NifNE has an a 2 P2 structure. The protein is responsible for the synthesis of FeMo-co (Paustian et aI., 1989). FeMo-co has been synthesized in vitro. The synthesis requires the participation ofNifB, NifNE and NifH proteins and molybdenum, homocitrate and Mg-A TP (Ludden et aI., 1993). The requirement ofNifB protein can be satisfied by adding a low molecular weight NifB cofactor (NifB-co) into the reaction mixture. NifB-co has been purified. It consists of significant amounts of iron and appears to be a small Fe-S cluster (Shah et ai, 1994). A model of FeMo-co has been proposed (Madden et aI., 1992):

o .

o

I/o

Fel/i slＯｲＢＧｎ］ｯｆ･ｾｘｓ＠ Fe--S

0

...•

H

ｈＯＩｚｾ＠

A

0

S-Fe,

ｓｾｆＨ＠

Dinitrogenase reductase consists of two identical polypeptides that are encoded by the nifH gene and a single [4Fe-4S] cluster. The newly synthesized NifH product, however, is inactive. The conversion of the inactive NifH polypeptides to the functioning dinitrogenase reductase requires the product of nij1\1 (Howard et aI., 1986). The molecular mass of dinitrogenase reductase is 68 kD. The role of dinitrogenase reductase is to transfer electrons to dinitrogenase where reduction of nitrogen occurs. In addition to NifM, the nifS and nijU gene products are required for the maturation of a fully functional Fe protein in A. vinelandii (Jacobson et aI., 1989a). In most free-living and symbiotic nitrogen fixing bacteria, nifHDK genes are organized as a single transcriptional unit. Multiple copies ofnift/DK or nifH do

Rhizobium-Legume Symbiosis - 203 exist. For example, R. leguminosarum bv. phaseoli has two nif HDK operons and one additional nijH gene (Quinto et aI., 1982, 1985). Azorhizobium caulinodans has two copies of nijH gene (Norel and Elmerich, 1987). The involvement of various nif genes in the biosynthesis of nitrogenase complex is presented in Fig. 4-5 (Roberts and Brill, 1981). nifA (activator), nifL (repressor)

/"'...

nijH

pro Fe protein

ｮＡｦｍＮｕｾ＠

modification

niJK.D

pro MoFe protein

ｾ

!

Active Fe protein

Regulation genes

Structural genes

ｾＡＮｶ＠ nijW,Z

Modification genes

modi ficalian

Active MoFe protein

"'.../ ni/F.J

Ferredoxin

Electron transport genes

+

Nitrollenase

Fig. 4-5. Functions of nif genes in the biosynthesis of nitrogenase. (Adapted from Roberts and Brill, 1981).

Rhizobial fix genes are organized into clusters: fixABCX, flXNOQP, fixLJ, and fixGHIS. The biological function of certain fix gene products has been determined. The fixABCX genes have been identified in B. japonicum, S. meliloli, R. leguminosarum biovars viciae and phaseoli (Earl et aI., 1987; Fuhrmann et aI., 1985; Gronger et aI., 1987; Michiels and Vanderleyden, 1993). The products of jixABCX may have a role in electron transfer (Gubler et aI., 1989). The flXGHIS genes have been identified in S. meliloli (Kahn et aI., 1989). The products of these genes are transmembrane proteins and may be involved in cation pumping. FixL and FixJ are the two-component regulatory system proteins (David et aI., 1988). In this system, FixL acts as an oxygen sensor that activates FixJ under low oxygen conditions. Activated FixJ promotes the transcription of the nifA andjixK genes whose products, in turn, activate transcription of other nif andjix genes (Monson et aI., 1993). The operonjixNOQP encodes membrane-bound, cytochrome c-containing heme/copper oxidase. Mutants in the jixNOQP gene cluster resulted in defective bacteroid development and symbiotic nitrogen fixation, suggesting

204 - Plant Pathogenesis and Resistance this oxidase complex is specifically required for the bacteroid respiration (Preisig et aI., 1993). The products and functions of rhizobia I nifandfix genes are listed in Table 4-5. Table 4-5. The nifandjix genes and their functions. Gene Product (kD) Function

Rhizobia

nifA

58.6

Activates expression of nif operons

nifB

50.8

nijD

54.1

nijE

50.2

nijF

19.1

Required for the synthesis of FeMo-co Structural gene for the ex subunit of the MoFe protein Required for the synthesis of FeMo-co Ferredoxin, electron transport

nijH 21.9 nif) 128.0 nijK

58.4

nijL nifM

55.2 30.6

nifN

49.2

nifQ nifS

20.7 43.2

nifT nijU

8.3

nifV

41.1

nifW

10.2, 13.5

nifX nifY nifZ

18.2 24.7 16.7

Reference

Kp Sm B·.I Rip Ac Av

Buchana-Wollaston et a!. (1981); Ditta et a!. (1987) Fischer and Hennecke (1987) Michielsetal. (1994) Nees et a!. (1988) Joerger and Bishop (1988)

Kp

Robson et a!. (1983)

Av

Paustian et al. (1989)

Kp

Deistung and Thorneley (\ 986) Robson et a!. (1983) Shah et a!. (1983)

Structural gene for the Fe protein Electron transport, Kp ferredoxin oxidoreductase Structural gene for the ｾ＠ subunit Kp of the MoFe protein Inhibits expression of nif operons Kp Maturation and activation of Kp the Fe protein Av Av Required for the synthesis of FeMo-co Av Uptake of molybdenum Cysteine desulfurase, Av maturation and activation of the Fe protein Unknown Kp Maturation and activation Av of the Fe protein Homocitrate synthase, Kp for the synthesis of FeMoco Protecting the MoFe protein Kp from O 2 damage Av Regulator of nifregulon Kp Unknown Av Maturation of the MoFe protein Kp Av

Robson et a!. (1983) Hill et a!. (1981) Howard et a!. (1986) Jacobson et a!. (1989a) Paustian et a!. (1989) Joerger and Bishop (1988) Jacobson et a!. (I 989a) Zheng et a!. (1993) Jacobson et a!. (1989b) Jacobson et a!. (1989a) Hoover et a!. (1987a,b) Paul and Merrick (1989) Jacobson et a!. (1989a) Kim and Burgess (1996) Gosink et a!. (\990) Jacobson et al. (1989b) Paul and Merrick (1989) Jacobson et a!. (1989a)

Rhizobium-Legume Symbiosis - 205 Table 4-5. (Continued) Gene Product (kD) Function

fixA

31.1

fixB

37.8

fixC

j'IXD j'IXG j'IXH fix! fix} fixK

47.2

Rhizobia

B'J Sm Ac B'J Flavoprotein Sm Ac Flavoprotein-Ubiquinone oxido- Sm reductase Ac

Regulator

Sm Sm Sm Sm Sm Sm

j'IXO

27.2

fixP

30.8

fixQ fixR fixS fixW fixX

5.5 30

Cation pump Sensor protein Activator and suppressor of nifandfix genes Regulator protein Sm Heme/copper-containing oxidase Rlv Bj Monoheme cytochrome c Rlv Bj Diheme cytochrome c Rlv Bj Membrane protein Bj B'J Regulator

24.9 10.9

Ferredoxin, electron transport

fixL fixN

22

55 60.9

Ac Sm Bj

Reference Gubler and Hennecke (1988) Earl et al. (1987) Arigoni et al. (1991) Gubler and Hennecke ( 1988) Earl et al. (1987) Arigoni et al. (1991 ) Earl et al. (1987) Kaminski et al. (1988); Arigoni et al. (1991); Goodman et al. (1994) Weber et al. (1985) Kahn et al. (1989) Kahn et al. (1989) Kahn et al. (1989) David et al. (1988) Batut et al. (1989) David et al. (1988) Schliiter et al. (1997) Preisig et al. (1993) Schliiter et al. (\ 997) Preisig et al. (1993) Schliiter et al. (1997) Preisig et al. (1993) Preisig et al. (1993) Thony et al. (1989) Arigoni et al. (1991) Earl et al. (1987) Dusha et al. (1987) Gubler et al. (1989)

Ac=Azorhizobium caulinodans; Av=Azotobacter vinelandii; Bj=Bradyrhizobiumjaponicum Kp=Klebsiella pneumoniae; Sm=Sinorhizobium meliloti

4.2.3.2 Regulation of nif gene expression The expression of the nif genes is regulated by a number of factors. In general, it is activated by microaerobic conditions and the nifA product. It is inhibited by high levels of O 2 and fixed nitrogen, including ammonia, nitrate, and amino acids, and the nifL product. In B. japonicum, NifA activates transcription of nif and fix genes during symbiosis in soybean nodules or in free-living microaerobic conditions. High O 2 tensions repress the nif andfix expression by regulating the activity ofNifA through oxidation of a metal cofactor. It has been shown that metal ions are essential for the activation of nif expression in vivo by NifA. Incubation with

206 - Plant Pathogenesis and Resistance the chelating agent o-phenanthroline abolished nifD activation by NifA. It is possible that the metal cofactor senses the oxygen conditions in the cell. Subsequently, the oxidized state of the NifA-metal complex influences the conformation ofNifA and its DNA-binding ability to the upstream activator sequences of nif and fix promoters, which are required for optimal activation (Morett et aI., 1991). In S. meliloti, transcription of nifA is also induced under microaerobic conditions. The expression of nifA is mediated by FixL and FixJ. The FixJL proteins belong to a two-component regulatory system and are responsible for sensing and transmitting the low-oxygen signal. Amino acid sequence analyses reveal that FixL and FixJ are homologous to a family of bacterial proteins that transduce environmental signals through a phosphotransfer mechanism (David et aI., 1988). FixL is an oxygen-binding hemoprotein and a kinase that phosphorylates FixJ (Gilles-Gonzalez et aI., 1991). A fragment of FixL, consisting of amino acid residues 127 to 260, binds heme and oxygen but has no kinase activity. The fragment at the C-terminal, beginning at residue 260, fails to bind heme but is active as a kinase. Thus, the FixL protein can be separated into a heme-binding oxygen-sensing domain and a kinase-active domain (Monson et aI., 1992). The expression of the R. meliloti nifA under microaerobic conditions is negatively regulated by ammonia and nitrate. The regulation is shown to be mediated through the FixL protein. A truncatedfix.! gene, the product ofwhich has been shown to induce nifA expression irrespective of the oxygen status of the cell, also circumvented the repressive effect of ammonia on nifA expression. These results suggest that the ammonia effect is mediated through the FixLJ regulatory cascade (Noonan et aI., 1992). 4.2.3.3 Other rhizobial genes involved in nitrogen fixation In addition to the nif and fix genes required for the synthesis of the nitrogenase complex, numerous genes are required for effective nitrogen fixation. The S. meliloti fdxN gene, which is part of the nifABfdxN operon, is required for symbiotic nitrogen fixation. The deduced amino acid sequence of FdxN is characterized by two cysteine motifs typical of bacterial ferredoxins. The Fixphenotype of an S. melilo!i fdxN:: [Tetracycl ine] mutant could be rescued by the R. leguminosarumfdxN gene. Site-directed mutagenesis revealed that cysteine residues at position 42 and 61 were essential for the activity of the S. meliloti FdxN (Masepohl et aI., 1992). In symbiotic nodules, a H2-uptake (hup) system is required to recycle the hydrogen gas evolved during the nitrogen fixation. There are 17 hup genes arranged in a region about 15 kb on pSym in R. leguminosarum bv. viciae strain UPM791 (Hidalgo et aI., 1990, 1992). Accumulation of transcripts specific for the hydrogenase structural genes (hupSL) paralleled that of nifA- and niftI-

Rhizobium-Legume Symbiosis - 207 specific mRNAs in the same cells, suggesting that induction of these hup and nif genes is triggered by the same signal or regulated by the same mechanism (Brito et aI., 1995). Bacteroids in N2-fixing nodules utilize C 4-dicarboxylic acids as an energy source. The uptake of these acids from host cells is regulated by del (C 4 diearboxylate lransport) genes. R. lrifolii and R. leguminosarum biovars viciae and lrifolii mutants defective in del genes are unable to transport C4 dicarboxylates and form ineffective nodules on their respective hosts (Finan et aI., 1983; Ronson et aI., 1981). Three del genes have been identified. The detA gene encodes a structural component necessary for C 4-dicarboxylate transport. The gene product consists of 444 amino acids with a molecular mass of 49 kD. It is highly hydrophobic with 68% apolar residues. The delB and dele genes encode positive regulatory elements (Ronson et aI., 1984). A nifA-regulated promoter sequence has been found upstream of the del structural gene, suggesting a coupling between the energy-demanding N 2-fixation and the import ofC 4-dicarboxylates (Ronson and Astwood, 1985). It has been reported that mutants with reduced levels of C 4-dicarboxylate transport in bacteroids have reduced N 2- fixation (Finan et aI., 1983). Thus, the presence of a functional C 4 -dicarboxylate transport system is essential for N 2-fixation. The B. japonieum hemB gene encodes a protein that is highly homologous to Ll-aminolevulinic acid (ALA) dehydrogenase from diverse organisms. The protein contains a Mg2+-binding domain similar to the one found in plant ALA dehydrogenases. Strains with a mutation at hemB fail to synthesize hemoglobin, suggesting that the gene product is essential for heme synthesis (Chauhan and O'Brian, 1993).

4.2.4 Plant (Nodulin) Genes in Nodulation and Symbiotic Nitrogen Fixation Nodulins are nodule-specific proteins encoded by plant genes during nodulation and nitrogen fixation. These proteins can be detected by nodule protein-specific antisera, gel electrophoresis of in vitro translational products of nodule RNA, and nodulin gene-specific cDNA probes. Depending on the time of appearance of these proteins during rhizobium-legume symbiosis, they may be divided into early and late nodulins. It is suggested that the genes encoding the early nodulins be designed as ENOD and those for late nodulins as NOD. The names ofthe ENOD and NOD genes are preceded with the initials of the plant genus and species names (Nap and Bisseling, 1990). Many nodulins, particularly late nodulins, have been described prior to this recommendation. Consequently, existing names continue to be used. Some of early and late nodulin genes are given in Tables 4-6 and 4-7. For a review ofnodulins, see Govers et al. (1987), Nap and Bisseling (1990), Franssen et al. (1992), Verma et al. (1992) ane De Bruijn et al. (1994).

208 - Plant Pathogenesis and Resistance Table 4-6. Early nodulin (ENOD) genes in symbiotic nitrogen fixation. Gene

Product (kD)

MtPRP4 MsCAl

62

SrCHI3 ENOD2

75

ENODJ ENOD5

6 14

ENOD7 ENOD12A ENOD12B

12 8 13

ENOD14 MtENOD16

6

NOD32 ENOD40

32 10

ENOD-GRP2 ENOD-GRP3 ENOD-GRP5

14 17 15

Function

Legume

Reference

Medicago truncatula Wilson et al. (1994) Coba de la Pena et al. Alfalfa (1997) Goormachtig et al. Acidic chitinase (111) Sesbania rostrata (1998) Alfalfa, Van de Wiel et al. Proline-rich protein (1990) Govers et al. pea, (1986); Franssen et soybean al. (1987); Chen et Sesbania rostrata al. (1998) Metal-binding protein Pea Scheres et al. (1990) Arabinogalactan Pea, broad bean Scheres et al. (1990); Friihling et al. (2000) protein Kozik et al. (1996) Unknown Pea Bauer et al. (1994) Proline-rich protein Alfalfa, pea Allison et al. (1993) Govers et al. (1991) Kozik et al. (1992) Metal-binding protein Pea Scheres et al. (1990) Medicago truncatula Greene et al. (1998) Phytocyanin-related compounds Broad bean Perlick et al. (1996) Chitinase Non-translatable RNA Alfalfa, Crespi et al. (1994) Papadopoulou et al. associated with plant soybean, growth; lateral root Medicago truncatula, (1996) initiation bean Proline-rich protein Carbonic anhydrase

Glycine-rich protein

Broad bean

Kiister et al. (1995) Schroder et al. (1997)

The early nodulins are involved in both the infection process and nodule organogenesis. Several lines of evidence indicate that the expression of early nodulin genes is activated by Nod factors. Treatment of vetch root with R. leguminosarum bv. viciae Nod factors results in cortical cell division (Spaink et aI., 1991; Van Brussel et aI., 1992). In these dividing cells, both ENOD12 and ENOD40 genes are induced (Vijn et aI., 1993). Transgenic alfalfa plants carrying an ENOD12 promoter fused to GUS expressed GUS activity after the plants were treated with Nod factors of S. melilot; (Pichon et aI., 1993). Similar results have been obtained from rice plants containing the promoter of MtNOD12 fused with GUS gene. Treatment of the transgenic rice roots with Nod factors induced MtENOD12-GUS expression in cortical parenchyma, endoderm is and peri cycle. In contrast, chitooligosaccharide backbone alone

Rhizobium-Legume Symbiosis - 209 failed to elicit such a response in the root tissues. These findings demonstrate that rice roots perceive Nod factors and that these lipochitooligosaccharides, but not simple chitin oligomers, act as signal molecules in activating the nodulin gene in cortical parenchyma (Reddy et aI., 1998). Table 4-7. Late nodulin (NOD) genes in symbiotic nitrogen fixation. Gene

Product (kD) Function

Legume

Reference

12

Leghemoglobin synthesis Alfalfa Glutamine synthetase Lupinus lute us. soybean

NOD-GRP} NOD-GRP4 NOD6 NOD}6 N24

15 \0 6 16 33

Glycine-rich protein

Broad bean

Allen et al. (1992) Boron and Legocki (1993); SenguptaGopalan et al. (1991) Schroder et al. (1997)

Pea Lotus japonicum Soybean

Kardailsky et al. (1993) Kapranov et al. (1997) Cheon et al. (1994)

N25 N22 N26

28

Alfalfa Alfalfa Soybean

Kiss et al. (1990) Allen et al. (1992) Weaver et al. (1994)

N28/32 N35

28-32 33

Unknown Unknown Peri bacteroid membrane protein Unknown Unknown Ion channel, peri bacteroid membrane transports Unknown Uricase II

N45

45

Unknown

Broad bean Soybean, moth bean (Vigna aconitifolia) Lupinus luteus L. angustifolius

LjNOD70

70

An enzyme catalyzes a Soybean reaction involving acetyCoA and a-keto acid Transport protein Lotus japonicus

KUster et al. (1994) Nguyen et al. (1985) Takane et al. (1997) Lee et al. (1993) Rice et al. (1993) Szczyglowski et al. (1989) Kouchi and Hata (1995)

NOD7}2

35

Lb

GS

26

N56

Protochlorophyllide Medicago reductase, synthesis of truncatula heme moiety of leghemoglobin

Szczyglowski et al. ( 1998) Wilson and Cooper (1994)

ManyearIy nodulin genes, includingENOD2, ENOD5, ENODJ 2, PRP4, and GRP, encode cell wall proteins and are involved in nodule morphogenesis. MtPRP4, found in Medicago truncatula, encodes a polypeptide of549 amino acids with aM, of62,000. The N-terminal22 amino acids serve as a membranetranslocating signal peptide. The remaining sequence of 527 amino acids consists of three repeating pentapeptides, PPVEK, PPVHK, and PPVYK, indicating it is a proline-rich cell wall protein. RNA gel blot experiments

210 - Plant Pathogenesis and Resistance detected MtPRP4 transcripts in symbiotic root nodules but not in roots, hypocotyls, or leaves. In situ hybridization experiments demonstrated that MtPRP4 expressed early in the development of the nodule meristem (Wilson et aI., 1994). Late nodulins are involved mainly in the nodular function. Leghemoglobins (Lbs) are involved in the transportation of oxygen and the regulation of oxygen tension in nodules. Glutamine synthetase (GS, EC 6.3.1.2), uricase, glutamate synthase (GOGAT, EC 1.4.1.13), and asparagine synthetase are involved in the assimilation of fixed nitrogenous compounds. Lbs are the red pigments commonly found in the root nodules that develop on leguminous plants. They are hemoproteins consisting of a heme and a peptide. The heme moiety is produced by the bacteroid and the globin peptide is encoded by the plant gene. The physiological function ofLbs is to facilitate O 2 diffusion within the nodule and into the bacteroids rapidly enough to support oxidative phosphorylation without damaging the functioning of nitrogenase. In soybean nodules, the Lbs may be chromatographically fractionated into four components: Lba, Lbb, Lbc, and Lbd, on a DEAE-cellulose column. These components are encoded apparently by multiply copies of Lb genes. The products may be subjected to post-translational modifications resulting in the formation of minor components. It has been suggested that the nitrogen-fixing activity in nodules is positively correlated with the amounts of Lbs present in the nodule (Uheda and Syono, 1982). The GmN56 gene is located in the infected host cells of soybean nodules. A clone containing GmN56 cDNA has been sequenced. The ORF is predicted to code for a peptide of565 amino acids. The deduced amino acid sequence has significant homology to the LeuA protein of E. coli and the NifV protein of A. vinelandii and K. pneumoniae. LeuA is 2-isopropylmalate synthase (EC 4.1.3 .12), the enzyme which catalyzes the formation of isopropylmalate from acetyl-CoA and a-ketoisovalerate. NifV is homocitrate synthase (EC 4.1.3.21), the enzyme which catalyzes the synthesis of homo citrate by condensing acetylCoA and a-ketoglutarate. Homocitrate is a component ofFeMo-co required for nitrogenase activity (Hoover et aI., 1987a,b). Thus, GmN56 may be involved in enzymatic reactions utilizing acetyl-CoA and a-keto acid as substrates. Whether the protein is involved in homocitrate formation remains to be determined (Kouchi and Hata, 1995). Nodulin-26 is a major peribacteroid membrane (PBM) protein in soybean nodules. The 26-kD protein spans the PBM six times with both N- and Ctermini facing the host cytoplasm (Miao et aI., 1992). It resembles the 27-kD tonoplast intrinsic protein (TIP) that occurs widely in plants. TIP has been shown to transport small metabolites between vacuoles and cytoplasm in seed storage tissue (Johnson et aI., 1990). This similarity suggests that nodulin-26 may have transport functions. Nodulin-26, purified from soybean nodules, is

Rhizobium-Legume Symbiosis - 211 readily incorporated into lipid bilayer, and it forms single channels. Thenodulin 26 channels transport both cations and anions (Miao and Verma, 1993; Weaver etal.,1994). Nodulin 35 is a nodule-specific uricase (uricase II). It is a homotetramer of a 33-kD polypeptide. The enzyme is localized in peroxisomes in the un infected cells of determinate nodules and is responsible for the biosynthesis of ureide. Two uricase genes, UR2 and UR9, have been cloned from soybean plants. They encode 309 amino acid proteins with 12 amino acid differences. UR9 is mainly expressed in root nodules although the transcript has been detected in roots, primary leaves and developing seeds at very low levels. In contrast, UR2 is expressed in many plant parts at low levels, including nodules. Thus, UR9 is a nodulin gene (Takane et aI., 1997).

4.3 EFFECTS OF DISEASES ON NODULATION AND NITROGEN FIXATION Nodulation and nitrogen fixation in symbiotic legumes evolve from a two-way communication between rhizobial and host genes. It is known that a genetic defect in either partner would result in the disruption of the nodulation process and act as an impediment to nitrogen fixation. It is also conceivable that biological stresses would interfere with communication between the rhizobia and host plants, resulting in the suppression of nodulation and a reduction in nitrogen fixing capacity. For a review of biological stresses on rhizobiumlegume symbiosis, see Bowden (1978) and Huang (1987).

4.3.1 Suppression of Binding between Rhizobia and Root Hairs The first step in nodule formation is the attachment of infective rhizobia to root hairs of legume roots. Lectins have been implicated in rhizobial attachment. Using an indirect immuno-fluorescence technique, lectin was detected on the tips of newly formed, growing root hairs and on epidermal cells located just below the young root hairs. It was not detected, however, on older, elongating root hairs. Inoculation of newly formed root hairs, epidermal cells under the young root hairs, and older root hairs with suspensions of R. leguminosarum (>10 6 cells/mL) resulted in nodulation of 90, 73, and 13% of the plants, respectively. These results demonstrate the importance of lectin in rhizobial symbiosis (Dfaz et aI., 1986). The binding of B.japonicum to cultured soybean cells also requires a bacterially synthesized galactose-binding lectin (Ho et aI., 1990). It is known that host-specificity in rhizobium-legume symbiosis is determined by a set of specific nod genes (see Section 4.2.2.3); the possible involvement of lectin in host specificity, however, can not be ruled out. Clover plants transgenic for a pea lectin can be nodulated by R. leguminosarum bv. viciae, which normally fails to nodulate clover (Dfaz et aI., 1989).

212 - Plant Pathogenesis and Resistance The effects of nematode infection on the binding of rhizobia to soybean roots have been investigated (Huang et aI., 1984). Soybean seedlings inoculated with juveniles of race 1 of Heterodera giycines, the soybean cyst nematode (SCN), were incubated with a suspension of B. japonicum prepared from cultures grown in a synthetic medium containing D-(1-3H) glucose. After washing to remove unbound rhizobia, the roots were oxidized, and the radioactivity of the resulting tritiated water was measured. Roots from SCN-inoculated seedlings had lower radioactivity on a per root or per unit weight basis than those from controls. Binding of B. japonicum to control soybean roots also was inhibited by pretreatment of roots with N-acetylD-galactosamine or D-galactose, the haptens of the 120 kD-soybean lectin, but not glucose. These results suggest that soybean lectin is involved in the binding of R. japonicum to soybean roots, and that the SCN infection suppresses the binding between roots and rhizobia. Scanning electron microscopy revealed that abundant rhizobia were on the surfaces of control soybean roots. Very few rhizobia were observed on root surfaces ofSCN-infected plants. The SCN had no apparent effect on either the numbers of root hairs or the surface area of the total root system one week after nematode inoculation when the binding experiments were conducted. Thus, the reduction in binding of rhizobia to SCN-infected soybean roots apparently was not due to the reduction in surface areas of infected roots. Instead it resulted from the interference of the nematode with soybean lectin metabolism (Huang et aI., 1984). 4.3.2 Suppression of Infection Thread Development Examination of sections of emerging nodules (12 days after rhizobial inoculation) from control and SCN-infected plants indicated that cellular differentiation had already begun. The meristems of nodules from control and SCN-infected plants had given rise to a peripheral uninfected tissue of cortical parenchyma cells and a central tissue invaded by the bacteria. Most of the cortical cells in nodules from control plants were still dividing, whereas some of those from SCN-infected plants had already differentiated into vascular elements. Although the basic cellular arrangement seemed to be similar between these two types of nodules under light microscopy, there were several noteworthy differences. The emerging nodules from control plants appeared to be more organized, with a central tissue developing evenly in all directions. Nodule development from SCN-infected plants was more or less disoriented; thick-walled sclerenchyma cells appeared early around the cortex and the central tissue, particularly at base of the nodule. The volume of enlarged, infected cells in the nodular central tissues was also smaller than that of similar cells in the control (Ko et aI., 1985).

Rhizobium-Legume Symbiosis - 213 Electron microscopy of the nodular central tissues revealed that infection by rhizobia proceeded similarly in both types of nodules. However, there were certain subtle differences. Infection threads ramified extensively in the central tissues of emerging nodules from control plants with rhizobia actively multiplying inside. Release of rhizobia from the infection threads into the cellular cytoplasm in the nodular central tissue was frequently observed. Localized dissolved areas were evident in the walls of the infection threads as the bacteria "budded off" individually in membrane vesicles from the infection threads. At this stage, the released rhizobia had not yet differentiated into swollen bacteroids (Fig. 4-6A). In similar nodular tissues of the SCN-infected soybeans, infection threads generally had large empty spaces, fewer rhizobia, and compact walls with no sign of weakening in any area. Many rhizobia in these infection threads were embedded in a thick matrix of polysaccharide mucilage and appeared shrivelled, losing their structural integrity (Fig. 4-6B) (Ko et aI., 1985).

4.3.3 Suppression of Nodulation One of the effects of biological stress on rhizobium-legume symbiosis is the suppression of nodulation (Table 4-8). Soybean mosaic virus (Beltsville isolate, SMV -B) infection offour soybean genotypes (G. max Hill and Essex; G. soja lines PI 424-005 and PI 378-693-B) resulted in significant decreases in mean top weight (gldry plant) and corresponding decreases in nodule mass (glfresh plant) at 53 days after virus inoculation as compared with the control (Orellana et aI., 1983). Similar results were obtained with the bean yellow mosaic virus (BYMV)-infected bean plants (Phaseolus vulgaris cv. Chalevoix). Significant decreases in top, root, and nodule weights of the BYMV-infected plants relative to controls were noted during the prebloom stage (25 days), the earliest pod stage (32 days) and the initial pod-fill stage (47 days). Most nodules decayed and dropped from the BYMV-infected plants prematurely (Orellana and Fan, 1978). Inoculation of G. max cv. Ransom with SCN significantly suppressed nodular development. There was a 26% reduction in nodular fresh weight in the nematode-infected soybean as compared to the control (Huang and Barker, 1983). Top growth of Siratro (Macroptilium atropurpureum), a tropical forage legume, was not affected by infestation with several Meloidogyne spp. Nodule weights in nematode-infected plants, however, were significantly increased compared with nematode-free controls (Lynd and Ansman, 1989).

214 - Plant Pathogenesis and Resistance

Fig. 4-6. Nodular tissues in soybean. Above. Tissues from control soybean plant. An infection thread (IT) is releasing rhizobium (BA) into the cytoplasm of infected soybean cells (X 4,100). Below. Tissues from a soybean cyst nematode-infected soybean plant. Infection thread (IT) are filled with degenerated rhizobia (DB) and mucilage (MU) (X 6, I 00). (Reproduced from Ko et aI., 1985, with permission from the American Phytopathological Society).

Rhizobium-Legume Symbiosis - 215 Table 4-8. Effect of plant diseases on nodulation. Host plant

Cultivar/line

Treatment

Glycine max

Hill

Control SMV Control SMV Control SMV Control SMV Control BPMV Control BPMV Control BPMV Control TRSV Control TRSV inoculated at day 8 and assayed at day 50 Control TRSV inoculated at day 18 and assayed at day 50 Control Heterodera glycines Control WCMV Control BYMV Control Meloidogyne spp.

Essex Glycine soja

PI 424-005 PI 378-693

Glycine max

Centennial Williams Peking

Glycine max

Harosoy

Glycine max

Tracy

Glycine max

Ransom

Trifolium pralense Ottawa Phaseolus vulgaris Charlevoix Macroptilium atropurpureum

Nodules 0.19· 0.07 0.38 0.17 0.10 0.07 0.55 0.12 1.47· 1.08 1.10 1.00 0.27 0.10 3.38· 0.05 1.00·

Reference Orellana et al. (1983)

Orellana et al. (1987)

Orellana et al. (1980) Orellana et al. (1980)

0.36 0.91

0.87 0.413· 0.307 440 b 300 0.55· 0.10 72.7c 24.0

Huang and Barker (1983) Khadhair et al. (1984) Orellana and Fan (1978) Lynd and Ansman (1989)

• g fresh wtlplant; b nodule no.lplant; c g dry wtlplant

4.3.4 Suppression of Leg hemoglobin Biosynthesis Leghemoglobins (Lbs) extracted from the nodules of two dissimilar yellow lupins inoculated with the same strain of R. lupin; were different as judged by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. When plants of a given Jine of yellow lupin were treated with two different rhizobial strains, the nodules had a Lb of similar chromatographic and electrophoretic profiles. These results indicate that the type ofLb produced in a given symbiotic Rhizobium-legume interaction is plant specific. This finding has been extended to soybean (Glycine max), red kidney bean (Phaseolus vulgaris), broad bean (Vicia/aba), and other legumes.

216 - Plant Pathogenesis and Resistance The efficiency of nitrogen fixation by nodules is influenced by various factors, including Lb concentrations. There is a positive correlation between the intensity of nitrogen fixation and the amount of Lb present in root nodules. Acetylene reduction by bacteroid suspensions, a nitrogenase- mediated reaction, is dependent upon Lb concentration (Virtanen et a\., 1947). The effects of plant diseases on the Lb content in legume nodules have been investigated (Table 4-9). Nodules harvested from nematode-infected soybeans had lower fresh weights per plant and lower specific nitrogenase activity (/lmoles of C 2H4 formed per gram of nodules per hour) as assayed by the acetylene reduction procedure. Lbs extracted from soybean nodules, purified in a Sephadex G-15 column, were separated by DEAE-cellulose column chromatography into four components: Lba, Lbb, Lbc, and Lbd. Lba from nematode-infected and control plants had similar ultraviolet and visible light spectra and gel electrophoresis profiles, as did the Lbb, Lbc, and Lbd. The ratio of LbclLba, however, was higher from nematode-infected soybeans than from control plants (Huang and Barker, 1983). Table 4-9. Effect of plant diseases on leghemoglobin content in nodules of legumes. Host plant

Cultivar

Pathogen

Soybean

Ransom

Control Heterodera glycines Control Meloidogyne incognita Heterodera cajani Control White clover mosaic virus

Cowpea

Red clover

• ｾｭｯｬ･ｳＯｧ＠

Ottawa

nodules; b mg/g nodules; c ｧＯｭ＠ｾ

Leghemoglobin 81.3" 47.2 8.05 b 3.90 5.10

l.35 c

Reference Huang and Barker (1983) Sharma and Sethi (1975) Khadhair et al. (1984)

0.55

nodules

Generally, LbC/Lba ratios are high when the nodules are immature. The LbC/Lba ratios for leghemoglobins obtained from nodules of control plants and plant parasitized by the nematode were 1.14 and 1.71, respectively. The former is in agreement with the ratio of 1.14 calculated from the data published by Appleby et a\. (1975). The factor(s) which contributed to the observed higher LbC/Lba ratio in nodules of nematode-infected plants has not been determined. Different Lb components are coded for by different plant mRNA, and the relative levels of these mRNA change during root nodule development (Verma et a\., 1979). Analysis of the in vitro translation products of mRNA from nodules of different ages has shown that Lbc is synthesized at a higher rate than Lba in young nodules. The reverse is a rule in mature nodules (Verma et a\., 1979). The ratio of LbC/Lba in soybean root nodules, therefore, remains high in the early stages of soybean growth and decreases during flowering and

Rhizobium-Legume Symbiosis - 217 fruiting (Fuchsman et al. 1976). Since nodules from nematode-infected soybeans had a higher Lbc/Lba ratio, this suggests that nodule development is impaired by nematode infection. The significant reduction in overall Lb content, however, indicates that nodules from nematode-infected plants are senescent. Although infection of soybean by the cyst nematode is known to limit size, the cause, whether due to impairment of nodule development or acceleration of nodule senescence, remains to be determined. Uhedaand Syono (1982) have demonstrated that Lba is more effective for oxygen binding and nitrogen fixation than Lbc. Therefore, the reduced nitrogen-fixing efficiency of nodules from nematode-infected plants may be attributed, in part, to its higher LbC/Lba ratio. The concentration of Lbs in the nodules from white clover mosaic virusinfected red clover was significantly lower than that in the nodules from virusfree controls. The decrease in Lb concentration was about 62% and occurred at 6 weeks after viral inoculation (Khadhair et aI., 1984).

4.3.5 Suppression of Nitrogenase Activity Specific nitrogenase activity is usually determined by the acetylene reduction assay using a gas-liquid chromatograph and expressed as flmoles of C 2H4 formed (or C 2H 2 reduced) per gram fresh weight of nodule per unit of time (Table 4-10). There was a 61 % reduction in nitrogenase activity in nodules from plants infected with Heterodera glycines compared to those from control soybeans (Huang and Barker, 1983). Increase in nodule specific N 2-fixing activity, however, has been reported in some legume-parasite interactions. For example, soybean cultivar Hill and PI 378-693 infected by SMV-B had 69 and 50% increases, respectively, in their nodule specific activity (Orellana et aI., 1983). Siratro (Macroptilium atropurpureum), a perennial legume, is known to tolerate heavy infestation by root-knot nematodes. Root-nodule weight (dry wt/plant) and specific nitrogenase activity (nmole C 2Hiplant/sec) in nematodeinfected plants were significantly increased compared with nematode-free plants. Nematode infestation did not significantly influence enzyme activity levels that govern the transformation of fixed-nitrogenous compounds within nodules. Infestation did, however, affect enzyme activity governing ureide transformation and glyoxylate metabolism. Ureide and pyruvate contents of nematode-free nodules were greater than those of nematode-infested plants. Nitrate reductase of nodule cytosol was 10 times higher in nematode-infested nodules compared with that from nematode-free nodules. It is possible that the increase in nitrate reductase offset the nematode effect on nitrogen fixation machinery (Lynd and Ansman, 1989).

218 - Plant Pathogenesis and Resistance Table 4-10. Effect of plant diseases on nitrogen-fixing activity. N 2-fixing activity

Host plant

Cultivar/line

Treatment

Glycine max

Hill

3.37" Control SMV-B 2.10 Control 4.98 SMV-B 1.88 Control 2.31 SMV-B 0.20 Control 6.12 SMV-B 2.01 56.1" Control BPMV 26.9 42.2 Control 19.9 BPMV 5.8 Control 2.2 BPMV Control 9.0" Tobacco ringspot virus 47 days 1.7 Control 2.17b Heterodera glycines 0.84 Control, 18.3" TRSV, inoculated at unifoliate stage 6.2 Control, 16.3 TRSV, inoculated at 10.2 trifoliate stage 16.3 c Control Meloidogyne spp. 9.6 Control 3.0" BYMV 0.8 Control 0.155" WCMV 0.08

Essex

Glycine soja

PI 424-005 PI 378-693

Glycine max

Centennial Williams Peking

Glycine max

Harosoy

Glycine max

Ransom

Glycine max

Tracy

Macroptilium atropurpureum Phaseolus vulgaris Trifolium pratense

Charlevoix Ottawa

Reference Orellana et al. (1983)

Orellana et al. (1987)

Orellana et al. (1978)

Huang and Barker (1983) Orellana et al. (1980)

Lynd and Ansman (1989) Orellana and Fan (1978) Khadhair et al. (1984)

4.3.6 Iron Metabolism and Nitrogen Fixation Phytoferritin is an iron-containing protein analogous to the ferritin in mammalian cells (Bienfait and Van der Mark, 1983). Accumulation of phytoferritin occurs primarily in nonphotosynthetic tissues functioning as a detoxicant against excess ferrous iron (Bienfait and Van der Mark, 1983). Phytoferritin also occurs in legume nodule meristems (Bergersen, 1963). Although the exact role ofthis protein in nodular development and function is not known, it is believed that nodular phytoferritin functions as storage for ferrous iron.

Rhizobium-Legume Symbiosis - 219 The occurrence of phytoferritin and its relationship to the effectiveness of soybean nodules has been investigated. Polyacrylamide gel electrophoresis and electron microscopy revealed that the accumulation of iron-protein in soybean nodules is influenced by nodule age, mutation in bradyrhizobia, and bradyrhizobial strain-soybean cultivar interactions. Iron-protein concentrations (Ilg/mg protein) were inversely related to heme concentrations (nmoles/mg protein), with correlation coefficients ranging from -0.98 in young nodules to -0.83 in mature ones. B. japonicum symbiotic mutants HS 129 and HS145 (Nod+Fix') produced nodules high in iron-protein. Electrophoresis of homogenate prepared from nodules on Lee 68 produced by B. japonicum HS129 yielded two different forms of the iron-proteins, 570 and 600 kD. The 570-kD iron-protein isolated by preparative PAGE behaved like horse-spleen-ferritin in responses to iron-stains, heat stability, UV absorption spectrum, iron unloading and reloading, and characteristic appearance in electron micrographs. These properties led to conclude that the 570-kD iron protein is phytoferritin. The nodule phytoferritin differed from horse-spleen-ferritin in electrophoretic mobility, serological properties, and molecular size. It was distinct from most other known phytoferritins in that it was composed of different subunit types (Ko et aI., 1987). Massive accumulation of phytoferritin in the plastids of cells in the nodular central tissues was found in the cyst nematode-infected soybean (Fig. 4-7) (Ko et aI., 1985). The accumulation of phytoferritin suggests that the metabolism of iron-containing compounds is affected by the presence of the cyst nematode.

4.4 CONCLUSIONS Because of the increase in world population, the need for protein-rich legumes for human consumption and animal feeds is increasing. Agricultural scientists have taken various measures to meet this challenge. Significant progress has been made at the molecular and biochemical levels, particularly, in elucidating the signal exchange between rhizobia-legume interactions. Numerous barriers, however, still limit maximum nitrogen fixation by legume-rhizobia symbiosis. Among these barriers, inhibition of nodulation by plant pathogens is a major threat to legume production. Unfortunately, research on dysfunction in symbiotic responses at physiological and biochemical levels is meager, and practically non-existence atthe molecular level. Unless strategies or tactics are developed to minimize the interference of the symbiosis by plant pathogens, the gain in N2 fixation realized by the advances in biochemistry, genetics, and agronomy could be negated.

220 - Plant Pathogenesis and Resistance

fig. 4-7. Effect of soybean cyst nematode on iron metabolism in soybean nodules. Lcft, a plastid from control central nodular tissue showing its stroma (S) containing osmiophilic droplets (00), cisternae (C), and lamellae (L) (X 46,000). Right, a plastid from a soybean cyst nematode-infected plant showing its stroma largely occupied by prominent crystalline arrays of phytoferritin, the iron-containing protein (X 46,000). (Reproduced from Ko et aI., 1985, with permission from the American Phytopathological Society).

Another barrier is the limited progress in genetic engineering of nitrogen fixation (Williams and Phillips, 1993; Bosworth et aI., 1994). The nifA gene plays a positive regulatory role in the expression of the nif regulon in S. meliloti. It is conceivable that additional copies of nifA and dctABD genes would increase alfalfa yield in the field. Results indicated that recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield ofwild-type strain RMBPC in the field where soil nitrogen and organic matter content was lowest. No increases was observed in other fields. Thus, recombinant strains can increase yields under certain field conditions (Bosworth et al., 1994).

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Rhizobium-Legume Symbiosis - 233 Reynolds PHS, Farnden K.IF (1979) The involvement of aspartate aminotransferase in ammonium assimilation in lupin nodules. Phytochemistry 18: 1625-1630 Rice S.I, Grant MR, Reynolds PHS, Farnden KJF (1993) DNA sequence ofnodulin-45 from Lupinus anguslifolius. Plant Sci 90: 155-166 Ritsema T, Geiger 0, Van Dillewijn P, Lugtenberg BJJ, Spaink HP (1994) Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv. viciae is essential for its biological function. J Bacteriol 176:7740-7743 Rivilla R, Sutton .1M, Downie .lA (1995) Rhizobium leguminosarum NodT is related to a family of outer-membrane transport proteins that includes TolC, PrtF, CyaE, and AprF. Gene 161: 27-31 Roberts GP, Brill W.I (1981) Genetics and regulation of nitrogen fixation. Annu Rev Microbiol 35:207-235 Robson R, Kennedy C, Postgate.lR (1983) Progress in comparative genetics of nitrogen fixation. Can.l Microbiol 29:954-967 Roche P, Debelle F, Maillet F, Lerouge P, Faucher C, Truchet G, Deenarie .I, Prome .I-C (1991) Molecular basis of symbiotic host specificity in Rhizobium meliloli: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals. Cell 67: 1131-1143 Rohrig H, Schmidt.l, Wieneke U, Kondorosi E, Barlier I, Schell J, .lohn M (1994) Biosynthesis of Ii po oligosaccharide nodulation factors: Rhizobium NodA protein is involved in N-acylation of the chitooligosaccharide backbone. Proc Natl Acad Sci USA 91 :3122-3126 Ronson CW, Astwood PM (1985) Genes involved in the carbon metabolism of bacteroids. In: Evans HJ, Bottomley P.I, Newton WE (eds) Nitrogen fixation research progress. Martinus Nijhoff, Dordrecht, pp 201-207 Ronson CW, Astwood PM, Downie.lA (1984) Molecular cloning and genetic organization ofC 4dicarboxylate transport genes from Rhizobium leguminosarum. J Bacteriol 160:903-909 Ronson CW, Lyttleton P, Robertson JG (1981) C4 -dicarboxylate transport mutants of Rhizobium Irifolii form inefficient nodules in Trifolium repens. Proc Natl Acad Sci USA 78:4284-4288 Rosenberg C, Boistard P, Denarie .I, Casse-Delbart F (1981) Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet 184:326-333 Rostas K, Kondorosi E, Horvath B, Simoncsits A, Kondorosi A (1986) Conservation of extended promoter regions of nodulation genes in Rhizobium. Proc Nat! Acad Sci USA 83: 1757-176 Rushing BG, Yelton MM, Long SR (1991) Genetic and physical analysis of the nodD3 region of Rhizobium meliloti. Nucleic Acids Res 19:921-927 Sadowsky M.I, Cregan PB, Gottfert M, Sharma A, Gerhold D, Rodriguez-Quinones F, Keyser HH, Hcnnecke H, Stacey G (1991) The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans. Proc Natl Acad Sci USA 88:637-641 Sanjuan.l, Carlson RW, Spaink HP, Bhat UR, Barbour WM, Glushka.l, Stacey G (1992) A 2-0methylfucose moiety is present in the lipo-oligosaccharide nodulation signal of Bradyrhizobiumjaponicum. Proc Natl Acad Sci USA 89:8789-8793 Schell MA (1993) Molecular biology of the LysR family of transcriptional regulators. Annu Rev MicrobioI47:597-626 Scheres B, van Engelen F, van der Knaap E, van de Wiel C, van Kammen A, Bisseling T (1990) Sequential induction of nodulin gene expression in the developing pea nodule. Plant Cell 2:687-700 Scheu AK, Economou A, Hong GF, Ghelani S, .lohnston A WB, Downie.lA (1992) Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems. Mol Microbiol 6: 231-238 SchlUter A, Patschkowski, Quandt .I, Selinger B, Weidner S, Kramer M, Zhou L, Hynes MF, Priefer UB (1997) Functional and regulatory analysis of the two copies of the flXNOQP operon of Rhizobium leguminosarum strain VF39. Mol Plant-Microbe Interact \0:605-616

234 - Plant Pathogenesis and Resistance Schofield PR, Watson 1M (1986) DNA sequence of Rhizobium trifolii nodulation genes reveals a reiterated and potentially regulatory sequence preceding nodABC and nodEF. Nucleic Acids Res 14:2891-2903 Scholl a MH, Elkan GH (1984) RhizobiumJredii sp. nov., a fast-growing species that effectively nodulates soybeans. Int J Syst Bacteriol 34:484-486 Schroder G, Friihling M, Piihler A, Perlick AM (1997) The temporal and spatial transcription pattern in root nodules of ViciaJaba nodulin genes encoding glycine-rich proteins. Plant Mol Bio133: 113-123 Schubert KR, Boland M1 (1984) The cellular and intracellular organization of the reactions of ureide biogenesis in nodules of tropical legumes. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research. PUDOC, Wageningen, pp 445-451 Schultze M, Kondorosi A (1998) Regulation of symbiotic root nodule development. Annu Rev Genet 32:33-57 Schultze M, Quiclet-Sire B, Kondorosi E, Virelizier H, Glushka IN, Endre G, Gero SO, Kondorosi A (1992) Rhizobium meliloti produces a family of sulfated lipo-oligosaccharides exhibiting different degrees of plant host specificity. Proc Natl Acad Sci USA 89: 192- 196 Schultze M, StaeheIin C, Rohrig H, lohn M, Schmidt I, Kondorosi E, Schell I, Kondorosi A (1995) in vitro sulfotransferase activity of Rhizobium melilotiNodH protein: Iipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure. Proc Natl Acad Sci USA 92:2706-2709 Schwedock I, Long SR (1989) Nucleotide sequence and protein products of two new nodulation genes of Rhizobium meliloti, nodP and nodQ. Mol Plant-Microbe Interact 2: 18 1- 194 Schwedock IS, Long SR (1992) Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa. Genetics 132:899-909 Schwedock IS, Liu C, Leyh TS, Long SR (1994) Rhizobium meliloti nodP and nodQ form a multifunctional sulfate-activating complex requiring GTP for activity. 1 Bacteriol 176:70557064 Scott DB, Young CA, Collins-Emerson 1M, Terzaghi EA, Rockman ES, Lewis PE, Pankhurst CE (1996) Novel and complex chromosomal arrangement of Rhizobium loti nodulation genes. Mol Plant-Microbe Interact 9: 187- 197 Segovia L, Young PW, Martinez-Romero E (1993) Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov. Int 1 Syst Bacteriol 43:374-377 Sengupta-Gopalan C, Gambliel H, Feder I, Richter H, Temple S (1991) Different modes of regulation involved in nodulin gene expression in soybean. In: Hennecke H, Vcrma DPSP (eds) Advances in molecular genetics of plant-microbe interactions, vol I. Kluwer Acad Publ, Dordrecht, pp 304-309 Shah VK, Stacey G, Brill WI (1983) Electron transport to nitrogenase: purification and characterization ofpyruvate:flavodoxin oxidoreductase, the nifJ gene product. 1 BioI Chern 258:12064-12068 Shah VK, Allen lR, Spangler NJ, Ludden PW (1994) in vitro synthesis of the iron-molybdenum cofactor of nitrogenase. Purification and characterization of NifB cofactor, the product of NifB protein. J BioI Chern 269: 1 154-1 158 Sharma NK, Sethi CL (1975) Leghaemoglobin content of cowpea nodules as influenced by Meloidogyne incognita and Heterodera cajani. Ind J Nematol 5: 113-114 Shearman CA, Rossen L, 10hnston A WB, Downie lA (1986) The Rhizobium leguminosarum nodulation gene nodF encodes a polypeptide similar to acyl-carrier protein and is regulated by nodD plus a factor in pea root exudate. EMBO J 5:647-652 Shelp BJ, Atkins CA (1984) Subcellular location of enzymes of ammonia assimilation and asparagine synthesis in root nodules of Lupinus albus L. Plant Sci Lett 36:225-230 Spaink HP (1995) The molecular basis of infection and nodulation by rhizobia: the ins and outs of sympathogenesis. Annu Rev Phytopathol 33 :345-368

Rhizobium-Legume Symbiosis - 235 Spaink HP (1996) Regulation of plant morphogenesis by lipo-chitin oligosaccharides. Crit Rev Plant Sci 15:559-582 Spaink HP, Weinman J, Djordjevic MA, Wijffelman CA, Okker JH, Lugtenberg BJJ (1989) Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein. EMBO.l 8:2811-2818 Spaink HP, Sheeley OM, Van Brussel AAN, GlushkaJ, York WS, Tak T, Geiger 0, Kennedy EP, Reinhold VN, Lugtenberg B1.I (1991) A novel highly unsaturated fatty acid moiety of lipooligosaccharid signals determines host specificity of Rhizobium. Nature 354: 126-130 Spaink HP, Wijfjes AHM, van der Drift KMGM, Haverkamp J, Thomas-Oates JE, Lugtenberg B11 (1994) Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum. Mol Microbiol 13:821-83 I Spaink HP, Wijfjes AHM, Lugtenberg B11 (1995) Rhizobium NodI and NodJ proteins playa role in the efficiency of secretion of Iipochitin oligosaccharides. J Bacteriol 177:6276-6281 Stacey G (1995) Bradyrhizobium japonicum nodulation genetics. FEMS Microbiol Lett 127: 1-9 Stacey G, Luka S, Sanjuan J, Banfalvi Z, Nieuwkoop A, Chun JY, Forsberg LS, Carlson R (1994) nodZ, A unique host-specific nodulation gene, is involved in the fucosylation of the Iipooligosaccharide nodulation signal of Bradyrhizobiumjaponicum. J Bacteriol 176:620-633 Stock .IB, Ninfa A.I, Stock AM (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53:450-490 Surin BP, Downie JA (1988) Characterization of the Rhizobium leguminosarum genes nodLMN involved in efficient host-specific nodulation. Mol Microbiol 2: 173-183 Surin BP, Watson JM, Hamilton WOO, Economou A, Downie JA (1990) Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum. Mol MicrobioI4:245-252 Sutton MJ, Lea EJA, Crank S, Rivilla R, Economou A, Ghelani S, Johnston A WB, Downie JA (1993) NodO: a nodulation protein that forms pores in membranes. In: Nester EW, Verma DPS (eds) Advances in molecular genetics of plant-microbe interactions, vol2. Kluwer Acad Publ, Dordrecht, pp 163-167 Sutton M.I, Lea E.lA, Downie JA (1994) The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes. Proc Natl Acad Sci USA 91 : 9990-9994 Szczyglowski K, Boron L, Szybiak-Str6zycka U, Legocki AB (1989) Characterization of cDNA clone coding for nodulin-45 from yellow lupin (Lupinus luteus). Plant Sci 65:87-95 Szczyglowski K, Kapranov P, Hamburger 0, De Bruijn FJ (1998) The Lotusjaponicus LjNOD70 nodulin gene encodes a protein with similarities to transporters. Plant Mol BioI 37:651-66 I Tajima S, Kouchi H (1997) Metabolism and compartmentation of carbon and nitrogen in legume nodules. In: Stacey G, Keen NT (eds) Plant-microbe interactions, vol 2. Chapman & Hall, New York, pp 27-60 Takana K-I, Tajima S, Kouchi H (1997) Two distinct uricase II (nodulin 35) genes are differentially expressed in soybean plants. Mol Plant-Microbe Interact 10:735-741 Thony B, Anthamatten 0, Hennecke H (1989) Dual control of the Bradyrhizobiumjaponicum symbiotic nitrogen fixation regulatory operon jixRnifA: analysis of cis- and trnas-acting elements . .I Bacteriol 171 :4162-416 Trinick M.I (1980) Relationships amongest the fast-growing rhizobia of Lablab purpure us, Leucaena leucocephala, Mimosa spp., Acacia!arnesiana and Sesbania grandiflora and their affinities with other rhizobial groups. J App Bacteriol 49:39-53 Truchet G, Roche P, Lerouge p, Vasse J, Camut S, de Billy F, Prome .I-C, Denarie J (1991) Sulphated Iipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature 351 :670-673 Uheda V, Syono K (1982) Effects ofleghemoglobin components on nitrogen fixation and oxygen consumption. Plant Cell Physiol 23:85-90

236 - Plant Pathogenesis and Resistance Van Brussel AAN, Bakhuizen R, Van Spronsen PC, Spaink HP, Tak T, Lugtenberg BJJ, Kijne J (1992) Induction of pre-infection thread structures in the leguminous host plant by mitogenic Iipooligosaccharides of Rhizobium. Science 257:70-72 Van de Wiel C, Norris JH, Bochenek B, Dickstein R, Bisseling T, Hirsch AM (1990) Nodulin gene expression and ENOD2 localization in effective, nitrogen-fixing and ineffective, bacteria-free nodules of alfalfa. Plant Cell 2: 1009-1017 Van Rhijn P, Vanderleyden J (1995) The Rhizobium-plant symbiosis. Microbiol Rev 59: 124-142 Van Rhijn P, Desair J, Vlassak K, Vanderleyden J (1994) Functional analysis of nodD genes of Rhizobium tropici CIAT899. Mol Plant-Microbe Interact 7:666-677 Van Spronsen PC, Bakhuizen R, Van Brussel AAN, and Kijne JW (1994) Cell wall degradation during infection thread formation by the root nodule bacterium Rhizobium leguminosarum is a two-step process. Eur J Cell Bioi 64:88-94 Vanaman TC, Wakil SJ, Hill RL (1968) The complete amino acid sequence of the acyl carrier protein of Escherichia coli. J Bioi Chern 243:6420-6431 Vazquez M, Davalos A, De las Peiias A, De Sanchez F (1991) Novel organization of the common nodulation genes: Rhizobium leguminosarum bv. phaseoli strains. J Bacteriol 173: 1250-1258 Verma DPS, Ball S, Guerin C, Wanamaker L (1979) Leghemog1obin biosynthesis in soybean root nodules. Characterization of the nascent and released peptides and the relative rate of synthesis of the major leghemoglobins. Biochemsitry 18:476-483 Verma DPS, Hu CA, Zhang M (1992) Root nodule development: origin, function and regulation ofnodulin genes. Physiol Plant 85:253-265 Vijn L Das Neves L, van Kammen A, Franssen H, Bisseling T (1993) Development of root nodules set in motion by Iipo-oligosaccharidc signal molecules. Science 260: 1764-1765 Virtanen AI, Erkama J, Linkola H (1947) On the relation between nitrogen fixation and leghemoglobin content of leguminous root nodules. II. Acta Chern Scand 1:861-870 Wandersman C, Delepelaire P (1990) ToIC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci USA 87:4776-4780 Weaver CD, Shomer NH, Louis CF, Roberts DM (1994) Nodulin 26, a nodule-specific symbiosome membrane protein from soybean, is an ion channel. J Bioi Chern 269:1785817862 Weber G, Reilander H, Piihler A (1985) Mapping and expression of a regulatory nitrogen fixation gene (jixD) of Rhizobium meliloti. EMBO J 4:2751-2756 Williams LE, Phillips DA (1993) Increased soybean productivity with a Rhizobiumjaponicum mutant. Crop Sci 23:246-250 Wilson RC, Cooper 18 (1994) A nodulin cDNA with homology to protochlorophyllide reductase. Plant Physiol 104:289-290 Wilson RC, Long F, Maruoka EM, Cooper 18 (1994) A new proline-rich early nodulin from Medicago truncatula is highly expressed in nodule meristcmatic cells. Plant Cell 6: 1265-1275 Wu HC, TokunagaM (1986) Biogenesis oflipoproteins in bacteria. Curr Top Microbiol Immunol 125:127-157 Young C, Collins-Emerson JM, Terzaghi EA, Scott DB (1990) Nucleotide sequence of Rhizobium loti nodI. Nucleic Acids Rcs 18:6691 Zaat SAl, Schripsema J, Wijffelman CA, van Brussel AAN, Lugtenberg BJJ (1989) Analysis of the major inducers of the Rhizobium nodA promoter from Vida sativa root exudate and their activity with different nodD genes. Plant Mol Bioi 13: 175-188 Zaat SAl, Wijffelman CA, Spaink HP, Van Brussel AAN, Okker RJH, Lugtenberg BJJ (1987) Induction of the nodA promoter of Rhizobium leguminosarum sym plasmid pRL 111 by plant flavonones and flavones. J Bacteriol 169: 198-204 Zheng L-M, White RH, Cash VL, Jack RF, Dean DR (1993) Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis. Proc Natl Acad Sci USA 90:27542758

Chapter 5 GROWTH REGULATORS AND PLANT TUMORIGENESIS

5.1 5.2

5.3

5.4

5.5 5.6 5.7 5.8 5.9 5.\0 5.11 5.12

Introduction Crown galls caused by Agrobacterium tumefaciens . 5.2.1 In vitro biosynthesis of auxins and cytokinins by Agrobacterium tumefaciens 5.2.2 Ti plasm ids of Agrobacterium tumefaciens . 5.2.3 Transformation of plant cells by Agrobacterium tumefaciens 5.2.3.1 Attachment of bacteria to plant cells 5.2.3.2 Recognition of plant signal molecules 5.2.3.3 Activation of vir genes. 5.2.3.4 Processing ofT-DNA for transfer 5.2.3.5 Intercellular transport ofT-DNA 5.2.3.6 Nuclear transport 5.2.3.7 Integration ofT-DNA into plant nuclear genome 5.2.4 Expression ofT-DNA in transformed cells and the auxin and cytokinin levels in crown gall tumors 5.2.5 Role of auxins and cytokinins in crown gall formation . 5.2.6 Use of Agrobacterium tumefaciens in plant genetic engineering Hairy roots caused by Agrobacterium rhizogenes 5.3.1 In vitro biosynthesis of cytokinins by Agrobacterium rhizogenes 5.3.2 Ri plasmids of Agrobacterium rhizogenes 5.3.3 Role of auxins and cytokinins in hairy root formation Olive knot caused by Pseudomonas syringae pv. savastanoi 5.4.1 Auxin production by Pseudomonas syringae pv. savastanoi 5.4.2 Production of cytokinins by Pseudomonas syringae pv. savastanoi . 5.4.3 Role of auxins and cytokinins in knot formation and development 5.4.4 Role of plAA in competitive fitness of Pseudomonas syringae pv. savastanoi . Fasciation diseases caused by Rhodococcus fascians Bacterial canker of almond caused by Pseudomonas amygdali Crown and root galls of gypsophila caused by Erwinia herbicola pv. gypsophilae Witches' broom diseases caused by Taphrina spp. Galls caused by Ustilago spp. Clubroot of crucifers caused by Plasmodiophora brassicae . Virus-induced tumors Conclusions References .

238 238 238 241 247 248 249 250 251 252 252 252 253 260 260 261 261 261 265 265 266 268 269 270 270 271 272 273 274 275 276 276 277

238 - Plant Pathogenesis and Resistance

5.1 INTRODUCTION Plant growth and differentiation are regulated by growth regulators. Pathogenic infection may cause a departure from normal levels of one or more growth regulators in the infected plant. Imbalance of growth regulation could alter the growth habit of the plant and result in symptoms such as stunting, overgrowth, epinasty, and premature leaf drop. The study of plant growth regulation in host-parasite interactions is complicated by the fact that (i) our understanding of biosyntheses and modes of action of growth regulators at the molecular level is limited; (ii) growth regulators generally do not act singly, but rather two or more act in concert; (iii) many plant pathogens produce growth regulators, making the determination of the relative amounts of growth regulators contributed by each partner to the host-parasite interaction difficult; and (iv) growth regulator activities are influenced by many metabolites of plant pathogens. Along with cutinases (Chapter 1), cell-wall-degrading enzymes (Chapter 2), and phytotoxins (Chapter 6), growth regulators are considered a major part of the pathogenic arsenal. In this chapter, discussion will be limited to the disturbance of growth regulators that result in tumorigenesis. For reviews of growth regulators and tumorigenesis, see Gelvin (1984), Surico (1986), Powell and Gordon (1989), Kado (1991), Yamada et al. (1991), Surico and lacobellis (1992), Hamill (1993), Yamada (1993), Gaudin et al. (1994), and Clare and McClure (1995).

5.2 CROWN GALLS CAUSED BY Agrobacterium tumefaciens 5.2.1 In vitro Production of Auxins and Cytokinins by Agrohacterium tumefaciens Tryptophan (Trp) is the primary precursor ofindole-3-acetic acid (lAA).1t is converted to IAA by various organisms via different pathways (Fig. 5-1). First, Trp may be transaminated to indole-3-pyruvic acid (lPyA), decarboxylated to indole-3-acetaldehyde (lAAld), and then oxidized to IAA. The reactions are catalyzed by tryptophan transaminase, indole-3-pyruvate decarboxylase and indole-3-acetaldehyde oxidase, respectively. Most plants synthesize IAA via this pathway (Sembdner et aI., 1980). Second, Trp may be decarboxylated to form tryptamine, followed by transamination to IAAld, and finally oxidized to IAA. The enzymes involved are tryptophan decarboxylase (EC 4.1.1.28), tryptamine transaminase, and indole-3-acetaldehyde oxidase, respectively. This pathway operates in many plant families (Sembdner et aI., 1980). Third, Trp is first converted to indole-3-acetamide (lAM) by tryptophan 2-monooxygenase (EC 1.13.12.3) and then to IAA by lAM hydrolase. This pathway functions primarily in prokaryotes, including Agrobacterium tumefaciens and Pseudomonas syringae pv. savastanoi (lnze et aI., 1984; Yamada et aI., 1985). The occurrence ofIAM and the activities of tryptophan monooxygenase and lAM

Plant Tumorigenesis - 239

H

t'Yptophao transaminase

Tryptophan

ｾｴｯｬ＠

decarboxylase

Ｇ［ＭＢＬｐｴｏｨｾ＠

monooxygenase

ｾＢｃｈｎＧ＠

CH,-:a::::H

peroxidase

ｃｈＢｾ＠

\

ｃｈＬﾷｾｎ＠

0 l0l-/ 0 l0l-/ 0 l0l-/ 0 l0l-/ H

H

Indole-3-pyruvic acid

"'"

H

H

Tryptamine

indoJepyruvate decarboxylase

/

tryptamine

transaminase

"" I

o

I

CH,·CHO

l0l-/

indolcacetamide hydrolase

H

Indole-3-acetonitrile

Indole-3-acetaldehyde

I

'\.

\

glucosinolase

cD

indole-3-ethanol oxidase

o l0l-/ /

(myrosinase)

\

CH 2-CH 20H

H

Indole-3-ethanol

H

Indole-3-acetic acid

CH 2

NOSOJ=

-C" ＧｓＮｾｄｇｉＢ｣ｯｳ･＠

H

Glucobrassicin

Fig. 5-1. Biosynthesis of indole-3-acetic acid. Most enzymes involved in these pathways have been characterized: tryptophan transaminase in Pseudomonas fluorescens (Oberhansli et aI., 1991), indolepyruvate decarboxylase in Erwinia herbicola (Brandl and Lindow, 1996), indole-3acetaldehyde oxidase in pea (Kutacek and Rovenska, 1991), tryptophan decarboxylase in Catharanthus roseus (Goddijn et aI., 1994), tryptophan 2-monooxygenase and indoleacetamide hydrolase in Agrobacterium tumefaciens (Inze et aI., 1984) and P. syringae pv. savastanoi (Yamada et aI., 1985), peroxidase and indole-3-acetaldoxime dehydratase in Chinese cabbage (Brassica campestris ssp. pekinensis) (Ludwig-MOller and Hilgenbergm 1990, Ludwig-MOller et aI., 1990), nitrile hydratase in Agrobacterium and Rhizobium (Kobayashi et aI., 1995), and nitrilases in Arabidopsis thaliana (Bartling et aI., 1994).

hydrolase have been demonstrated also in trifoliata orange (Poncirus trifoliata) (Kawaguchi et aI., 1993) and squash seedlings (Rajagopal et aI., 1994). Finally, IAA can be synthesized from indole-3-acetaldoxime (lAAdox), a naturally occurring compound. IAAdox is converted to indole-3-acetonitrile (IAN) by indole acetaldoxime dehydratase (EC 4.2.1.29) and then to IAA by nitrilase (EC 3.5.5.1). Alternatively, IAN may be first converted to lAM by nitrile hydratase and finally to IAA by lAM hydrolase. Several higher plants (e.g., maize,

240 - Plant Pathogenesis and Resistance

Arabidopsis thaliana), fungi (e.g., Taphrina spp.), and bacteria (e.g., Agrobacterium and Rhizobium) synthesize IAA via IAN (Yamada et aI., 1990; Wright et aI., 1991; Normanly et aI., 1993; Bartling et aI., 1994; Kobayashi et aI., 1995). A. tumefaciens. therefore, uti Iizes two pathways to produce IAA: Trp - lAM - IAA route (Inze et aI., 1984) and IAAdox - IAN - lAM - IAA route (Kobayashi et aI., 1995). The genes encoding tryptophan-2-monooxygenase and lAM hydrolase, the enzymes which catalyze the first pathway, reside on the Ti (tumor-inducing) plasmid. Nitrile hydratase, the enzyme involved in the second route, has been purified from cells of A. tumefaciens and Rhizobium spp. It has a molecular mass of 102 kO and is made up of four identical subunits (Kobayashi et aI., 1995). The gene encoding nitrile hydratase, however, has not been cloned. It is not known whether the gene is chromosome- or plasmidborne. Consequently, the role ofIAA in tumorigenesis by A. tumefaciens will be discussed in the context of the Trp - lAM - IAA biosynthesis route. VirulentA. tumefaciens strains C58 (contains TiC58 nopaline plasmid) and B6 (contains TiB6 octopine plasmid) produce copious quantities of IAA in culture media containing tryptophan (Table 5-1). The plasmid-free, avirulent mutant 101293-3 synthesizes reduced amounts of IAA (Liu et aI., 1982). Insertion ofnopaline or octopine Ti plasm ids into mutant 101293-3 restores its virulence and increases its ability to produce increased levels of IAA. When these transform ants are cured of their Ti plasm ids, virulence and ability to produce increased levels of IAA are concomitantly lost. A Tn5 mutagenized TiC58 plasmid, deficient in the ability to synthesize increased levels of IAA, has been inserted into mutant 101293-3. The resulting tran sform ants 101293-3 (TiC58::Tn5) remain avirulent and fail to produce increased levels of IAA. These results indicate that genes involved in the synthesis of IAA are located on the Ti plasmid (Liu et aI., 1982). Cytokinins are N 6-substituted purines that, in the presence of auxins, induce cell division. The structures of some of the cytokinins are given in Fig. 5-2. Two pathways have been proposed for the biosynthesis of isopentenyladenine (iP) and its derivatives, the key components of cytokinins: synthesis via tRNA and synthesis involving 5'-AMP. In the first pathway, dimethylallyl pyrophosphate (OMAPP):tRNA transferase transfers the isopentenyl group to tRNA. The transferase is encoded by the chromosome-borne miaA gene (Morris et aI., 1993). Upon autolysis or excision, isopentenyladenine (iP) is released from the isopentenylated tRNA (Fig. 5-3) (Letham and Palni, 1983). In the second pathway, OMAPP:5'-AMP transferase (isopententyl transferase) transfers the isopentenyl group to 5'-AMP to form isopentenyladenosine 5'-monophosphate which is then converted to iPA (Fig. 5-4). The enzyme is encoded by ipt (isopentenyl transferase). The gene is located on TONA of the nopaline Ti plasmid or TL-ONA of the octopine Ti plasmid. iPA

Plant Tumorigenesis - 241 Table 5-1. In vitro production of IAA and cytokinins by strains of Agrobacterium tumefaciens. Strain

Growth regulator type/level

C58 (Virulent)

IAA t-ZR c-ZR t-msZR IAA t-ZR c-ZR t-msZR IAA t-Z t-ZR iPA IAA t-Z t-ZR iPA t-Z iP/iPA t-Z t-ZR iP/iPA

NT1 (Avirulent)

B6 (Virulent)

B6-37 (Avirulent)

C58 (Virulent) T -37 (Virulent)

Assay method Reference

1,400Ilg/L 0.3 1.1 0.2 670 0.0 0.0 0.0 730llgiL 2.9 0.12 0.28 520 0.1 0.23 0.19 2 ng/mLlA(,()()unit 0.4

GC/HPLC Liu and Kado (1979); GC/MS McCloskey et al. (1980)

RIA

Weiler and Spanier (1981)

RIA

Akiyoshi et al. (1987)

44 2 2

t-Z = trans-zeatin; t-ZR = trans-zeatin riboside; c-ZR = cis-zeatin riboside; iP = isopentenyl adenine; iPA = isopentenyl adenosine; t-msZR = trans-methylthiozeatin riboside

and iP thus produced are subjected to hydroxylation by cytokinin hydroxylase and other modification to form a family of cytokinins (Fig. 5-5). A. tumefaciens produces cytokinins in culture media (Table 5-1). Strain B6 produces W-isopentenyladenine (Hahn et aI., 1976). Strain C58, but not its plasmid-free mutant NT1, produces trans-zeatin riboside in culture media (McCloskey et aI., 1980).

5.2.2 Ti Plasmids of Agrobacterium tumefaciens kb) in crown gall The involvementofa large-sizeA. tumefaciens plasmid ＨｾＲＰ＠ induction was first reported by Van Larebeke et al. (1974). They have shown that the conversion of a crown gall-inducing strain to non-tumorigenicity is correlated with the concomitant loss of a large plasmid. Subsequently, they have shown that the acquisition of the tumor-inducing ability by non-oncogenic Agrobacterium is a result of plasmid transfer (Van Larebeke et aI., 1975). Thus, when the Ti plasmid is present in A. tumefaciens, the strain is oncogenic. The introduction of the Ti plasmid into Agrobacterium-related bacteria, such as the nodule-inducing bacterium Rhizobium leguminosarum biovar. trifolii, confers the tumor-inducing ability on the bacteria (Hooykaas et aI., 1977). These results

242 - Plant Pathogenesis and Resistance

ＨｲＺ［ｾ＠

H

H

ｈｏＧｾｊ＠

t-( cis-Zeatin

cis-Zeatin riboside

trans-Zeatin riboside

trans-Zeatin

ex} ｾ＠

H CH,OH N-CH,-CH, -C'

'rn,

H

2-Methylthiozeatin

N6_(l\.2-lsopentenyl) adenine

2-Methylthiozeatin riboside

N6_(l\.2-lsopentenyl) adenosine

Dihydrozeatin

Zeatin-O-(3-D-glucoside (R = H) Zeatin riboside-O-(3-D-glucoside (R - ribosvl)

Dihydrozeatin riboside

Kinetin

Fig. 5-2. Some of the N 6 -substituted purines with cytokinin activity.

demonstrate that virulence determinants are on the Ti plasmid_ Introduction of the Ti plasmid into distantly related bacteria such as E. coli, however, does not result in tumor-inducing strains (Hille et aI., 1983). This finding indicates the fact that the Ti plasmid alone is insufficient to cause plant tumors. Other factors, most likely the ones determined by chromosomes, are necessary for in planta oncogenicity.

Plant Tumorigenesis - 243

)=A,. ,. 5'

IsopentenyI pymphosphate

Autolysis! excision

tRNA

IsopentenyIated tRNA

N6-IsopentenyI adenine iP

Fig. 5-3. Biosynthesis of isopentenyladenine from isopentenyl pyrophosphate and tRNA. (Adapted from Morris et aI., \993).

One of the characteristic features of crown gall cells is their ability to proliferate autonomously in the absence of auxins and cytokinins required for the growth of normal plant cells. Genes encoding these growth regulators are iaaM (also known as awe1 or tms 1), iaaH (also known as awe2 or tms2), and ipt (also known as cyt or tmr). They are located on Ti plasm ids. The iaaM gene encodes tryptophan monooxygenase, which catalyzes the conversion of Ltryptophan to indole-3-acetamide; iaaH encodes indole-3-acetamide hydrolase, which converts indole-3-acetamide to indole-3-acetic acid.

CH,

CH,

'\/ C

II

+

isopentenyl transferase

CH

P-P-O.H2C/

5'-Adenosine monophosphate (5'-AMP)

IsopentenyI pyrophosphate (IPP)

N6-Isopentenyladenosine 5'-monophosphate (iPA 5'-phosphate)

Fig. 5-4. Biosynthesis of isopentenyladenosine from isopentenyl pyrophosphate and 5'-AMP. (Adapted from Morris et aI., \993).

244 - Plant Pathogenesis and Resistance 5'·Adenosine monophosphate (5'-AMP)

lsopentenyl pyrophosphate

RNA

Isopentenylated

RNA

N6-lsopentenyl cYlokinin adenosine 5'· hydroxylase phosphate ----=----''---•• Ribosylzeatin 5'-monophosphate

!

5'-nucleotidase

N6-lsopentenyl

!

cytokinin

5' -nue Ieot!·d ase

1

hydroxylase. Zeatin riboside _

adenosine adenosine autolysis! excision

ｾ＠

adenosine nucleosidase

nucleosidase

Dihydrozeatin riboside Methylthiozeatin riboside Zeatin riboside- (J.pD-glucoside

cytokinin

N6.lsopentenyl _ _ｨＮＺｹ､｟ｲｯｸｬ｡ｳ･Ｍｾ＠ adenine

Zeatin _ _ _....... Dihydrozeatin Methylthiozeatin Zeatin-()-j3-D-glllcoside Zeatin-7-glucoside Zeatin-9- 'Iucoside

Fig. 5-5. Biosynthesis of cytokinins.

The ipt gene encodes DMAPP:5"-AMP transferase, which catalyzes the production of cytokinin isopentenyladenine (iP) and isopentenyladenosine (iPA). The gene possesses a eukaryotic promoter that expresses only upon transformation into plant cells. It is not expressed in culture media. Strains harboring nopaline Ti plasm ids (e.g., pTiC58) also produce cytokinin transzeatin (t-Z) and trans-zeatin riboside (t-ZR) in culture media and transformed plant tissues, indicating the presence of additional genes for cytokinin biosynthesis that are not regulated by the eukaryotic promoter (Regier and Morris, 1982). The trans-zeatin secretion (tzs) locus has been subsequently identified (Beaty et aI., 1986). The tzs gene is located on the Vir region of the nopaline Ti plasmid. Its expression is regulated by the VirANirG and is responsive to certain plant phenolics (John and Amasino, 1988; Powell et aI., 1988). The ORF has 729 bp and would code for a polypeptide of243 amino acids with a predicted molecular weight of27,598. Extensive homology exists between tzs and ipt, indicating that tzs may possess DMAPP:5"-AMP transferase activity (Beaty et aI., 1986). When expressed in E. coli, large quantities oft-Z and iP are produced by transformed bacteria, indicating that the production ofthese two cytokinins is associated with the presence of the tzs gene. These results also indicate that tzs confers on A. tumefaciens the ability to produce t-Z and DMAPP:5"AMP transferase activity. The other characteristic of crown gall cells is the ability to synthesize a group of compounds known as opines, normally absent in plant cells. The synthesis is under the control of bacterial genes and used by the bacterium for its growth. The specific type of opines formed by crown gall cells depends on

Plant Tumorigenesis - 245 the type of Ti plasm ids carried by the A. tumefaciens bacterium. Thus, Ti plasm ids may be classified as octopine, nopaline, leucinopine, and succinamopine plasm ids (Fig. 5-6). All Ti plasm ids, with the exception of octopine plasmids, contain a segment of DNA known as T-DNA that can be transferred and integrated into plant genomes. The T-DNA of the nopaline Ti plasmid pTiC58 consists of24,782 bp flanked by two 25 bp border sequences (Gielen et aI., 1999). The entire nopaline-type plasmid pTi-SAKURA has 206,479 bp (Suzuki et aI., 2000). Octopine plasm ids contain two segments of Ti plasmid DNA that can be independently transferred to plant cells during tumor induction (Fig. 5-7). The segment of the octopine Ti plasmid that is oncogenic is called the left-transferred DNA (TL-DNA). The other segment without oncogenic properties is called the right-transferred DNA (T R-DNA). TDNA in nopaline, leucinopine and succinamopine plasm ids are oncogenic and are partially homologous to the TL-DNA of octopine plasm ids. H,N, , C-NH-(CH,h-CH-COOH HN' ｾｈ＠ I

HJC-CH-COOH

H2N,

I

NH

0, CH-CH,-CH-COOH

ｾｈ＠

HJC

Nopoline

Leucinopine

I

I

HJC-CH-COOH

CH,-(CHOH),-CH,OH

Lysopine

l=D I I

COOH

ｾ＠ｬ =;:yCOOH I NH

CHrCH-COOH

Histopine

Mannopine

HOOC-(CH,h-CH-COOH

Succinamopine

NH

N H

NH I

HOOC-(CH,h-CH-COOH

O. : C-(CH,h-yH-COOH H,N NH

ｾ＠ C-CH'-yH-COOH

H,N I

HOOC-(CH,h-CH-COOH

Octopine

H,N-(CH,l.-fH-COOH

HJC.

HN··C-NH-(CH,h-YH-COOH NH

HOOC

CH,-CH,-COOH

Cucumopine

HOH'CpGIUCO Se CH,OH-(CHOH)

ｾｎｈ＠

if1

CH,OH-(CHOHl.-i H,

O.

HOOcyNyO (cH,h-CONH,

ｾ＠

HO HO

ｾｶ＠

ｾ｢ｈｉ＠

CH,OH

0

0

o--p=o I

Agropine

Agropinic acid

OH

--(,(COOH

'll;c( HOOC

Cue urn opine lactam

Agrocinopine A

Fig. 5-6. Structures of selected opines produced by various strains of Agrobacterium tumefaciens. (Adapted from Dessaux et aI., 1993).

Most Ti plasmids are stably maintained in the bacteria despite of their large size. The major regions of the Ti plasmid encoding functions relevant to tumor formation are T-DNA and the vir region. The vir region is about 35 kb in length and may be divided into eight distinct loci: virA - virH (Stachel and Nester, 1986). Loci virA, virG, and virF contain

246 - Plant Pathogenesis and Resistance

.

illall icm.\/ ipl On.I· nO.I'

ｾＢＧＷ＠

ｾ＠

Izs A

8

G

C

n

...............ｾ＠

H

...... .........

............

,

\

:

I' Ｌｾ＠

"\ \

:,','

',,'

................. _", -_

'"

E

..... , \

I

"

fro

-DNAOpine' " // catabolism' ,'or; Conjugativ '/---:'" IranSrer. ,,' Rephc3tor '

pTiC58

" ..... '\ age arc oee

..

Replicalor

ｾ＠

,

Fig. 5-7. Genetic maps of single T-DNA (pTiC58) and bipartite T-DNA (pTiAch5) plasmids in Agrobaeterium tumefaeiens. pTiC58 is a nopaline-type plasmid. The genes encode tryptophan monooxygenase (iaaM), indoleacetamide hydrolase (iaaH), isopentenyl transferase (ipt), nopaline synthase (nos), are located on the T-DNA. pTiAch5 is an octopine-type plasmid. It carries octopine synthesis gene (oes) and synthesizes several opines of the octopine family in infected plants. oes, iaaH, iaaM, and ipt are located on the T L-DNA. Octopine-type plasmids also carry genes for mannopine synthesis (mas) and agropine (ags). These genes are located on the T R-DNA. The functional maps of the vir region of pTiC58 and pTiAch5 have been determined by Rogowsky et al. (1990) and Birot and Casse-Delbart (1988), respectively.

one gene for each locus. The remaining loci contain multiple genes. virA is constitutively expressed and noninducible; virB, virC, virD. and virE are expressed only upon activation by plant signals; and virG is both constitutively expressed and inducible (Stachel and Nester, 1986). The products of the vir genes are involved in the T-DNA transfer process, but the DNA in the vir region is not integrated into the genome of the transformed plant cells. Strains with the mutation in virA, virB, virD, and virG lose the ability of oncogenicity of the bacterium. Mutations at virC and virE attenuated the virulence. The TDNA, on the other hand, is physically integrated into the transformed plant nuclear genome and is responsible for the phenotype ofthe transformed cells. T-DNAs of octo pine and nopaline plasm ids share extensive DNA homology, and the conserved region contains six protein-coding genes: genes 1,2,4,5,6", and 6h • Mutations in genes I and/or 2 induce attenuate tumors with an abundance of shoots, ind icating that these genes encode functions suppressing shoot formation in wild-type tumors. Ti plasm ids with mutations in gene 4 induce attenuate tumors that typically show root proliferation, indicating that the gene suppresses root formation in tumors containing complete T-DNA. It is well established that shoot formation results from high cytokinin/auxin ratios and that root formation is favored by high auxin/cytokinin ratios. Thus, genes

Plant Tumorigenesis - 247 and 2 encode enzymes involved in the biosynthesis of auxins and gene 4 encodes enzymes for cytokinin biosynthesis. The functions of genes 63 and 6b also have been investigated. The gene 63 determines a permease system for the excretion of octopine and related opines from transformed plant cells (Messens et aI., 1985). The gene 6b is an oncogene capable of inducing tumor formation in a limited number of plant species such as Nicotiana glauca and Kalanchoe tubiflora (Hooykaas et aI., 1988). T -DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes have been cloned. Tobacco (N tabacum) leaf discs infected with A. tumefaciens carrying the cloned gene produce shooty calli on hormonefree Murashige-Skoog medium. Southern hybridization has demonstrated that the T-DNA segment is integrated into plant genomes. Some of these immature shoots developed into mature shoots with morphological abnormalities. When leaf discs from these mature plants are placed on the same medium, numerous shoots develop from the wounding sites, indicating that the transgenic plants possess a high regenerative potential (Wabiko and Minemura, 1996). The coding region of T-DNA gene 5 has been expressed in E. coli. It encodes a 26-kD protein. The biosynthesis of this protein correlates with an increase in conversion of tryptophan to indole-3-lactate (ILA). Expression of chimeric gene 5 constructs in transgenic tobacco result in overproduction of ILA that enhances shoot formation in undifferentiated tissues and increases the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. ILA also inhibits the auxin induction of the gene 5 promoter and competes with IAA for in vitro binding to purified cellular auxin binding proteins. Thus, ILA autoregulates its own synthesis and, thereby, modulates a number of auxin responses in plants (Korber et aI., 1991). Several other regions of the Ti plasmid have been identified and their functions assigned: the ori region is required for the replication of the plasmid, the age region is necessary for arginine catabolism, the occ region is essential for octopine catabolism, and the tra region is needed for conjugal transfer of the plasmid between bacteria. These loci, however, are nonessential for oncogenicity.

5.2.3 Transformation of Plant Cells by Agrobacterium tumefaciens The process of crown gall tumorigenesis may be divided into the following steps: attachment of agrobacteria to plant cells, recognition of signal molecules and activation of vir gene expression, formation of single stranded T-DNA, conjugation of T-DNA with protein to form T-complex, the export of Tcomplex into the cytoplasm of the host plant cell, transport of the T-complex through the nuclear pore, and integration of T-DNA into the genome of transformed cells. The subject has been reviewed (Zupan and Zambryski, 1995; Sheng and Citovsky, 1996; Gelvin, 2000).

248 - Plant Pathogenesis and Resistance

5.2.3.1 Attachment of bacteria to plant cells Attachment of agrobacteria to the surface of the plant cell is the first step in tumorigenesis. Mutants of A. tumefaciens that are unable to attach are either highly attenuated or avirulent (Douglas et aI., 1985; Cangelosi et aI., 1987; Thomashow et aI., 1987). Three classes of attachment-deficient mutants have been isolated. One class of mutants has been obtained by Tn5 transposon mutagenesis. All chromosomal Tn5 insertions leading to the avirulent, attachment-defective phenotype are localized in a ll-kb portion of the chromosome region where chvA and chvB (chromosome virulence) are located (Douglasetal., 1985). ThechvBgeneencodesa235-kD inner membrane-bound protein that binds UDP-glucose and serves as an intermediate in catalyzing the synthesis of a cyclic P-l,2-D-glucan. The glucan is a neutral, small polysaccharide (17-20 glucose residues) (Puvanesarajah et aI., 1985). The chvA gene, on the other hand, is involved in the synthesis of the transport protein that exports the cyclic glucan into the periplasm and extracellular media (O'Connell and Handelsman, 1989). chvA mutants of A. tumefaciens are avirulent and attachment deficient. The bacterial cells ofchvA mutants contain approximately the same amount of intracellular glucan as the cells of the virulent strains. The mutants, however, release into the culture medium only 2% of the glucan released by the virulent strains. Introduction of a cosmid carrying the wild-type chv region restores virulence, attachment and secretion of glucan to chvA mutants. Thus, chvA gene encodes a protein involved in export ofP-l ,2-glucan (O'Connell and Handelsman, 1989). The exact role of P-l ,2-glucan in attachment and virulence, however, is not fully understood. The second class of attachment-deficient mutants fails to synthesize cellulose fibrils and polypeptides that are responsible for attachment of bacteria to plant cells. The loci cel and att are responsible for the biosynthesis of these components. They are located on the bacterial chromosomal DNA (Robertson et aI., 1988; Matthysse, 1987). Two operons required for cellulose synthesis have been identified. One operon contains celABC whereas the other contains ceIDE. The celA gene is homologous to the cellulose synthase (bscA) gene of Acetobacter xylinum and the celC gene is homologous to endoglucanase genes ofA. xylinum and Erwinia chrysanthemi. The remaining genes have no significant homology to other genes. Transposon mutagenesis shows that celC and celE are required for cellulose synthesis in A. tumefaciens (Matthysse et aI., 1995). The att operon contains nine ORFs (attA,A}BCDEFGH). The altA} and attB ORFs show homology to genes encoding the membrane-spanning proteins from gram-negative bacteria. The altA, and attE show homology to the genes encoding ATP-binding proteins (Matthysse et aI., 1996). The functions of other genes have not been determined.

Plant Tumorigenesis - 249 The third class of mutants are devoid of succinoglycan. The production of this acidic, extracellular polysaccharide is controlled by the pscA (polysaccharide composition) gene (Thomashow et aI., 1987). Results of genetic complementation and DNA hybridization experiments indicate that the pscA gene is structurally and functionally related to the exoC locus of Rhizobium meliloti (Marks et aI., 1987). ExoC mutants produce ineffective nodules whereas pscA mutants are nononcogenic or give rise to attenuated tumor formation. The pscA gene encodes phosphoglucomutase, an enzyme essential for the biosynthesis of cellulose and succinoglycan (Uttaro et aI., 1990). The role of cellulose and succinoglycan in attachment and virulence is unclear. Mutants deficient in the biosynthesis of succinoglycan or cellulose have been found to be virulent (Matthysse, 1983; Cangelosi et aI., 1987). 5.2.3.2 Recognition of plant signal molecules Wounding is a prerequisite for crown gall tumorigenesis. Signal molecules capable of activating vir genes and initiating tumorigenesis are synthesized de novo following wounding (Fig. 5-8). These signal molecules may be divided into two groups: simple phenolic compounds and flavonol glycosides. The first group is represented by acetosyringone, a-hydroxyacetosyringone, vanillin, catechol, gallic acid, sinapic acid, and many other structurally similar compounds and their glycosides (Stachel et aI., 1985; Bolton et aI., 1986; Spencer et aI., 1990; Delmotte et aI., 1991). The second group includes kaempferol-3-glucosyl galactoside and quercetin glycosyl galactoside (Zerback et aI., 1989). Several monosaccharides, including arabinose, glucose, fucose, galactose, and xylose, act synergistically to the phenolic compounds and promote maximum vir expression (Cangelosi et aI., 1990; Shimoda et aI., 1990). For a review of chemical signaling between Agrobacterium and plant hosts, see Spencer and Towers (1989), Gelvin (1992), Winans (1992), and Baker et al. (1997). Most monocots are highly resistant to A. tumefaciens infection. One hypothesis for the resistance is that monocots fail to produce vir gene inducers and T-DNA transfer cannot be initiated. This hypothesis is supported by the observation that the monocot Dioscorea bulbifera produces no detected phenolic compounds and is nontransformable with A. tumefaciens. This plant, however, can be transformed by agrobacteria preincubated with wound exudates collected from a dicot plant (Schafer et aI., 1987). The hypothesis, however, is challenged by the isolation and characterization of ethyl frulate (C'2H,P4) from nontransformable wheat (Triticum monococcum). This molecule is more active for vir gene induction at low concentrations than acetosyringone. It also is produced in quantities sufficient for significant vir induction as assayed with a vir::lacZ fusion gene. Thus, the resistance of

250 - Plant Pathogenesis and Resistance monocots to agrobacteria may result from a block of the T-DNA transfer process subsequent to vir gene induction (Messens et aI., 1990).

2'.4',4-Trihydroxy-3.5-dimethoxy chalcone

HO,,r--

froH

ｗｾ＠ Kacmpferol 3-0-(2"-0P-D-glucopyranosyl) -I)..

alacto

ranoside

Quercetin 3-0-(2"-0P-D-glucopymnosyl)

D alacto

ranoside

OH

°

2',4'.4-Trihydroxy-3-methoxy chalcone

Fig. 5-8. Signal molecules produced by wounded plant cells that activate vir gene expression. (Stachel et aI., 1985; Zerback et aI., 1989; Messens et aI., 1990; Spencer et aI., 1990).

5.2.3.3 Activation of vir genes VirANirG is a two-component sensory system in A. tumefaciens (Stachel et aI., 1986; Winans, 1991; Charles et aI., 1992; Lee et aI., 1995, 1996). When the VirA protein, which is constitutively expressed by virA and resides in the bacterial membrane, senses and interacts with the plant signal molecule, information is transferred through phosphorylation to the virG product, a 27-kD protein in the cytosol. The activated VirG protein, in turn, activates all of the vir genes by binding to a 12-pb conserved sequence called the "vir box", which

Plant Tumorigenesis - 251 is located upstream of each of the vir genes (Leroux et aI., 1987). Mutations in either virA or virG completely block the response to plant signals. The VirA protein is a polypeptide of829 amino acids with a M.-of92,000. It has a typical leader sequence, indicating that a portion of the protein crosses the plasma membrane. The N-term inal half of VirA is periplasm ic, whereas the C-terminal is cytoplasmic (Melchers et aI., 1989). The C-terminal half of V irA contains the kinase activity and is autophosphorylated (Jin et aI., 1990a; Morel et aI., 1990). VirA senses the plant-signalling compounds and transduces the signal to VirG in the form of ATP transphorylation from VirA-kinase to VirGo The VirG protein is a DNA-binding protein that binds nonspecifically at low concentrations. A model for the interaction between the VirA and VirG proteins and the vir genes is illustrated in Fig. 5-9.

Fig. 5-9. A diagrammatic representation ofthe perception of plant signal molecules by VirA and the activation ofVirG. The activated VirG in turn activates all ofthe vir genes (Reproduced from Nester and Gordon, 1991, with permission from Springer-Verlag, New York).

5.2.3.4 Processing of T -DNA for intercellular transport VirD, and VirD2 are encoded by the 5' half of the virD locus. Both proteins participate in the processing ofT-DNA for transfer. VirD, is a 21-kD protein with DNA-relaxing activity. VirD2 is a strand-specific and sequence-specific endonuclease (Yanofsky et aI., 1986; Jayaswal et aI., 1987; Filichkin and Gelvin, 1993; Scheiffele et aI., 1995). It nicks the Ti plasmid at 25-bp sequences that border the T-DNA, resulting in the formation of a singlestranded T-DNA (ssT-DNA), known as the T-strand (Stachel et aI., 1986). VirD2 is covalently bound to the T-strand at the 5' terminus (Herrera-Estrella et aI., 1988; Diirrenberger et aI., 1989; Howard et aI., 1989). The binding protects the 5'-terminus of the T-strand from exonuc1eolytic degradation (Diirrenberger et aI., 1989). VriC, can enhance T-strand production when VirD,

252 - Plant Pathogenesis and Resistance and VirD 2 are limiting (De Vos and Zambryski, 1989) and overdrive aT-DNA transfer enhancer (Toro et aI., 1989). The T-strand also is coated along its entire length with a 69-kD ssDNA binding protein, the product of virE2 (Das, 1988; Citovsky et aI., 1988; Citovsky et aI., 1989; Gelvin, 1998). The binding protein is highly specific to ssDNA, with little or no binding activity toward either dsDNA or ssRNA (Das, 1988). The protein binds ssDNA, forming VirE2 protein-ssDNA complex with a binding site of28-30 nucleotides (Citovsky et aI., 1989). This complex, known as "T-complex", is stable at high salt and resistant to exo- and endonucleases. Thus, the VirE2 protein acts to protect the T-strand during transfer (Citovsky et aI., 1988).

5.2.3.5 Intercellular transport ofT-DNA After activation, virB operon synthesizes 11 membrane-bound proteins. Some ofthem are involved in "T-pilus"formation. T -complex may be transferred from Agrobacterium to the plant cell through this pilus (Kado, 1994; Lai and Kado, 1998). 5.2.3.6 Nuclear targeting VirD 2and VirE2contain nuclear localization signal sequences (NLS). The NLS resides in the C-terminal region of VirD2 can localize the nucleus of plant nuclei (Tinland et aI., 1992; Koukolikova-Nicola et al. 1993). Similarly, VirE2 protein contains two NLS regions that can target linked reporter proteins to plant cell nuclei (Citovsky et aI., 1992). Since both VirD2 (which is covalently bound to the T-strand at the 5'-terminus) and VirE2 (which is a part of the Tcomplex) are transported to the plant cell along with the T-DNA, they may be involved in the targeting of the T-complex to plant nucleus. 5.2.3.7 Integration ofT-DNA to plant nuclear genome The precise mechanism of T-DNA integration into the plant genome is not known. It is possible that plant DNA is invaded and denatured by the T-strand followed by second T -strand repair synthesis (Tinland, 1996). Alternatively, the T-strand may be converted to an extrachromosomal double-stranded form prior to integration (De Neve et aI., 1997). The integration of the 5' end of the Tstrand into plant DNA is generally precise, suggesting that VirD2 may participating in the T-DNA integration (Durrenberger et aI., 1989; Tinland et aI., 1995). The genes involved in agrobacterial tumorigenesis are summarized in Table 5-2. The sequence of events from the contact between susceptible plant cells and agrobacteria to the gall formation is summarized in Fig. 5-10 and Fig. 5-11.

Plant Tumorigenesis - 253 Contact between susceptible plant cells and agrobacteria

ｾ＠

C'hv A, C'hvB, AIt, PscA

Recognition of plant signals by agrobacteria

ｾ＠

VirAlVirG

Transcriptional activation of vir genes

ｾ＠

VirDI' VirD2' VirC'1

Generation ofT-strand

ｾ＠

VirE2

Assembly ofT-complex

ｾ＠

VirEJ, VirBnl

!Transport ofT-complex through bacterial and plant cell membranes

ｾ＠

VirD2' VirE2

Targeting T-com(:1lex to plant nucleus

ｾ＠

VirD2' VirE2

Integration ofT-DNA into plant genome

ｾ＠

transcription ofiuuM. iuuH, ipl

Production of auxin and cytokinin ｾｳｴｩｭｵｬ｡ｯｮ＠ of cell division and enlargeme Formation of crown galls Fig, 5-10, Sequence of events in the Agrobacterium-plant cell interaction,

5.2.4 Expression of T -DNA in Transformed Cells and the Auxin and Cytokinin Levels in Crown Gall Tumors After integration of the T-DNA into the genome of the plant cell, the T-DNA encodes several enzymes that are involved in the biosynthesis of auxin and cytokinin, The iaaM gene encodes tryptophan monooxygenase, which converts tryptophan to indole-3-acetamide, The iaaH gene encodes indole-3-acetamide hydrolase, which converts indole-3-acetamide to indole-3-acetic acid, The ipt gene encodes a 27-kD isopentyl transferase, which transfers an isopentyl group from isopentyl pyrophosphate (IPP) to 5 -AMP to form N6-isopentyladenosine 5'-monophosphate (iPA) (Buchmann et aI., 1985). The host cells can convert iPA to trans-zeatin and trans-ribosylzeatin, Cells of willow (Salix viminalis) transformed with the ipt gene from A, tumefaciens grow in tissue culture as undifferentiated callus without shoot induction, Transformed calluses contain high levels of 9-p-D-ribofuranosyl zeatin [(9R-5'P)Z] and its monophosphate, indicating the presence of the functional isopentyl transferase enzyme, Shoot differentiation in many plant species can be induced by high levels of cytokinins. The absence of shoot differentiation in willow is apparently unrelated to a lack of zeatin-type cytokinin in the transformed callus. Perhaps the riboside form of cytokinins are 1

254 - Plant Pathogenesis and Resistance CYTOPLASM

----,1---lIoIIyringae pv. tomato, pv. atropurpurea, and pv.

310 - Plant Pathogenesis and Resistance glycinea. Thus, the locus is highly conserved in coronatine producers (Ma et ai., 1991; Moore et ai., 1989). Stimulation of ethylene production is one of many physiological changes to plants caused by coronatine. Application of coronatine or coronafacoylvaline to bean leaves stimulates ethylene biosynthesis and induces chlorotic response in treated tissues. This stimulation is concomitant with an increase in 1aminocyclopropane-I-carboxylic acid (ACC), the immediate precursor of ethylene. The stimulation of ethylene production is inhibited by aminoethoxyvinylglycine, an inhibitor of ACC synthesis. Furthermore, the coronamic acid moiety of coronatine is structurally similar to ACC:

I-Aminocyclopropane-I-carboxylic acid

Coronamic acid

These observations indicate the possibility that part or all of the increased ethylene is the breakdown product of coronatine. Alternatively, coronamic acid may be converted to ACC and serve as precursor of ethylene synthesis. The question of whether part or all of the increased ethylene comes from the breakdown of coronatine has been investigated (Kenyon and Turner, 1992). Application of tobacco leaves with coronatine at a concentration of 15 pmol cm2 induced the formation of 44 pmol ethylene cm- 2 leaf during a 4-h incubation period. Thus, the ethylene released cannot be derived solely from the applied coronatine. When 14C-methionine is supplied to leaf discs in the presence of coronatine, more 14C-ethylene is liberated than in the absence of the phytotoxin. This provides further evidence that the ethylene released from coronatinetreated tissue is derived from methionine. The activity of ACC synthase, the enzyme that converts S-adenosyl-L-methionine to ACC, increased in PhaseD/us aureus hypocotyls during a 6-h treatment with coronatine (Kenyon and Turner, 1992). These results indicate that ethylene is synthesized de novo by the methionine pathway and that coronatine may enhance the activity of ACC synthase. In conclusion, it is unlikely that coronatine itself is converted directly to ethylene. Methyl jasmonate, a plant signal molecule, is structurally similar to that of coronafacic acid:

0p H

COOCH,

H

;}

Coronafacic acid

COOH

Methyljasmonate

Phytotoxins - 311 Recent reports indicate that coronatine and coronafacic acid mimic the function of methyl jasmonate (Feys et aI., 1994; Weiler et aI., 1994; Greulich et aI., 1995; Koda et aI., 1996). For example, these compounds induce accumulation of a proteinase inhibitor in tomato leaves and inhibit root growth in Arabidopsis seedlings (Feys et aI., 1994). They also promote cell expansion in potato tubers and senescence in oat leaves (Greulich et aI., 1995; Koda et aI., 1996). A significant difference does exist. Methyljasmonate does not cause chlorosis in tomato leaves, a symptom typical of coronatine treatment (Palmer and Bender, 1995). E. Fumonisins Fumonisins are metabolites produced by Fusarium moniliforme and other related species, including F. proliferatum. F. anthophilum. F. dlamini. F. napiforme. F. nygamai (Nelson et aI., 1992, 1993). They are also produced by Alternaria alternata f. sp. lycopersici (Chen et aI., 1992). Fumonisin BI is a mycotoxin, and its toxicity to animals and human is well documented (Gelderblom et aI., 1988; Rheeder et aI., 1992; Ross et aI., 1992). It is also toxic to higher plants. The adverse effects include the formation of adventitious roots on excised tomato shoots (Bacon et aI., 1994), electrolyte leakage from cells of jimsonweed (Abbas et aI., 1992) and duckweed (Lemna pausicostata) (Tanaka et aI., 1993), and growth inhibition of corn callus (Van Asch et aI., 1992). Fumonisin B 1 is a diester of propane-I ,2,3-tricarboxylic acid on a backbone of2-am ino-12, 16-d imethy 1-3,5,10,14, 15-pentahydroxyicosane (Bezu idenhout et aI., 1988). ｃｏｈ＠

ｾ

HOOC

o

o NHR, 81 82 83 AI A2

RI

R2

R3

H H H COCH3 COCH l

OH H OH OH H

OH OH H OH OH

Fumonisins

The biosynthetic pathway to fumonisin production is not well characterized. Fumonisins may be condensed from alanine and linoloyl-CoA. Incorporation of intact L_ 13 C- and 2H-alanine into fumonisin BI has been demonstrated (Branham and Plattner, 1993). The methyl groups on C-12 and C-16 are derived from L-methionine (Plattner and Shackelford, 1992):

312 - Plant Pathogenesis and Resistance Alanine + Linoloyl-CoA----- C 20 Backbone I

,

: transfer of methyl groups from methionine

: to ('-12 and ('-16 by methyltransferase

C 20 Backbone with C-12 and C-16 methylated

! hydroxylation at CoS. C-IO. C-14 and C-IS I

t

C20 Backbone with C-12 and C-16 methylated and C-5, C-I 0, C-14 and C-15 hydroxylated : esterification at C-14 and (,-15 with : propane 1.2.3-tricarboxylic acid

t

Fumonisin BI

The phytotoxic effects of five fumonisins and some of their hydrolysis products have been compared on detached leaves of tomato. The results indicate that fumonisins B I, B2, and B3 cause significantly more necrosis than other compounds tested. On corn and tomato seedlings, fumonisin BI is more toxic than B2 and B3 (Lamprecht et aI., 1994). Following a single application of 10 flg offumonisin B I , stem cuttings of tomato cultivar 'Ace' respond by forming callus within 24-48 hr and adventitious roots in 72-96 hr (Bacon et aI., 1994). The mechanism for the induction of adventitious roots is not known. F. AAL-toxins AAL-toxin is produced by Alternaria alternata f. sp. lycopersici, the causal fungus of tomato stem canker. The phytotoxin consists of several fractions. Fraction TA is l-amino-l1, 15-dimethyl-heptadeca-2,4,5, 13, 14-pentol with propane-l ,2,3-tricarboxylic acid linked at C-13 (TAl) or C-14 (TA2)' Fractions TB and TC have the same structure as T A with the exception that fraction TB lacks the hydroxyl group at C-5 and TC lacks the hydroxyl groups at C-4 and C-5. Fractions TO and TE have the same structures as TB and TC, respectively, with the exception that I-amino group is acetylated (Bottini and Gilchrist, 1981; Bottini et aI., 1981; Caldas et aI., 1994). The biosynthetic pathways for AAL-toxin production have not been elucidated. It is known that glycine is incorporated into the C-l and nitrogencontaining groups and the methyl groups on C-ll and C-15 are of methionine origin (Winter et aI., 1996). Results with a detached leaf bioassay indicate that AAL-toxin causes necrosis on susceptible tomato plants. Electrolyte leakage, however, has not been detected prior to the onset of necrosis, indicating that the plasma membrane is not the primary target site of AAL-toxin. Oxaloacetate, threonine, and methionine at a concentration of 34 mM protect tomato against the AALtoxin-induced necrosis. Amino acids in the glutamate, serine, and aromatic families are ineffective in toxin protection. Orotic acid, an intermediate in the pyrimidine biosynthesis, also provides protection against AAL-toxins. These

Phytotoxins - 313 results indicate that AAL-toxin may act as an antimetabolite in the metabolism ofL-aspartate and pyrimidine (Gilchrist, 1983).

TAl

H

OH

TA2

H

OH

OH ..ooc..cH2-CHCOOH-CH2..cOOH OH OH OH ..ooc..cHr CHCOOH..cH 2..cOOH

OH OH

H H

..ooc..cH2-CHCOOH..cH r COOH OH OH ..ooc..cH2-CHCOOH-CH2-COOH

H

H

..ooc..cHr CHCOOH-CH2..cooH

H

H

TDI TD2

COCH) OH

H

COCH) OH

H

TEl

COCH) H

H

TE1

COCH) H

H

OH

-ooc..cH2..cHCOOH..cH2-COOH

OH -()()C.CH2..cHCOOH-CH 2..cooH OH

OH -OOC..cH2-CHCOOH..cH2-COOH

..ooc..cH2-CHCOOH-CH 2-COOH OH

OH -()()C.CHr CHCOOH-CH2..cooH

AAL-toxins

Carbamyl aspartate is the first committed step of pyrimidine biosynthesis in higher plants (Fig. 6-3). Aspartate carbamoyltransferase (ACTase, EC 2.1.2.3) catalyzes the condensation reaction between carbamyl phosphate and aspartate leading to the formation of carbamyl aspartate. Assay of ACTase activity from tomato (a host) and mung bean (a nonhost) reveals that the enzyme from susceptible tomato is most sensitive to AAL-toxin whereas the enzyme from mung bean is the least sensitive to the toxin. It has, therefore, been suggested that ACTase is a molecular site of action of AAL-toxin (Gilchrist, 1983; Gilchrist and Harada, 1989). Results of other studies, however, do not support the theory that AAL-toxin AAL-toxin is inhibits ACTase. In tomato suspension-cultured cells, 1 ｾｍ＠ sufficient to inhibit uptake of [3H]-uridine and [3H]-thymidine and cell growth, but the inhibition of pyrimidine uptake apparently does not induce pyrimidine shortage since net synthesis of RNA or DNA is not affected. Thus, inhibition of uri dine and thymidine uptake probably is notthe cause of growth inhibition. Toxin sensitivity of cell cultures is not affected by supplementing cultures with pyrimidine or aspartate (Fuson and Pratt, 1988). In jimsonweed, orotic acid does not alleviate the effect of AAL-toxins on chlorophyll content or electrolyte leakage (Abbas et aI., 1992). Thus, AAL-toxins do not act on ACTase.

314 - Plant Pathogenesis and Resistance aspartate carbamoyhransrerase

OCOH ｾ＠

+

t

AAL-toxin

..

CH{0H)CH{CH')2

Fig. 6-7. Proposed biosynthetic pathways for AK-, AF-, and ACT-toxins. (Adapted from Feng et aI., 1990; Kohmoto et aI., 1995).

The tangerine pathotype of A. citri causes brown spots on Dancy tangerine and Emperor mandarin. It produces ACT-toxins in culture media. Chemically, they are ester derivatives of9, I O-epoxy-9-methyl-(2E,4Z,6E)-decatrienoic acid, which is identical to the one in AK-toxins (Fig. 6-7) (ltoh et aI., 1993; Kohmoto et aI., 1993, 1995). Among 67 citrus plants examined, 28 species, cuitivars, and lines susceptible to the tangerine pathotype ofA. citri are sensitive to the ACTtoxins (Kohmoto et aI., 199 I). The structural elucidation of AF-, AK-, and ACT-toxins provides an insight to the structure-activity relationship. It appears that high toxicity is due to the decatrienoic acid moiety common to these phytotoxins and that host specificity

356 - Plant Pathogenesis and Resistance is determined by the remaining moiety. The similarity among the decatrienoic acid moieties of AK-, AF-, and ACT-toxins and AOE of the HC-toxin indicates that the mode of action of these four phytotoxins may be similar. The biosynthetic pathways for AK-, AF-, and ACT-toxins are not fully known. The (8R,9S)-9, I 0-epoxy-8-hydroxy-9-methyl-(2E,4Z,6E)-decatrienoic acid moiety has been shown to be condensed from six molecules of acetic acid (Nakatsuka et aI., 1990). Time-course study indicates that decatrienoic acid is produced earlier than AK-toxin in cultures. Addition of synthetic (±)-decatrienoic acid into culture medium increases AK-toxin I production. [83H]decatrienoid prepared from 8-oxo-decatrienoic acid with NaB3H4 can be efficiently incorporated into AK-toxin I (Fig. 6-7) (Feng, et aI., 1990). The other moiety of AK-toxin, N-acetyl-p-methyl-phenylalanine, has been isolated from the culture media (Kohmoto et aI., 1995). The Japanese pear pathotype of A. alternata is genetically unstable in its AK-toxin production (Tsuge et aI., 1986). Field isolates of AK-toxin producers often lose both the ability to produce the phytotoxin and pathogenicity during culture on media. The possible participation of plasmid DNA in AK-toxin production has been examined. Circular DNA plasm ids have been found in some field isolates, but their presence has not been correlated to phytotoxin production. Possible involvement of mycoviruses in AK-toxin production has also been investigated by studying the presence of double-stranded RNA (dsRNA) genomes, which are characteristic of my co viruses. However, dsRNAs have been found in mycelia of both AK-toxin producers and toxin-deficient mutants, and no correlation between the presence of dsRNA and phytotoxin production could be established (Hayashi et aI., 1988). 6.2.3.2 Cyclic polyketides A. Substituted benzenes Zinniol, [I ,2-bis(hydroxymethyl)-3-methoxy-4-methyl-5-(3-methyl-3-butenyloxy) benzene], is a penta-substituted benzene produced by Alternaria zinniae, A. dauci, A. tagetica, A. macrospora, A. carthami, A. porri, and A. solani. These fungi cause leafspotand seedling blight on their respective hosts (Barash et aI., 1981; Cotty et aI., 1983; Cotty and Misaghi, 1984; Starratt, 1968). [3H]Zinniol has been shown to bind to carrot protoplasts and microsomes, but the H+ -A TPase in protoplasts and the Caz+-ATPase activity in the microsomes are not affected by the binding. A marked stimulation of Ca 2+ influx by zinniol, however, has been observed in protoplasts. In addition, the action of a calcium channel blocker (e.g., verapamil) is inhibited by the phytotoxin. Thus, zinniol is considered as a calcium channel agonist (Thuleau et aI., 1988).

Phytotoxins - 357

Zinniol

B. Substituted phenols and related compounds A Discula sp. that causes anthracnose of flowering dogwood (Cornusjlorida) produces several phenolic compounds, including 4-hydroxybenzoic acid, 4hydroxy-3-(3'-methyl-2'-butenyl)benzoic acid, and isosclerone. They are toxic to dogwood and several sorghum and weeds species (Venkatasubbaiah and Ch i1ton, 1991).

ｾＩＧ＠

Q0 00

OH

4-Hydroxy benzoic acid

OH

OH

p-Hydroxy Tyrosol benzaldehyde

6-Methylsalicylic acid

4-Hydroxy-3-(3'-methyl2'-butenyl)benzoic acid

Isosclerone

Tubakia dryina, a pathogen of red oak (Quercus rubra), produces isosclerone and 3- and 6-hydroxyisosclerone. These phenolics cause necrosis on leaves of red oak as well as on several weed species (Venkatasubbaiah and Chilton, I 992b). Tyrosol and p-hydroxybenzaldehyde are phytotoxins produced by Botryosphaeria obtusa, a pathogen of frogeye leaf spot of apple (Venkatasubbaiah and Chilton, 1990). 6-Methylsalicylic acid is a phytotoxin produced by a rhubard pathogen, Phoma sp. (Venkatasubbaiah and Chilton, 1992c). A gene encoding 6-methylsalicylic acid synthase has been isolated from a genomic library of Penicillium patulum. The ORF has 5322-bp coding a protein of 1774 amino acids with a molecular mass of 190,731 (Beck et aI., 1990). Drechslera indica, a pathogen of common purslane (Portulaca oleracea) and spiny amaranth (Amarathus spinosus) produces two cyclic polyketide phytotoxins. Curvulin has a molecular formula of C12HI 4 0 S and a molecular weight of238. O-methylcurvulinic acid has a molecular formula ofC II H I2Os and a molecular weight of224:

358 - Plant Pathogenesis and Resistance

ｦｴ ,

CHO

Curvulin

OH

ｾ＠

0 CH,

""'"

COOH

/./

O-Methyl curvulinic acid

Both polyketides are phytotoxic to purslane and spiny amaranth (Kenfield et aI., 1989b). C. Macrocyclic polyketides Curvularin and ap-dehydrocurvularin are phytotoxins produced by Alternaria cinerariae, a pathogen causing damping-off and leaf spot of ornamental cinerariae (Senecio cruentus). Both compounds cause stem necrosis and vascular necrosis of zinnias (Zinnia e/egans) and Canada thistle (Cirsium arvense) (Robeson and Strobel, 1981). They are macrocyclic octaketides synthesized from eight molecules of acetate in a head-to-tail arrangement with molecular formulae of CI6H200S and CI6HISOS' respectively (Arai et aI., 1989; Ghisalberti et aI., 1993; Li et aI., 1992; Yoshizawa et aI., 1990): HO

Curvularin

af3·DehydroclIrvlllarin

Curvularin and ap-dehydrocurvularin exhibit cytotoxic activity towards sea urchin embryogenesis. At a concentration of 2.5 flg/mL, curvularin totally blocks cell division, specifically disordering microtubule organizing centers in centrosomes, inducing barrel-shaped mitotic spindles, and preventing the development of asteral rays and the movement of chromosomes (Kobayashi et aI., 1988). ap-Dehydrocurvularin at a concentration of 1.2 flg/mL causes miniature spindles in the mitotic apparatus and prevents microtubule assembly (Kobayashi et aI., 1988). Brefeldin A was first isolated from Penicillium brefeldianum and subsequently from Nectria radicola, Curvularia lunata, and C. sublata. It has a molecular formula of CI6H2404 and is synthesized from eight acetate units (Handschin et aI., 1968):

ｾｏｈ＠

8 CH,CoOlt----

H

ｾ＠

q' H

----

ｾ＠

ｾｈｏｉ［＠

I

.&

HO

H

OH ----

-...:::'

""

OH

H Brefeldi" A

Phytotoxins - 359 Brefeldin A has been used extensively in the study of structure and function of Golgi apparatus and the secretion of macromolecules in animal cells (Fujiwara et aI., 1988; Lippincott-Schwartz et aI., 1991). The effects of brefeldin A on plant cells have also been investigated (Henderson et aI., 1994; Driouich et aI., 1993; Satiat-leunemaitre and Hawes, 1993; Piro et aI., 1999). In suspension-cultured cells of sycamore maple (Acer pseudoplatanus), brefeldin A at the concentrations of2.5 and 7.5 J.lg/mL causes swelling of the ER cisternae and induces the formation of large clusters of Golgi stacks. The phytotoxin also causes the accumulation of dense vesicles in the cytoplasm. These vesicles contain large amounts of xyloglucan. All of these structural changes disappear within 120 min after removal ofbrefeldin A. In vivo labeling experiments using [3H]leucine demonstrate that protein secretion, but not synthesis, is inhibited by approximately 80% in the presence of the phytotoxin. Brefeldin A also affects secretion of polysaccharides as there is a 50% drop in incorporation of [3H]xylose and eH]fucose into cell wall hemicelluloses (Driouich et aI., 1993). In oat coleoptile segments, brefeldin specifically inhibits the incorporation of labeled glucose into cellulose and cell wall polysaccharides without altering the incorporation of 14C-leucine and 14C_ glycine into cell wall proteins and glycoproteins (Piro et aI., 1999). D. Substituted quinones and related compounds Substituted quinones Ascochitine, an ortho-quinone-methide, is a toxic metabolite produced by Ascochyta pisi (Lepoivre, 1982), A. fabae (Oku and Nakanishi, 1963), and A. hyalospora (Venkatasubbaiah and Chilton, 1992a). It inhibits germination of rape seed at 200 J.lg/mL (Oku and Nakanishi, 1963), increases permeability of pea leaf discs at 100 J.lg/mL (Lepoivre, 1982), and causes necrosis on detached leaves ofa number of weeds (Venkatasubbaiah and Chilton, I 992a). It also has antifungal activity against the germination of conidia of Pyricularia oryzae and Cochliobolus miyabeanus (Oku and Nakanishi, 1963). Feeding experiments with sodium [1_ 13 C]_ and [1,2- 13C]acetate and [Me- 13 C]methionine have demonstrated that the skeleton of ascochitine is derived from a single hexaketide chain. It consists of six acetate units joined head to tail and three C 1 units from S-adenosyl-L-methionine (Colombo et aI., 1980). L·mcthioninc

CH,COOH -------------

OH Ascochitine

360 - Plant Pathogenesis and Resistance

F arnesylquinone Isolate El-l of Bipolaris hieolor, which was isolated from a diseased leaf of finger millet, produces four toxic compounds in culture media: cochlioquinones A and B, stemphone, and isocochlioquinone A (Miyagawa et aI., 1994). B. oryzae (=Coehlioholus miyabeanus) produces cochlioquinones A and B (Canonica et aI., 1980). Cochlioquinone A is also produced by Helminthosporium leersii (Barrow and Murphy, 1972). Incorporation experiments using [1 ,2- 13 C]acetate have demonstrated that 12 acetate molecules were incorporated into cochlioquinone. Six molecules are synthesized into a farnesyl unit, and the remaining molecules form the acetogenin moiety whose secondary methyl groups derive from methionine (Fig. 6-8) (Canonica et aI., 1980).

:y&yy

CH3CC)()H ...

ｾｏｓａ＠

:

,

0

1

6: o

,: :

o A. R1=H, R,=OAc, R3=OH B, R"R,=o, R3=H Cochlioquinones

0

i

1

,

ｾ＠

.-,,-

. .

cychzatlOn

decarboxylation. hydroxylation

-------------.

Farnesyl pyrophosphate

Fig. 6-8. A proposed biosynthetic pathway of cochlioquinones by Cochliobolus miyabeanus. (Adapted from Canonica et aI., 1980).

Naphthoquinones Several Fusarium spp. produce naphthoquinone pigments in cultures. Some of these pigments have antibacterial, antifungal, insecticidal, and phytotoxic activity. For a review, readers are referred to Parisot et a\. (1990), F. solani and F. oxysporum isolated from roots of diseased citrus trees produce phytotoxic naphthoquinones, including fusarubin, anhydrofusarubin, javanicin, norjavanicin, marticin, isomarticin, 9-0-methylfusarubin, 9-0-methylanhydrofusarubin, and 5-0-methyljavanicin (Tatum and Baker, 1983; Tatum et a\., 1985). Naphthoquinones are synthesized from seven molecules of acetate. The

Phytotoxins - 361 heptaketide is cyC\ized to form fusarubinoic acid, which serves as the precursor offusarubin and its related compounds (Fig. 6-9).

000

ｈｾ＠

oeOOH

o

JvO H,ro

ｾ＠

", ｟ＭｾＧ＠

0

Ｉｾ＠

OH 0 Dihydrofusarubin

.,/

,

OHO./

______________

OOH · . . An hy drOJ3vamcm

••

..' 1'1'1'1'

ＺＧｾ＠

·····'W H,CO

OHO '" Fusaru b " mOle aCI' d " ..........

OHO. Anhydrofusarubm

H'COW ,/ \, COW co'¢YY OHO

;:,....

I I

, •••••

/

\,

, ..

0

..,

0

OH

OH 0 Norjavanicin

H,.

OH V

;:,... OH

0

H

J

I I 0

lavanicin

OHO

0

'7

;:,. .

OH

I

CH,OH

I

OHO

OH

I I

V

;:,. .

0

OH

0

Fusarubin

0

0 Novarubin

Fig. 6-9. A proposed biosynthetic scheme for fusarubin and related naphthoquinones. (Adapted from Pari sot et aI., 1990).

The effect of dihydrofusarubin and isomarticin on the cytology of leaves of rough lemon (Citrus jambhiri) seedlings has been investigated (Achor et aI., dihydrofusarubin alone or in 1993). At a concentration of 100 ｾｧＯｭｌＬ＠ combination with isomarticin (80:20), causes vein necrosis, collapse of spongy mesophyll cells and phloem, depletion of starch, swelling of chloroplasts and disruption of cellular organization. At the ultrastructural level, the phytotoxins disrupt outer membranes of chloroplasts, disorganize granal stack, and vesiculate interstromallamellae (Achor et aI., 1993). Naphthoquinones have marked inhibitory effect on the pollen germination of Pinus thunbergii (Kimura et aI., 1988).

Anthraquinones Cereospora betieo/a, the causal agent of sugar beet leaf spot, produces a series of phytotoxic compounds, including the red cercosporin (see next section, p.363) and the yellow Cereospora betieola toxins. Chemical structures of the

362 - Plant Pathogenesis and Resistance yellow compounds have been determined by three research groups. lalal et al. (1992) named two yellow phytotoxins cebetin A and cebetin B. Cebetin A is a conjugate of octaketide-derived xanthone and anthraquinone containing, respectively, a chlorine atom and an epoxide. Cebetin B exists as a dimeric magnesium chelate with a molecular formula of(C3IH210,3Cl'2CH30H)2Mg2' Both cebetins A and B at a concentration of I JlglmL are lethal to sugar beet cells in suspension cultures in the presence of light. Milat et al. (1992) characterized structures of two yellow phytotoxins from C. beticola and named beticolins 1 and 2. The structure of beticolin 1 has basic features similar to that of cebetin A (Ducrot et aI., 1994b). Several minor beticolins have also been characterized (Ducrot et aI., 1994a). Arnone et al. (1993) named the yellow phytotoxin as Cercospora beticola toxin (CBT). CBT has a molecular formula of C62H42CI2Mg2026 and a structure similar to that of cebetin B. The minor difference in these structures indicates thatthe production ofC. beticolatoxins may be strain- or culture condition-dependent. Little is known about the biosynthesis ofCBT. Results from incorporation of [1-'3C]acetate and [1 ,2- 13 C]acetate indicate a possible route as shown in Fig. 6-10 (Arnone et aI., 1993).

• CH;COON. ____

ｾｯ｟Ｍﾩ＠

0000

000

00

®

OH

OHO

0

Anthraquinone

OH

....

Ｎｾ＠

····r··

OH

J

Xanthone

,

OH

...

dimerizalion ---------

Chlorinated anthraquinone-xanthone conjugate

Cercospora beticola toxin

Fig. 6-10. Biosynthetic pathway for Cercospora beticola toxin. (Adapted from Arnone et aI., 1993).

Perylenequinones Perylene is an aromatic hydrocarbon with the following skeleton:

Phytotoxins - 363 o

o Perylene

3, 1O-Perylenequinone

4,9-Dihydroxy-3,10 perylenequinone

Many perylenes exist as 3, I O-perylenequinone and 4,9-dihydroxy-3, 10perylenequinones, Cercosporin (+)-Cercosporin is a non-host-specific toxin first isolated from Cercospora kikuchii, the pathogen responsible for purple speck disease of soybean (Yamazaki and Ogawa, 1972). It has subsequently been isolated from C. apii, C. canescens, C. nicotianae, C. oryzae. C. personata. C. ricinella. and C. zinniae. The toxin has a molecular formula ofC29H2601O and a molecular weight of534. Its absolute configurations of the asymmetric carbons at C-14 and C-17 and the axial chirality have been established as Rand R, respectively. (+)Isocercosporin [4,9-dihydroxy-2, II-dimethoxy-l, 12-(2-hydroxypropyl)-6, 7methylene dioxy-perylene-3, 1O-quinone] is a diastereomer of (+)-cercosporin isolated from Scolecotrichum graminis, the causal agent of leaf streak disease of orchardgrass (Tabuchi et aI., 1991). It has the asymmetric carbons in Sand the axial chirality in R configuration. (+)-14-Acetylisocercosporin, which has an acetyl group on C-14 of (+)-isocercosporin and a molecular formula of C3 ,H280", has also been isolated from S. gram in is (Tabuchi et aI., 1994b). At a concentration of20 llg/mL, (+ )-isocercosporin has higher toxicity than either (+ )-cercosporin or (+)-14-acetylisocercosporin on lettuce root growth (Tabuchi et aI., 1994b). Feeding experiments indicate that acetate are incorporated efficiently into cercosporin. Labeled carbons from [1-'3C]- and [2-I3C]acetate were found to be arranged alternatively, indicating that the molecular skeleton is formed by condensation of acetate units. These results indicate that one acetate and six malonates are polymerized via the polyketide route, followed by decarboxylation, hydroxylation, and O-methylation. Finally, the methylene dioxybridge is formed between two identical units to form cercosporin. The methoxyl and methylenedioxy groups apparently come from formate (Fig. 6-11) (Okubo et aI., 1975). Four rice cultivars differing in resistance to Cercospora oryzae were tested for sensitivity to purified cercosporin. The most susceptible rice cultivar 'Labelle' has proven to be the most sensitive to the phytotoxin and the most resistant cultivar 'Red Rice' is able to grow in the presence of cercosporin at

364 - Plant Pathogenesis and Resistance concentrations that are completely toxic to 'Labelle'. 'Red Rice' cells contain about one-tenth as much cercosporin as cells of 'Labelle', indicating that resistance cells have a mechanism for excluding, exporting, modifying, or detoxifying the phytotoxin (Batchvarova et aI., 1992). Lipid peroxidation caused by cercosporin has been demonstrated in vitro (Cavallini et aI., 1979) and in vivo (Daub, 1982b). The generation of singlet oxygen and superoxide by cercosporin has been demonstrated by Daub and Hangarter (1983). In an electron spin resonance analysis of tobacco cell membranes treated with cercosporin, a marked increase in the ratio of saturated to unsaturated fatty acids, an increase in the membrane phase transition temperature, and hence, a decrease in membrane fluidity were noted, all leading to electrolyte leakage and cell death (Daub and Briggs, 1983). Quenchers of singlet oxygen delay cell death caused by cercosporin (Daub, 1982a), and the superoxide generated by cercosporin in vitro is scavenged by superoxide dismutase (Daub and Han garter, 1983). Thus, the effect of cercosporin on membrane lipids is mediated by singlet oxygen and superoxide. OH

OH

OH

0

15

I'

OH

OH

\\\........ (+)-Cercosporin, 011 R ｾ＠ 0H (+)-14-Acetylcercosporin. R ｾ＠ COCH Acetate

CHrCO-S-CoA Acetyl-CoA

ｾ

110

OH

I

J

\

ｾｃｯｬ＠

0

u

t

Ir co,

:

ｾｏ＠

coo! tllrCO-S-COA ________ MaIonyl-CoA

ｾ＠

OH

0

0

0

COOIl

0

0 (+)-Isocercosporin. R ｾ＠ H (+)-14-Acetylisocercosporin, R ｾ＠ COCH

J

Fig. 6-11. Biosynthesis of cercosporin. (Adapted from Okubo et aI., 1975; Tabuchi et aI., 1991, 1994b).

The fungi that produce cercosporin are resistant to the phytotoxin. Examination of redox potential at the cell surface reveals that resistant fungi are

Phytotoxins - 365 able to reduce more tetrazolium dyes than are sensitive fungi. Addition of reducing agents ascorbate, cysteine, and reduced glutathione at concentrations that do not react with cercosporin to growth media decrease cercosporin toxicity for sensitive fungi. Thus, cercosporin resistance results from the production of reducing power at the surfaces of resistant fungal cells, leading to reduction and detoxification of the cercosporin molecule (Sollod et aI., 1992). The biology of cercosporin has been reviewed recently by Daub and Ehrenshaft (2000).

Stemphyltoxins and altertoxins Stemphylium botryosum var. lactucum is a leaf spot pathogen of lettuce. This pathogen produces several reduced perylenequinone phytotoxins, stemphyltoxins I to IV, and stemphyperylenol (Fig. 6-12).

Stemphyltoxin I

Stemphyltoxin II (Alterto.in II)

Stemphyltoxin III

Stemphyltoxin IV

011

Stemphyperylenol

Altertoxin I

(Dih droaller r lenol

Alterlo.in III

Alterperylenol (Allejehin)

Fig. 6-12. Structures of toxic perylenequinones. (Adapted from Hradil et aI., 1989; Robeson et aI., 1984).

Stemphyltoxin II [( II R, 12R, 12aS, 12bR)-4,9, 12b-trihydroxy-3, 1O-dioxo1,2,3,10,11,12,12a, 12b-octahydro-ll, 12-epoxyperylene] has a molecular formula ofC2oHI406 and a molecular weight of350. Similar to stemphyltoxin II, stemphyltoxin I has a hydroxyl group at C-l and a molecular formula of C2oH1407, and stemphyltoxin III is dehydrogenated at C-J and C-2 and has a

366 - Plant Pathogenesis and Resistance molecular formula of C 20 H 1206· Stemphyltoxin IV is an epoxidized (at C-l and C-2) form of stemphyltoxin III with a molecular formula of C2o H I2 0 7 • Stemphyperylenol [(1 R,6bR,7R, 12bR)-1 ,4,7,1 O-tetrahydroxy-3,9-dioxo-l,2,3, 6b,7,8,9,12b-octahydroperylene] has a molecular formula ofC2oHI606 and a molecularweightof352. Biosynthesisofstemphyltoxinsand stemphyperylenol probably occurs via a head-to-head and head-to-tail coupling of two pentaketide-derived moieties, respectively (Arnone et aI., 1986). Several Alternaria species also produce perylenequinone toxins. A. cassiae produces stemphyltoxin II and stemphyperylenol. In addition, this pathogen also produces stemphyltoxin-related compounds, altertoxin I and alterperylenol (Hradil et aI., 1989). Alteichin, a phytotoxin produced by A. eichorniae, a pathogen of water hyacinth, has the same structure as alterperylenol (Robeson et aI., 1984). Dihydroalterperylenol produced by A. alternata (Stack et aI., 1986; Stinson et aI., 1982) has the same structure as altertoxin I (Okuno et aI., 1983). Structures of these perylenequinone toxins are given in Fig. 6-12. Stemphyltoxins II and III and altertoxin III give a positive mutagenicity test in the Ames Salmonella typhimurium assay and thus are suspected carcinogens (Davis and Stack, 1991; Stack et aI., 1986).

6.2.4 Heterocycles as Phytotoxins Heterocycles are compounds containing a ring that is made up of carbon and at least one other kind of atom. The most common heterocyclic rings found in phytotoxins are those with one oxygen and those with one nitrogen atom. 6.2.4.1 Heterocycles containing oxygen atom Pyrans and pyrones are six-membered heterocyclic compounds in which one carbon atom in the benzene rings is substituted with oxygen. Pyrans have two hydrogen atoms at C-2 or C-4 position. Microbial pyrans and their derivatives have been reviewed (Dickinson, 1993). Pyrones have a ketone at C-2 or C-4 position and are named (X- and y-pyrones, respectively. Furans are fivemembered rings with one oxygen atom and furanones are those furans with one ketone: 0

H2

OH, 0 0

Pyran-2-H

0

Pyran-4-H

6

Cl

y-pyrone

a-Pyrone

0

o

0

0 0

Furan

d

0

0

Furanone

Phytotoxins

- 367

A. Phytotoxins containing a-pyrone ring Pestalopyrone and convolvulopyrone Pestalotiopsis oenotherae, a pathogen of evening primrose, produces an apyrone phytotoxin pestalopyrone. Chemically, it is 6-( J'-methylprop-l'-enyl)-4methoxy-2-pyrone with a molecular formula of C IO H I2 0 3 and a molecular weight of 180:

Pestalopyrone

Pestalopyrone causes necrosis on leaves of evening primrose, prickly sida (Sida spinosa), Johnsongrass (Sorghum halepense), morning glory (Ipomoea sp.), lambsquarter (Chenopodium album), and Agrostis alba. The pathogen has the potential to be used as a bioherbicide (Venkatasubbaiah et aI., 1991). Convo Ivu lopyrone, 3-( 4-methoxy-3-methyl-a-pyrone-6-yl)-2-methyl-2butenoic acid, is produced by Phomopsis convolvulus, a causal agent of anthracnose of field bindweed (Convolvulus arvensis). It has a molecular formula ofClIHI 2 0 s with a molecular weight of225:

:' -0X

HOOC

ｾ＠

o

0

Convolvulopyrone

Convolvulopyrone has weak herbicidal activity against field bindweed (Tsantrizos et aI., 1992).

Phomapyrones Phomapyrones are metabolites of a weakly virulent isolate of Phoma lingam, the causal agent of the blackleg disease of crucifers:

ｾ Phomapyrone A

o

I

Ｚ＠

o

0

Phomapyrone B

ｾＺ＠

I 0

0

Phomapyrone C

368 - Plant Pathogenesis and Resistance These metabolites differ from epipolythiodioxopiperazine sirodesmins, which are produced by the virulent isolates of the pathogen (see Section 6.2.1.2.B). The phytotoxicity of these compounds has not been established. The crude culture extracts from which phomapyrones were isolated, however, were phytotoxic to Brassica species (Pedras et aI., 1994).

Solanapyrones and alternaric acid Alternaria solani, the causal organism of early blight disease of tomato and potato, produces two types of a-pyrone-containing toxins: solanapyrones and alternaric acid. Solanapyrone A has a molecular formula of C ls H 220 4. Its apyrone ring is substituted with a methoxyl and an aldehyde group at C-13 and C-14, respectively. Administration of sodium [1- 13 C]acetate enhances the resonances for C-2, C-4, C-6, C-8, C-I 0, C-II, C-13, and C-1S of solanapyrone A. Similarly, the signals of aldehyde and methoxy carbons are enhanced by [SI3CH 3]methionine. Thus, the building blocks of solanapyrone A are acetate and methionine, and the synthesis may involve the intramolecular Diels-Alder reaction (Oikawa et aI., 1989; Oikawa et aI., 1994): CHO

CHO

CHO

2(',

S CH , COOH --------

H

H

H

Solan.pyrone A

Alternaric acid has a molecular formula ofC21H300s. Feeding experiments using [1-14C]_ and [2- 14 C]acetate have demonstrated the incorporation of nine acetate units into the molecule. Alternaric acid is synthesized from two polyketide chains (one is made of C I4 and C4 units and the other C I2 and C 6 units) rather than one single chain (Tabuchi et aI., 1994a). A possible biosynthetic pathway for alternaric acid proposed by Tabuchi is given in Fig. 6-\3.

ACRL-toxins Certain strains of Alternaria citri cause brown spot disease of rough lemon (Citrusjambhiri) and Rangpur lime (C.Umonia). These strains produce ACRLtoxins. For 67 Citrus species, cultivars, and hybrids tested, there is a correlation between susceptibility to the pathogen and sensitivity to ACRL-toxins (Kohmoto et aI., 1991). ACRL-toxin I has the molecular formula ofC19H3006. between susceptibility to the pathogen and sensitivity to ACRL-toxins

Phytotoxins 9 CI1COOH

;.... o

0

0

0

0

......

Acetate

0

ｾｓＭＦｊｚＫ＠

o

-

Q

..................

0

0

0

0

0

0

C12 and G. Polykelides

｛ｾｬ＠

- '-"-' -.1

[

OH Proalternaric acid I

,

ｾ＠

'"

0

. ........,

;..........

(10£)-10, l1-Dideoxy-1 0, 11".. 10,1 I-Dideoxy-6.19-dihydroalternaric acid \... ',' dehydro-6, 19-dihydroalternaric acid

ｏｈ＠

Enz-S

t

ＮＭｾ＠

ｾ

- 369

/'

1O-Deoxy-6, 19-dihydroalternaric acid

,.

0

7OH/'

------. COOH Alternaric acid

Fig. 6-13. Proposed biosynthetic routes to alternaric acid. (Adapted from Tabuchi et a!., 1994a).

Structurally, it is 6-(2,4,8-trihydroxy-3, 7,9-trimethyl-undeca-5,9-dienyl)-4hydroxy-5,6-dihydro-2-pyrone (Gardner et aI., 1985; Kohmoto et aI., 1985). Five other toxic analogs have also been characterized (Kono et aI., 1985a): OH

o ACRL·toxin

The first structural change induced by ACRL toxin and detected by electron microscopy was in the mitochondria of susceptible rough lemon. At a concentration of 1 I1g/mL, the phytotoxin causes swelling of the mitochondria, reduction in numbers of cristae, and reduction in electron density of the matrix. Nearly all mitochondria are affected 6 hr after phytotoxin treatment. These observations indicate that the initial site of phytotoxin action is in the

370 - Plant Pathogenesis and Resistance mitochondria (Kohmoto et aI., 1984). Further studies on isolated mitochondria confirm that at a concentration of 1 ｾｧＯｭｌＬ＠ the ACRL toxin induces a significant increase in O 2 uptake with exogenous NADH as the substrate. Toxin-induced respiration in mitochondria is not inhibited by salicylhydroxamate, an inhibitor of cyanide-resistant respiration, but is inhibited by antimycin A and NaN 3 , inhibitors of mitochondrial electron transport chain. These results indicate that ACRL toxin does not stimulate cyanide-resistant respiration, that exogenous NADH is oxidized by a NADH dehydrogenase, and that electrons are transferred via cytochromes to oxygen. Oligomycin, an inhibitor ofH+-ATPase in the mitochondrial membrane, decreases O2 uptake in NADH oxidation, but the effect is canceled by ACRL toxin, indicating that the toxin-induced increase in oxygen consumption is an act of uncoupling action. The increase in O2 consumption caused by ACRL resembled that caused by uncouplers, such as 2,4-dinitrophenol or carbonylcyanide-m-chlorophenylhydrazone. ACRL toxin has no effect on mitochondria isolated from resistant hosts, such as 'Dancy' tangerine, 'Emperor' mandarin, and grapefruit (Akimitsu et aI., 1989).

Neovasinin Neocosmospora vasinfecta is a pathogen of root- and fruit-rot and seedling damping-off of pepper, peanut, soybean, bean, and coconut. Isolate NHL2298 produces several a-pyrone-containing metabolites in cultural media. They are neovasipyrones A and Band neovasinin. Neovasipyrones are derived from a hexaketide chain and five C 1 units from methionine and are the precursors of neovasinin (Fig. 6-14) (Furumoto et aI., 1995; Nakajima et aI., 1992b). o

000000

ＶｃｈＬｏﾷｾ＠

•••••• -

ｯｾ＠

A

000

,

!

mcth)'lmion

i OH

Neovasifuranones

ｾ

o

OH

I

ｯ＠

0

dchydrntion. cyclbJllion

9'

- ..... .

HO

Neovasinin

Neovasi

rone

Fig. 6-14. A proposed biosynthetic pathway ofneovasinin and related compounds. (Adapted from Furumoto et aI., 1995).

Phytotoxins

- 371

Neovasinin is highly toxic to soybean, one of the host plants of N vasinfecta. It induces chlorosis at 1 /!g/plant and necrosis at 2 /!g/plant. Neovasipyrones are not toxic to soybean (Furumoto et aI., 1995).

Isocoumarins and coumarins Isocoumarins are derivatives of benzopyrone. The benzene ring and the attached 3-carbon pyrone ring arise as a unit via cinnamic acid of the general phenylpropanoid pathway. Septoria nodorum, a pathogen of wheat, produces a series of toxins belonging to the isocoumarin family. They are mellein (Devys et aI., 1974); methylmellein; and 4-, 5- and 7-hydroxymellein (Devys et aI., 1980, 1992, 1994). Botryosphaeria obtusa, a pathogen causing frogeye leaf spot on apple, also produces mellein and 4- and 5-hydroxymellein (Venkatasubbaiah and Chilton, 1990). Mellein has also been isolated from culture media of Phoma tracheiphila, the fungus causing mal secco disease of citrus. It is a non-host-specific toxin. At the concentration of 100 /!g/mL, mellein induces wilt in tomato cuttings (Parisi et aI., 1993): OH

0

Mellein

6-Hydroxymellein has been isolated from culture filtrates of Aspergillus terreus, Gilmaniella humicola, and Pyricularia oryzae. It is produced also by Tubakia dryina, the pathogen causing red oak (Quercus rubra) leaf spot, and a Discula sp. that causes anthracnose on dogwood (Venkatasubbaiah and Chilton, 1991, 1992b). Monocerin was first isolated as a metabolic product of Exserohilum monoceras (Aldrige and Turner, 1970). It was subsequently isolated from E. turcicum (=Drechslera turcica), a pathogen of maize and Johnsongrass (Sorghum halepense) (Cuq et aI., 1993; Robeson and Strobel, 1982), and from Fusarium larvarum, an entomophagous fungus (Claydon et aI., 1979).

ｈｃｏｾ＠ ' , : 7 H,CO

0

I

0

:::::,.. /CH, CH, 'CHJ Monocerin

ｈＧｃｏｾ＠

.

ｾＨＩ＠

OH

,:7

0

0 HO ..•• H

De-()-methyldiaporthin

Monocerin is an isocoumarin with a molecular formula ofC'6H2006 and a molecular weight of 308 (Cuq et aI., 1993). It has antifungal activity against

372 - Plant Pathogenesis and Resistance

Erysiphe graminis (Grove and Pople, 1979), insecticidal activity against blowfly (Calliphora erythrocephala) (Claydon et aI., 1979), and phytotoxic activity against Johnson grass (Robeson and Strobel, 1982). Drechslera siccans is a pathogen of perenn ial ryegrass (Lotium perenne) and oat (Avena sativa). It produces isocoumarin de-O-methyldiaporthin (Hallock et aI., 1988). Phytophthora infestans produces coumarin and umbelliferone in a synthetic minimal medium. Feeding experiments confirm that P. infestans is able to incorporate L-phenylalanine and p-coumaric acid into coumarin and umbelliferone (Austin and Clarke, 1966). The biological function of these coumarins is not known. B. Phytotoxins containing y-Pyrone ring Cochliobolus spicifer, a pathogen ofleafspot disease in wheat, produces three metabolites containing a y-pyrone ring: spiciferones A, B, and C. Spiciferone A, a major metabolite, is phytotoxic. It produces blotchy spots on cotyledons of wheat. At a concentration of 100 Ilg/mL, spiciferone A inhibits 70% of the germination of lettuce seeds (Nakajima et aI., 1989, 1991 a). Spiciferone C is weakly toxic, while spiciferone B is not toxic to wheat cotyledons. Bioassays of spiciferones and their derivatives indicate that a methyl group at C-2 and a ketone at C-7 are essential to the phytotoxicity (Nakajima et aI., 1993a). Feeding experiments indicate that spiciferone A is derived from a hexaketide and two Cl units (Nakajima et aI., 1993b): 6 CH,COONa

ｾ＠

ｾ＠

000000

---S-mz

ｾｏ＠

0

OH

0

ｾｏ＠

'8.2, is synthesized in large quantities (10 to 12% of total cellular protein) by suspension-cultured tobacco 'Wisconsin 38' grown in media under osmotic stress (25 g NaCIIL). Osmotin is a TL protein because it resembles thaumatin in molecular weight, amino acid composition, and the presence of a signal peptide on the precursor protein. Fifty-five percent amino acids in the known osmotin sequences are identical to that ofthaumatin (Singh et aL, 1987). Zeamatin, a seed protein consisting of206 amino acids with aM,. of 22,077 from maize, is also a TL protein. It has a 52% sequence homology with that of thaumatin II (Richardson et ai, 1987). Proteins similar to zeamatin have been subsequently detected in the grains of other plants, including barley, flax, oats, sorghum, and wheat (Hejgaard et aL, 1991; Vigers et aL, 1991). A polypeptide consisting of202 amino acids with a calculated M,. of21 ,461 has been isolated from soybean (Glycine max) leaves. The protein has an pI of 4.6. It has 64% amino acid identity with thaumatin and 71 % with zeamatin (Graham et aL, 1992). A 29-kD protein is the most abundant soluble protein in ripe cherry fruit (Prunus avium). Antibodies to the protein have been used to screen a cDNA library from ripe cherry. A clone encoding a polypeptide of 245 amino acid residues subsequently has been isolated. The predicted mature protein has a sequence similar to thaumatin (Fils-Lycaon et aL, 1996). A cDNA clone encoding a thaumatin-like protein has been isolated from a flower meristem cDNA library of A rabidopsis thaliana. The deduced amino acid sequence consists of243 amino acids with a calculated molecular mass of 25,948 and an pI of9.6 (Hu and Reddy, 1995) Spraying the leaves of young potato plants with salicylate induces the appearance of eight proteins. One of them has a M,. of 21,000 and is identified as a TL protein by amino acid sequence (Pierpoint et aL, 1990). The first indication that certain PR proteins are homologous to thaumatin came from the study ofPROB 12, a cDNA clone ofa TMV-infected 'Samsun NN' tobacco (Cornelissen et aL, 1986a). Sequencing of the cDNA reveals an ORF for a protein of226 amino acids with a predicted M,. of21 ,596. The amino acid sequence has 65% homology to that of thaumatin II. The Ala-Ala sequence, which represents the site of cleavage of the signal peptide, is

Pathogenesis-Related Proteins - 637 conserved in the tobacco protein. The tobacco protein, however, lacks the Cterminal extension of the thaumatin precursor. Because of its homology to thaumatin, this PR protein is considered to be a TL protein. Similar TL proteins have been isolated from TMV- or TNV-infected tobacco (Stintzi et aI., 1991; Woloshuk et aI., 1991; Pierpoint et aI., 1992). Several PR proteins have subsequently been identified as TL proteins (Table 10-4) ( Pierpoint et aI., 1987; Cusack and Pierpoint, 1988; Rodrigo et aI., 1991; Stintzi et al., 1991). When 'Rutgers' tomato is infected with the citrus exocortis viroid, a 23-kD PR protein (P23) is induced. The N-terminal sequence of this protein is homologous to the salt-induced tomato osmotin NP24, indicating the thaumatin-Iike nature ofP23 (Rodrigo et aI., 1991). P23 protein accumulates in vacuoles and has antifungal activity. At 50 f.lg/mL, purified P23 protein inhibits 80% of the growth of Trichothecium roseum, 75% of F. oxysporum f. sp. lycopersici, and 60% of Phytophthora citrophthora (Rodrigo et aI., 1993). cDNA clones for P23 have been isolated and Southern analysis shows that at least two genes can encode P23 or P23-related proteins. Sequence analysis has revealed significant differences in both coding and downstream untranslated regions between the cDNA sequences for P23 and NP24 (Rodrigo et aI., 1993). Infection of potato (Solanum commersonii) cell cultures with Phytophthora infestans activates the expression of three osmotin-like protein genes (Zhu et aI., 1995). Barley (Hordeum vulgare) cv. Alva challenged with an incompatible race of mildew (Erysiphe graminis f. sp. horde i) produces a PR protein with a M, of 19,000 and an pI of3.4. Amino acid sequence analysis indicates that this PR protein is a TL protein (Bryngelsson and Green, 1989). Inoculation of wheat with a nonhost pathogen Erysiphe graminis f. sp. hordei (barley powdery mildew) results in activation of several defense genes, including one with an ORF for a polypeptide of 173 amino acids and a calculated M, of 17,605. The PR protein is considered to be a TL protein since its amino acid sequence has 40-50% homology with those of thaumatin II, osmotin, and zeamatin (Rebmann et aI., 1991a). Four cDNA clones encoding TL-proteins have been isolated from oat (Avena sativa) infected by an incompatible isolate of the oat stem rust fungus (Pucciniagraminis f. sp. avenae). Each cDNA clone contains an ORF predicted to encode a 169-amino-acid peptide and a signal peptide of29 amino acids at the N-terminus. The amino acid sequences reveal >80% identity among the four cDNA clones. High levels of these gene transcripts accumulate 42 to 48 hr after inoculation with the incompatible isolate of the oat stem rust pathogen or an isolate of wheat stem rust (P. graminis f.sp. trifici). Plants infected with a compatible isolate accumulate relatively lower amounts of the transcripts (Lin et aI., 1996).

638 - Plant Pathogenesis and Resistance Table 10-4. Thaumatin-Iike proteins as pathogenesis-related proteins in plant-pathogen interactions. Plant

Eliciting pathogen

!If,

Barley

Incompatible race of Erysiphe graminis f. sp. hordei Puccinia graminis f. sp. avenae isolate Pga-IH

19,000 3.4

Bryngelsson and Green (1989)

15,400 15,400 15,300 15,600 26,700 26,700 27.000 15,700

Lin et al. (1996)

Oat (Avena sativa)

Potato (Solanum commersonii)

Phytophthora infestans

Rice (Oryza sativa)

Pseudomonas syringae pv. syringae TMY 24,000

Tobacco (Nicotiana tabacum) cv. SamsunNN Tobacco hybrid (N. debneyi x N. glutinosa) Tomato leaves Wheat (Triticum aestivum) leaves

pI

6.2 6.2 6.2 4.1 6.7 8.0 5.7 4.8

Localization Reference

Zhu et al. (1995)

Reimmann and Dudler (1993) Cornelissen et al. (1986a)

TNY

21,000 6.5 Extracellular Pierpoint et al. (1992) 5.7

Citrus exocortis viroid Erysiphe graminis f. sp. hordei

23,000 17,600

Rodrigo et al. (1991) Rebmann et al. (199Ia)

Genes encoding pathogen-induced thaumatin-like protein have also been cloned from rice and sequenced (Reimmann and Duller, 1993)

10.2.5 Protease Inhibitors as PR Proteins During plant growth and development, the protein turnover rate is high since certain proteins needed at one stage of life cycle might have to be eliminated at another by proteolysis (Vierstra, 1996). The process is catalyzed by proteases and is regulated by a number of factors, including protease inhibitors. Protease inhibitors are small polypeptides with molecular masses in the range of5 to 25 kD. In addition to their roles in regulating proteolytic activities, they are important in protecting tissues from degradation by foreign proteases. Several protease inhibitors have been found in plants. Some inhibit serine proteinase while others inhibit cysteine proteinase (Ryan, 1990). In tomato, two classes of protease inhibitors have been reported. Protease inhibitor I consists of small proteins having a M, of about 8,000 and a reactive site directed toward chymotrypsin. Protease inhibitor II have a molecular mass of about 12 kD and two reactive sites specific to trypsin and chymotrypsin. Protease inhibitor I is synthesized as a pre-proprotein of III amino acids

Pathogenesis-Related Proteins - 639 containing an N-terminal signal peptide of 23 residues followed by a highly charged region of 19 amino acids and a mature protein of69 residues (Graham et aI., 1985a). The mature protease inhibitor II has 123 residues after removal of a signal peptide of25 amino acids (Graham et aI., 1985b). Upon inoculation of two inbred tomato lines differing in susceptibility to Pseudomonas syringae pv. tomato, proteinase inhibitor II mRNA was found to accumulate more rapidly in the resistant than in the susceptible tomato line (Pautot et aI., 1991). In 'Russet Burbank' potato, the proteinase inhibitor I gene encodes a preproprotein of 107 amino acids, including a pre-sequence of23 amino acids and a pro-sequence of 13 amino acids. There is a 77% identity between the mature proteinase inhibitor I in potato and tomato (Cleveland et aI., 1987). The potato proteinase inhibitor II ORF encodes a peptide of 154 amino acids, including a signal peptide of31 amino acids. The mature protein has an 80% identity with tomato proteinase inhibitor II (Sanchez-Serrano et aI., 1986). In 'Samsun NN' tobacco, a proteinase inhibitor is found in TMV-induced hypersensitive tissues. This inhibitor is highly active against four different serine endoproteinases of fungal and bacterial origin but poorly active against trypsin and chymotrypsin of animal origin. The inhibitor has a AI. of 6,000. Its amino acid composition and N-terminal sequence is highly homologous to a potato inhibitor I protein (Geoffroy et aI., 1990). Suspension-cultured tobacco 'Wisconsin 38' cells treated with an elicitor prepared from Phytophthora parasitica var. nicotianae produce a proteinase inhibitor. The inhibitor has a AI. of 10,500 and is specific to trypsin but not chymotrypsin (Rickauer et aI., 1989). A cDNA clone, bsil, has been isolated from barley (Hordeum vulgare) coleoptiles challenged with an incompatible Septoria nodorum (= Stagonospora nodorum). [t encodes a cysteine-rich polypeptide of 89 amino acids with a relative molecular mass of 9405, including a putative N-terminal secretory signal sequence of22 amino acids. The exported mature protein has 67 amino acids with a AI. of 7153. BsiI shares 41 % identity and 49% similarity with a Bowman-Birk-type proteinase inhibitor from maize (Stevens et aI., 1996). Inhibitors of cysteine proteinases, known as cystatins, have also been isolated from various plant species, including rice (Abe et aI., 1987), corn (Abe et aI., 1996), and avocado (Kimura et aI., 1995). Biosynthesis of cystatins has been shown to be developmentally regulated (Abe et aI., 1987). Consequently, they are not considered to be PR proteins. When transgenic tomato plants express rice cystatins, the size of females of Meloidogyne incognita and Globodera pallida and the frequency with which they parasitize are reduced compared to the normal plants (Urwin et aI., 1995; Atkinson et aI., 1996).

640 - Plant Pathogenesis and Resistance

10.2.6 Endoproteinases as PR Proteins Tomato leaves infected by the citrus exocortis viroid produce several PR proteins (Granell et aI., 1987). One of these proteins, P69, is an alkaline endoproteinase. It has a M, of 69,000, an optimal pH of 8.5-9.0, and an pI of 9.0. P69 selectively degrades the large subunit of Rubisco. This finding is in agreement with histopathological observations that degeneration of chloroplasts is the common feature of tissues infected by viroids (Vera and Conejera, 1988). P69 is located in vacuoles and in intercellular spaces (Vera et aI., 1989). Whether the PR protein has other roles in infected tissues remains to be investigated. Tomato plants sprayed with ethephon (an ethylene-releasing compound) or salicylic acid also accumulate P69. It is possible that viroid infection stimulates ethylene production which in turn mediates P69 production (Vera and Conejera, 1989).

10.2.7 Peroxidases as PR Proteins A cDNA encoding a peroxidase (EC l.l1.1. 7) has been cloned from wheat (Triticum aestivum) infected with Erysiphe graminis. The predicted protein consists of 312 amino acids of which the first 22 form a putative signal sequence. The protein has a calculated pI of 5.7 and shares 57% sequence identity with the turnip peroxidase (Rebmann et aI., 1991 b). Infiltration of rice seedlings with the nonhost pathogen Pseudomonas syringae pv. syringae induces systemic resistance to Pyricularia oryzae (Smith and M6traux, 1991). A clone containing an ORF encoding a polypeptide of317 amino acids with the first 25 amino acids as a signal sequence has been isolated from the rice eDNA library. Sequence comparison reveals that the rice polypeptide has 62% homology with the turnip peroxidase and 76% with the pathogen-induced wheat peroxidase (Reimmann et aI., 1992). Peroxidases catalyze a number of reactions that fortify plant cell walls (Kolattukudy et aI., 1992). These reactions include the incorporation of phenolics into cell walls and lignification and suberization of plant cell walls.

10.2.8 Ribonuclease-Like PR Proteins An intracellular PR protein with an estimated M, of 17,500 and pI of 4.7 has been detected in cell cultures of parsley (Petroselinum crispum) treated with an elicitor from Phytophthora megasperma f. sp. glycinea (Somssich et aI., 1986). cDNA encoding the PR protein subsequently has been cloned. The ORF of 465 nucleotides encodes a polypeptide of 155 amino acids with a calculated Mr of 16,515 (Somssich et aI., 1988). This PR protein has 70% amino acid sequence identity with an IS-kD ribonuclease isolated from a callus cell culture of ginseng (Panax ginseng), indicating that the parsley PR protein is a ribonuclease-like protein (Moiseyev et aI., 1994, 1997).

Pathogenesis-Related Proteins - 641 The betv1 gene fam iIy of birch (Betula verrucosa) encodes a group of 17-kD polypeptides, including constitutively expressed pollen allergens and pathogenactivated PR proteins (Breiteneder et aI., 1989; Swoboda et aI., 1994). The Bet v 1 PR proteins have 70 to 88% amino acid identity to the pollen allergens and are homologous to ginseng ribonuclease, indicating the PR proteins are ribonuclease-like. Enzyme assays confirm that Bet v 1 proteins possess nuclease activity specific for RNA but not single- or double-stranded DNAs (Bufe et aI., 1996; Swoboda et aI., 1996). The promoter of an apple Ypr 10 gene encoding the major allergen Mal d 1 protein has sequence homology with the genes encoding PR protein 10, suggesting Mal d 1 is a pathogenesis-related protein (Piihringer et aI., 2000). In potato, cDNA encoding proteins STH-2 and STH-21 have been cloned. Both STH-2 and STH-21 mRNAs are found to accumulate in potato tubers and stems upon wounding or treatment of a mycelial homogenate of Phytophthora infestans. Nucleotide sequence analysis reveals that both clones contain an ORF coding for a peptide of 155 amino acids. These proteins have high similarity with the sequences from the parsley and Betvl proteins (Matton and Brisson, 1989; Matton et aI., 1990). Other PR proteins having amino acid sequences similar to ginseng ribonuclease are PR-p 16.5 from Lupinus albus (Pinto and Richardo, 1995), DRRG49c from pea (Chiang and Hadwiger, 1990), and PvPRl from bean (Walter et aI., 1990)(Table 10-5). Table 10-5. Ribonuclease-like proteins as pathogenesis-related proteins in plant-pathogen interactions. Plant Bean (Phaseo/us vulgaris) Lupinus a/bus

Eliciting pathogen M,

Colletotrichum 16,500 Iindemuthianum Colletotrichum 16,500 g/oeosporioides Parsley (Petroselinum Elicitor from 17,500 crispum) Phytophthora megasperma f. sp. g/ycinea Pea (Pisum sativum) Fusarium solani 16,800 CV. Alaska Potato (So/anum Mycelia of 17,000 tuberosum) cv. Phytophthora Kennebec inJestans Tobacco transformed TMV, TEV, 18,000 with apple YprlO Botrytis cinerea gene Sorghum (Sorghum Cochliobolus bie%r) cv. DKI8 heterostrophus

pi

Localization Reference

Walter et al. (1990) 5.2- Extracellular Pinto and Richardo (1995) 5.6 4.7 Intracellular Somssich et al. (1988)

4.4 6.1 Intracellular

Chiang and Hadwiger (1990) Matton and Brisson (1989) Piihringer et al. (2000) Lo et al. (1999)

642 - Plant Pathogenesis and Resistance 10.2.9 Thionins and Plant Defensins as PR Proteins Thionins are low-molecular-weight proteins (M. ｾ＠ 5,000) occurring in seeds, stems, roots, and leaves of certain mono- and dicotyledonous plants. They have 45 to 54 amino acids and six to eight of them are cysteine residues. They may be divided into two major groups based on their structure: thionins and ythionins. Thionins have cysteine residues conserved at positions 3, 4, 16,27,33, and 41 and a tyrosine residue at position 13 (with few exceptions) (Florack and Stiekema, 1994). Sequence analysis of cDNAs indicates that these proteins are synthesized as precursor proteins with three distinct domains: an N-terminal signal peptide, the mature thionin, and a C-terminal acidic domain (Ponz et aI., 1986, Rasmussen and Rasmussen, 1993). y-Thionins have eight cysteine residues conserved at positions 3, 15,21,25,37,46,48, and 52 and a glycine residue at position 13 (Broekaert et aI., 1995). For a review of their distribution, biosynthesis, and biological function, readers are referred to Bohlmann and Apel (1991), Bohlmann (1994), Florack and Stiekema (1994), Broekaert et al. (1995, 1997), and Garda-Olmedo et al. (1998). The fact that biosynthesis ofthionins can be triggered by pathogens and that thionins exhibit toxicity for plant pathogens indicates that thionins are PR proteins (Table 10-6). Transcriptional activation of thionin genes by plant pathogens has been demonstrated in several host-parasite interactions. Leaf thionin mRNA increases upon infection of barley with spores of Erysiphe graminis f. sp. hordei (Bohlmann et aI., 1988). Infection of barley coleoptiles with Septoria nodorum also results in a rapid transcriptional activation of leaf thionin genes (Titarenko et aI., 1993). Not all thionin genes are pathogeninducible. Between the two thionin genes Thi2.1 and Thi2.2 in Arabidopsis thaliana seedlings, no potent elicitors have been found for the Thi2.2 gene. The expression of the Thi2.1 gene is highly inducible in seedlings by a compatible pathogen Fusarium oxysporum f. sp. matthiolae (Epple et aI., 1995, 1997). Somethionins possess antimicrobial activity. For example, thionins from wheat and barley exhibit toxicity toward a number of plant pathogenic bacteria and fungi (Fermandez de Caleya et aI., 1972; Bohlmann et aI., 1988). Expression of the gene encoding thionin from barley endosperm, under the CaMV35S promoter, confers to transgenic tobacco enhanced resistance to Pseudomonas syringae pv. tobaci (Carmona et aI., 1993). y-Thionins were isolated first from seeds of wheat and barley (Colilla et aI., 1990; Mendez et aI., 1990) and subsequently from seeds of Aesculus hippocastanum (Hippocastanaceae), Clitoria ternatea (Fabaceae), Dahlia merckii (Asteraceae), Heuchera sanguinea (Saxifragaceae), Brassica napus, B. rapa, Sinapis alba, and Arabidopsis thaliana (Brassicaeae) (Terras et aI., 1993; Bohlmann, 1994; Broekaert et aI., 1995; Osborn et aI., 1995). They were also isolated from Helianthus annuus (Asteraceae) inflorescence (Urdangarin et aI., 2000). Due to their structural and functional similarity to those of insect and

Pathogenesis-Related Proteins - 643 mammalian defensins, these y-thionins are also named plant defensins (Terras et aI., 1995). Table 10-6. Thionins as pathogenesis-related proteins in plant-pathogen interactions. Localization Reference

Plant

Eliciting pathogen

M,

pi

Barley (Horde un vulgare) leaves Barley coleoptiles

Erysiphe graminis f. sp. hordei Septoria nodorum

5,000

Arabidopsis thaliana seedlings

Fusarium oxysporum f.sp. matthiolae

5,000

Extracellular Bohlman et al. (1988) Titarenko et al. (1993) Epple et al. (1995, 12.4 1997)

Most of plant defensins are constitutively expressed. Some are induced in response to biotic stress (Table 10-7). Two genes, pI39 and pI230, are induced in pea pods upon inoculation with either a compatible (Fusarium solani f. sp. pisi) or an incompatible (F. solani f. sp. phaseoli) pathogen (Chiang and Hadwiger, 1991). They encode proteins with predicted M.. of 8,200 and 8,000, respectively. Both proteins contain a signal peptide and are cleaved to mature proteins of about 5 kD. The mature proteins contain about 17% cysteine residues and share similarities with amino acid sequences ofthionins. Table 10-7. Plant defensins as pathogenesis-related proteins in plant-pathogen interactions. Plant

Eliciting pathogen

Arabidopsis thaliana

Alternaria 5,000 brassicicola Fusarium solani 5,000 f. sp. pisi and phaseoli 5,000 Alternaria brassicicola, Botrytis cinerea

Pea (Pisum sativum) cv. Alaska Radish (Raphanus sativus) leaves

M,

Localization

Reference Penninckx et al. (1996) Chiang and Hadwiger (1991 )

Extracellular

Terras et al. (1995)

In seeds of radish (Raphanus sativus), the defensins Rs-AFPl and Rs-AFP2 are located in the cell wall and released during seed germination. Both Rs-AFPs have antifungal activities against a broad spectrum of filamentous fungi. The N-terminal regions of the proteins show homology with the amino acid sequences of the pea y-thionins (Terras et aI., 1992a). Both the cDNAs and the genomic regions encoding the Rs-AFP precursor proteins have been cloned. Transcripts hybridizing with an Rs-AFPl cDNA-derived probe are present in near-mature and mature seeds. They are barely detectable in healthy, uninfected leaves but accumulate systemically at high levels after localized infection by

644 - Plant Pathogenesis and Resistance Alternaria brassicicola. The induced leaf proteins Rs-AFP3 and Rs-AFP4 have been purified and shown to be homologous to the seed proteins Rs-AFPl and Rs-AFP2. A chimeric Rs-AFP2 gene under the control of the CaMV 35S promoter enhance resistance to the foliar pathogen A. longipes in transgenic tobacco (Terras et aI., 1995).

10.2.10 Lipid-Transfer Proteins as PR Proteins Lipid-transfer proteins (LTPs) are basic, 9-kD proteins with the ability to bind and transfer a variety of lipids between membranes in vitro. They are located extracellularly in a variety of tissues from mono- and dicotyledonous plants. Several roles of LTPs in vivo have been proposed: participation in cutin formation, defense reactions against plant pathogens, symbiosis, and the adaptation of plants to various environmental conditions (Arondel and Kader, 1990; Kader, 1996, Arondel et aI., 2000). Several lipid-transfer proteins have antibiotic activity against bacterial and fungal pathogens. The sugar beet LTPs exhibit a strong in vitro antifungal activity against Cercospora beticola (Kristensen et aI., 2000) (Table 10-8). Biosynthesis ofLTP can be elicited by biotic stresses. For example, the barley LTP mRNA levels were significantly increased in young etiolated leaves when they were inoculated with Erysiphe graminis (Molina et al. 1993). Table 10-8. Lipid-transfer proteins as pathogenesis-related proteins in plant-pathogen interactions. Plant

Eliciting pathogen

Barley leaves

Erysiphe graminis, Rhynchosporium secalis Xanthomonas campestris pv. vesicatoria

Pepper leaves

Radish seeds Sugar beet leaves

M,

Localization

Reference Garcia-Olmedo et at. (1995) Jung and Hwang (2000)

9,000 9,300

Terras et at. (1992b) Extracellular Kristensen et at. (2000)

10.2.11 a-Amylases as PR Proteins Activity of a-amylase (EC 3.2.1.1) is greatly enhanced in leaves of tobacco infected with TMV. Two a-amylases have been isolated and purified from TMV-infected tobacco leaves. They have a molecular mass of 44 kD and an pI of 4.5. Both amylases display some features of tobacco PR proteins: an apoplastic localization, an acidic isoelectric point, high level of resistance to protease action, and a low level of expression in healthy leaves (Heitz et aI., 1991). The role of a-amylases in TMV-infected tissues is not known.

Pathogenesis-Related Proteins - 645

10.2.12 PR Proteins with Unknown Biological Function PR-1 proteins were among the first described PR proteins in TMV-infected tobacco plants. The biological function of this protein family remains elusive. Genetic analysis has demonstrated that tobacco PR-1 a, 1b, and 1c isoforms are encoded by separated genes. The nucleotide sequences of the genes encoding PR-1 proteins obtained from both 'Samsun NN' and 'Xanthi-nc' have >90% homology. The ORFs of the PR-1 genes each encode a polypeptide sequence of 168 amino acid in length with a hydrophobic domain of30 amino acids at the N-terminus. This domain corresponds to a signal polypeptide sequence which is required in protein sorting but is cleaved from the proprotein during the formation of the mature PR-1 polypeptide of 138 amino acids (Cornelissen et aI., 1986b). PR-1 s are isolated from intercellular fluids of infected leaf tissues and culture media of elicited suspension-cultured cells or protoplasts, indicating they are secreted out of cells. In situ hybridization analysis, however, indicates that crystal idioblasts, specialized cells in tobacco, express the PR-1 gene at the mRNA level. Transgenic plants that constitutively express a chimeric gene encoding an acidic PR-1 b isoform also accumulate PR-1 protein in the extracellular spaces and within crystal idioblast vacuoles. The accumulation of PR-1 protein in vacuoles of crystal idioblasts indicates that these cells sort proteins in a unique manner (Dixon et aI., 1991). Although PR-1 isoforms are acidic, a genomic clone encoding the basic PR-1 protein has been isolated and characterized (Payne et aI., 1989). Upstream sequences ofthe gene encoding the PR-l a protein have been fused to reporter genes. The constructs have been used to transform tobacco tissues. A sequence of 689 bp or longer, but not shorter than 643 bp, induces the expression of the reporter gene in TMV-inoculated or SA-treated leaves, indicating the TMV- and SA-responsive elements are located between -643 and -689 in the PR-l a promoter (Van de Rhee et aI., 1990). The cis-acting elements for regulating gene expression ofthe tobacco PR-1 a protein gene have been analyzed in transgenic tobacco. The 5'-flanking 2.4-kb fragment from the PR-1 a protein gene was joined to the bacterial GUS gene and introduced into tobacco cells by Agrobacterium-mediated gene transfer. Promoter activity was monitored by quantitative and histochemical assay ofPglucuronidase activity in leaves of regenerated transgenic plants. The level of p-glucuronidase activity was clearly increased by treatment with salicylic acid, wounding, or TMV infection. Cytochemical studies of the induced pglucuronidase activity revealed tissue-specific and developmentally regulated expression of the PR-l a gene after stress, chemical treatment, or pathogenic attack. A series of 5'-deleted chimeric genes was constructed and transformed into tobacco plants. Transgenic plants with a O.3-kb fragment of the 5'-flanking region of the PR-1 a gene had the same qualitative response as those with the 2.4 kb fragment upon treatment with SA or TMV infection. Thus, the O.3-kb DNA

646 - Plant Pathogenesis and Resistance fragment was sufficient to allow the regulated expression of the PR I a gene (Ohshima et aI., 1990). Proteins homologous to tobacco PR-l proteins have been isolated from various plant tissues, including several host-parasite interactions. A polypeptide serologically related to the tobacco PR-l proteins has been isolated from the root tissues of maize (Zea mays). The protein has 140 amino acid, a M. of 14,970, an pI of 4.2, and 66-68% amino acid identity with the tobacco PR-l protein and 55% identity with the tomato P14 protein (Gillikin et aI., 1991). A clone containing an ORF encoding a protein homologous to tomato P14 and tobacco PR-l proteins has been isolated from a cDNA library prepared from maize seeds two days after germ ination. Sequence analysis ofthe corresponding genomic clone revealed that it was encoded by a single copy gene in maize. The accumulation of its mRNA increases after infection by a maize pathogen Fusarium moniliforme (Casacuberta et aI., 1991). A barley cDNA clone (PRb-l) corresponding to an mRNA differentially induced in resistant and susceptible barley cultivars by powdery mildew infection has been isolated and characterized. The deduced amino acid sequence revealed a24-amino acid signal and 140-aminoacid ｰ･ｴｩ､ＨｾＱＵ＠ kD). There is a close homology to PR-l-Iike proteins of maize, tobacco, tomato, and Arabidopsis thaliana. Increased expression of the PRb-1 gene was observed in resistant but not near-isogenic susceptible barley plants following Erysiphe gram in is inoculation or treatment with ethylene, salicylic acid, methyl jasmonate, or 2,6-dichloroisonicotinic acid (Mouradov et al., 1993). MtPR-l is a cDNA clone isolated from a cDNA library constructed from roots of Medicago truncatula grown in aeroponic conditions and inoculated with Rhizobium meliloti. It is 890-bp long, containing a 5' untranslated region of 27 bp, an ORF of 519 bp, and a 3' untranslated region of 344 bp. The ORF encodes a deduced precursor protein of 173 amino acid residues (molecular mass = 19.8 kD), consisting of a mature protein of 139 amino acid residues (molecular mass = 15.8 kD and pI 6.5) and a putative signal sequence of 34 amino acid residues. Sequence analysis shows 58 and 57% identity with maize and barley PR-l proteins, respectively (Szybiak-Strozycka et aI., 1995). Three basic 14-kD proteins (PI4a, P14b, and P14c) have been isolated from tomato (Lycopersicon esculentum cv. Baby) leaves infected with Phytophthora infestans. Serology and amino acid sequence comparisons show that the three proteins are members of the PR-l proteins. They exhibit antifungal activity against P. infestans. The biological function of these proteins is not known (Niderman et aI., 1995). Tobacco PR-4 is another group of PR proteins with unknown biological function. Inoculation of leaves of'Samsun NN' tobacco with TMV induces the formation of an intracellular, 20-kD antifungal protein. The protein causes lysis of the germ tubes of Trichoderma viride and Fusarium solani. Analysis of the

Pathogenesis-Related Proteins - 647 protein and the corresponding cDNA reveals that the protein contains a Nterminal chitin-binding domain that is present in the class I chitinases and the putative wound-induced proteins (WINI and WIN2) of potato. It also contains a C-terminal domain with high identity with potato WINI and WIN2. The protein is synthesized as a pre-proprotein and is processed into the mature protein by the removal of the N-terminal signal peptide and C-terminal sequence (Ponstein et aI., 1994). In addition to tobacco PR-l and PR-4 proteins, there are several PR proteins with unknown biological functions. For example, a gene encoding a PR protein is rapidly activated in leaves of 'Datura' potato inoculated with certain races of Phytophthora inJestans or treated with a fungal elicitor. The gene occurs in multiple copies and encodes a polypeptide with aM.of25,054 and an pI of5.5. The coding sequence of the gene and the deduced amino acid are similar to the corresponding sequences of a 26-kD heat shock protein from soybean. The biological function of the PR protein has not been determined (Taylor et aI., 1990).

10.3 BIOSYNTHESIS OF PR PROTEINS 10.3.1 Elicitation ofPR Protein Accumulation by Elicitors 10.3.1.1 Biotic elicitors PR proteins have been detected in more than 20 host-parasite interactions. In addition to plant-pathogenic viruses and fungi, bacteria, nematodes, viroids, and insects also elicit PR protein biosynthesis (Bronner et aI., 1991; ConradsStrauch et aI., 1990; Hammond-Kosack etal., 1989; Vera and Conejera, 1988). Besides leaves, roots (Benhamou et aI., 1991 a; Tahiri-Alaoui et aI., 1990, 1993), calli (Antoniw et aI., 1981), Agrobacterium tumeJaciens-transformed tissues (Antoniw et aI., 1983), protoplasts (Grosset et aI., 1990; Jouanneau et aI., 1991), and suspension-cultured cells (Suty et aI., 1995) also synthesize PR proteins under certain biotic stresses. A. Exogenous elicitors Components of fungal cell walls and certain fungal metabolites are potent elicitors ofPR protein biosynthesis (Table 10-9). A glucan preparation obtained from mycelial walls of Phytophthora megasperma f. sp. glycinea elicits phytoalexin biosynthesis in soybean (Ayers et aI., 1976) and a number of other plants (Kombrinkand Hahlbrock, 1986; Zhu and Lamb, 1991). This preparation also elicits PR protein accumulation in cultured parsley cells (Petroselinum crispum) (Kombrink and Hahlbrock, 1986) and cultured peanut cells (Arachis hypogaea) (Herget et aI., 1990) but not in tobacco (Kopp et aI., 1989). These results indicate that the elicitor has certain degrees of host specificity.

648 - Plant Pathogenesis and Resistance Table 10-9. Biotic elicitors ofPR-protein induction. Elicitor

Plant

PR proteins elicited

Reference

ChitoRice (Oryza sativajaponica) oligosaccharide cv. Nakdongbyeo Cryptogein Tobacco cells Ethylene Arabidopsis thaliana

Chitinases (I)

Kim et al. (1994)

PR-bl Chitinase (I)

Sunflower (Helianthus annuus) endo-PG, endo-PL Tobacco leaves Oxalic acid Sunflower (Helianthus annuus) Garlic (Allium sativum) Salicylic acid

PR-I,2,3,5

Suty et al. (1995) Verburg and Huynh, (1991); Jung et al. (1995)

p-I,3-Glucanase PR-I,2,3,5

Paiva et al. (1993) Jung et al. (1995)

Chitinases

Van Damme et al. (1993); Van Huijsduijnen et al. (1986b); Pinto and Richardo (1995) Lotan and Fluhr (1990)

･ｮ､ｯＭｾｉ＠

,4-

Xylanase

Tobacco 'Samsun NN'

PR-I

Lupinus a/bus

PR-IO

Tobacco leaves

p-I,3-Glucanase

Elicitin is a class of 10-kD proteins secreted by various Phytophthora spp. It induces localized necrosis and phytoalexin production in tobacco (see Chapter 7). In addition to phytoalexins, certain elicitins, such as cactorein (produced by P. cactorum), also elicit PR protein production (Dubery et aI., 1994). A number of exoenzymes produced by plant pathogens are elicitors of PR protein biosynthesis. ｅｮ､ｯＭｾｉ＠ ,4-xylanase purified from culture filtrates of Trichoderma viride, and endo-PG and endo-PL prepared from Erwinia carotovora subsp. carotovora are potent elicitors ofPR protein ｾＭｉ＠ ,3-glucanase synthesis in tobacco leaves (Lotan and Fluhr, 1990; PaIva et aI., 1993). Oxalic acid, a toxin produced by Sclerotinia sclerotiorum, is an efficient inducer ofPR-1 ,2,3, and 5 proteins in sunflower (Helianthus annuus) (Jung et aI., 1995). Cholera toxin is produced by the gram-negative bacterium Vibrio cholerae. The toxin has a molecular weight of 84,000 and is composed of one A subunit (M.=27,000) and five B subunits (M.=11,600). The A subunit is synthesized as a single polypeptide but is usually proteolytically nicked into two fragments, Al (M.=22,000) and A2 (M,.=5,000). The Al can activate the signalling pathway dependent on heterotrimeric GTP-binding proteins (Mekalanos et aI., 1983). Tobacco plants have been transformed with a chimeric gene encoding the Al subunit of cholera toxin regulated by a light-inducible wheat Cab-1 promoter. These transgenic plants show reduced susceptibility to Pseudomonas

Pathogenesis-Related Proteins - 649

tabaci, accumulate high levels of salicylic acid, and constitutively express PR protein genes encoding PR-I and the class II isoforms of PR-2 and PR-3. Microinjection of tobacco plants with cholera toxin also induces the expression of a PR-I-GUS reporter gene. These results indicate that cholera toxin-sensitive G-proteins are involved in the expression of a subset ofPR genes (Beffa et aI., 1995). B. Endogenous elicitors Salicylic acid (SA) is a phenolic compound found in plants (see Fig. 7-2 in Chapter 7). It has diverse biological functions in higher plants, including induction of PR proteins and disease resistance (Raskin 1992a,b; Klessig and Malamy, 1994). Endogenous levels of SA in 'Xanthi-nc', but not 'Xanthi', tobacco increase at least 20-fold in TMV-infected leaves. The increase coincides with the synthesis of PR proteins in leaves of 'Xanthi-nc' tobacco. Exogenous SA also elicits PR protein accumulation and resistance (Malamy et aI., 1990). For example, spraying tobacco plants with SA induces the synthesis ofPR- I proteins and resistance to alfalfa mosaic virus (Van Huijsduijnen et aI., 1986). Salicylic acid also induces chitinases in leaves and bulbs of garlic (Allium sativum)(Van Damme et aI., 1993). Leaf discs of sunflower (Helianthus annuus) floated on a solution containing aspirin (acetylsalicylic acid) produce PR-l, 2, 3, and 5 proteins in the intercellular spaces (Jung et aI., 1993). Seedlings treated with elicitors of Colletotrichum lagenarium have chitinase activity 2 to 10 times higher than in control tissues after 24 hr. This elicitation is correlated with increase in ethylene levels in treated tissues (Roby et aI., 1986). Arabidopsis thaliana sprayed with a 1 mg/mL solution of 2chloro-ethylphosphonic acid, an ethylene generator, accumulates chitinase (Verburg and Huynh, 1991). Negative results, however, have been reported. For example, spraying wheat seedlings with the ethylene generator at the concentration of 30 mM has no effect on P-l ,3-glucanase biosynthesis (Sock et aI., 1990).

10.3.1.2 Abiotic elicitors Abiotic agents such as UV light (Green and Fluhr, 1995), air pollutants (Schraudner et aI., 1992), heavy metals (Asselin et aI., 1985), and chemicals such as polyacrylic acid (Antoniw and White, 1980; Gianinazzi, 1984) and 2,6dichloroisonicotinic acid (Kragh et aI., 1991; Kogel et aI., 1994; Nielsen et aI., 1994a; Conrath et aI., 1995) are potent elicitors of PR protein biosynthesis (Table 10-10). UV-B (280 to 320 nm) induces PR-l accumulation in irradiated area of tobacco leaves. The mechanism of induction is not fully understood. Treatment ofleaves with antioxidant (N-acetyl-L-cysteine or pyrrolidine dithiocarbamate) blocks the induction ofPR- I synthesis by UV -B. Similarly, leaves treated with

650 - Plant Pathogenesis and Resistance cycloheximide fail to accumulate PR-I protein by UV-B induction. The inhibition ofPR-l induction by antioxidants and the protein synthesis inhibitor indicates that PR-I protein is synthesized de novo and is mediated by active oxygen species. The possible involvement of active oxygen species in PR-I induction is further supported by the observations that leaves treated with rose bengal, a photodynamic inducer that forms singlet oxygen upon irradiation with light, accumulate PR-I protein. This induction of PR-I accumulation is inhibited by antioxidant N-acetyl-L-cysteine (Green and Fluhr, 1995). Table 10-10. Abiotic and chemical elicitors ofPR-protein induction. Elicitor

Plant

Darkness Tobacco 2,6-DichloroArabidopsis thaliana isonicotinic acid Tobacco

PR proteins elicited

Reference

Basic PR-1 PR-I, ＬｾＭＱ＠ ,3-glucanase, thaumatin-like protein PR-1

Sessa et al. (1995) Uknes et al. (1992)

Barley ｾＭｉ＠ ,3-Glucanase Sugar beet (Beta vulgaris) Chitinase (IV) Epoxiconazole Ozone

UV-B irradiation UV -C irradiation

Wheat (Triticum aestivum) P-I,3-glucanase, CV. Star chitinase Tobacco ｾＭｉ＠ ,3-Glucanase (I), chitinase (I) Tobacco (Nicotiana PR-I tabacum) cv. Samsun NN Tobacco (Nicotiana PR-1 tabacum) cv. Xanthi-nc

Conrath et al. ( 1995) Kragh et al. (1991) Nielsen et al. (1994a) Siefert et al. (1996) Ernst et al. (1992), Thalmair et al. (1996) Green and Fluhr (1995) Yalpani et al. (1994)

Treatment of leaves with a single pulse of 0 3 (0.15 ｾｌｬＬ＠ 5 hr) elevates P1,3-glucanase and chitinase activities in 03-sensitive and 03-tolerant tobacco cultivars. In the sensitive cultivar Bel W3, the activity of P-I ,3-glucanase increases 40- to 75-fold and chitinase increases 4-fold within 5 to 10 hr after exposure. In the tolerant cultivar Bel B, the activities of P-I ,3-glucanase and chitinase increase 30- and 3-fold, respectively (Schraudner et ai., 1992). In tobacco, accumulation of a basic PR-I protein transcript is induced by dark treatment. The gene encoding the protein has been cloned and sequenced. It contains an ORF for 179 amino acids. The transcript is also induced by physiological levels of ethylene, but such an induction takes place only in the light (Eyal et aI., 1992). Several chemicals have been shown to elicit PR protein synthesis:

Pathogenesis-Related Proteins - 651

ｾ＠ CIANJla Polyacrylic acid

2,6-Dichloroisonicotinic acid

Probenazole (3-Allyloxy-1 ,2-benzisothiazole-l, I-dioxide

Polyacrylic acid, an elicitor of interferon synthesis in animals, elicits the accumulation of PR proteins in 'Xanthi-nc' tobacco as well as resistance to TMV and TNV, and partial resistance to potato viruses X and Y and Pseudomonas syringae (Antoniw and White, 1980; Ahl et aI., 1985; Dumas et aI., 1987). Probenazole (3-allyloxy-l ,2-benzisothiazole-1 , I-dioxide) induces resistance in rice against rice blast fungus. A cDNA clone has been isolated from a probenazole-treated rice plant that encodes the protein PBZl (Midoh and Iwata, 1996). Sequence analysis reveals a significant homology at the amino acid level between the predicted PBZI and a group ofPR proteins such as pea pI49 (Fristensky et aI., 1988), Betv I (Breiteneder et aI., 1989), and PcPR I-I (Somssich et aI., 1988). 2,6-Dichloroisonicotinic acid (INA) and its methyl derivative induce both local and systemic resistance in a number of plants. These compounds have no antifungal activity compared to fungicides but induce the accumulation of various PR proteins (Metraux et aI., 1991; Wasternack et aI., 1994; Van Kan et aI., 1995). Several heavy metals such as silver, cadmium, manganese, and barium exhibit some degrees of PR protein elicitation (White et aI., 1986).

10.3.2 Regulation ofPR Protein Biosynthesis In tobacco, the synthesis of the PR-l proteins in tobacco is regulated by the level ofmRNA, and high levels ofPR mRNAs are accumulated only after the plants are elicited. Thus, transcriptional activation is the primary mechanism for PR protein biosynthesis (Carr et aI., 1985). Several compounds, including ethylene, salicylic acid, and 2,6-dichloroisonicotinic acid, have been implicated as mediators of PR protein accumulation. Leaves or seedlings of melon (Cucumis melo) treated with elicitors from Colletotrichum lagenarium have chitinase activity 2 to 10 times higher than in control tissues 24 hr after treatment. This elicitation is correlated with simultaneous increase in ethylene levels in treated tissues. In the presence ofaminoethoxyvinyl-glycine, an ethylene biosynthesis inhibitor, both elicitorinduced ethylene and elicitor-induced chitinase are inhibited. This inhibition can be overcome by exogenous ethylene. Application of I-aminocyclopropaneI-carboxyl ic acid, the precursor of ethylene, also triggers chitinase activity (Roby et aI., 1986). Similar effects of ethylene on PR protein biosynthesis have been demonstrated in other plants. For example, ethylene treatment (-20 JlLlL

652 - Plant Pathogenesis and Resistance air for 2 days) of'Havana 425' tobacco markedly increases the P-l ,3-glucanase content of leaves (Felix and Meins, 1987). Ethylene treatment (10 nLlmL, 48 hrs) of primary leaves of Phaseolus vulgaris induces chitinase activity (Boller et aI., 1983). Thus, ethylene is involved in the signal transduction pathway leading to the activation of PR genes. A combination ofmethyljasmonate and ethylene synergistically induce PR1 and PR-5 mRNA accumulation in tobacco seedlings. The combination also synergistically activates the PR-5 promoter fused to a GUS reporter gene. The ethylene/methyl jasmonate responsiveness of the PR-5 gene promoter is localized on a fragment that exhibits responsiveness to several other inducers, including salicylic acid and fungal infection. A protein kinase C inhibitor, 1-(5-iso-quinolinylsulfonyl)-2-methylpiperazine, blocks ethylene induction of PR-l mRNA accumulation but not PR-5 mRNA, indicating that these two PR genes appear to have at least partially separate signal transduction pathways (Xuetal.,1994). Several lines of evidence indicate that ethylene is not the only mediator of PR protein biosynthesis. First, spraying wheat seedlings with the ethylene generator chloroethylphosphonic acid has no effect on PR protein biosynthesis (Sock et aI., 1990). Second, the epinastic (Epi) mutant of tomato (Lycopersicon esculentum) cultivar VFN8 overproduces ethylene, resulting in extreme epinasty, swelling of stem and petioles, and a shortened root system. These characteristics are similarto those of tomatoes infected with the citrus exocortis viroid where PR proteins are accumulated (Garcia Breijo et aI., 1990). Comparative studies reveal no enhanced PR protein production in the Epi mutant compared to its parent cultivar VFN8, indicating that ethylene may not be involved in the synthesis of PR proteins (Belles et aI., 1992). Third, both TMV and endo-p-l ,4-xylanase elicit PR protein synthesis in tobacco leaves. The ethylene biosynthesis inhibitors, AVEG and silver thiosulfate, inhibit PR protein accumulation elicited by TMV inoculation but not by xylanase. These results indicate that PR protein accumulation elicited by TMV is mediated by ethylene whereas xylanase induces an ethylene-independent pathway for PR protein synthesis (Lotan and Fluhr, 1990). Thus, a complex regulatory network is involved in the activation of PR proteins. Induction of resistance to TMV in 'Xanthi-nc' tobacco by SA was first reported by White (1979). The induction of resistance is concom itant with the production ofPR proteins. The mechanism by which PR proteins are induced is not clear. A SA-binding protein has been isolated and identified as a catalase. The binding of SA to the catalase inactivates the enzyme, resulting in an increase in intracellular levels of active oxygen species which is known to play a role in induction ofPR protein gene expression (Chen et aI., 1993). 2,6-Dichloroisonicotinic acid (INA) induce systemic acquired resistance in cucumber against Colletotrichum lagenarium and other pathogens (Metraux et

Pathogenesis-Related Proteins - 653 aI., 1991). Treatment of barley leaves with INA induces the accumulation of an unidentified basic protein of 45 kD and a 6-kD thionin (Wasternack et aI., 1994). In tomato, INA induces mRNAs encoding various PR proteins (Van Kan et aI., 1995). In sugar beet (Beta vulgaris), INA induces resistance against Cercospora beticola (Nielsen et aI., 1994a). In this case, transcripts encoding chitinase and P-l,3-glucanase are not found in the INA-treated tissues, indicating that INA-induced resistance is independent of PR protein accumulation. INA also binds to catalase, inhibits the enzyme activity, and induces PR-l gene expression (Conrath et aI., 1995). In contrast to ethylene, auxin and cytokinin inhibit chitinase and P-l,3glucanase biosynthesis in tobacco (Mohnen et aI., 1985; Felix and Meins, 1987; Shinshi et aI., 1987; Grosset et aI., 1990). The chitinase content of cloned tobacco pith tissues subcultured on hormone-free medium increases five-fold to about 8% of the total soluble protein during a 7-day period. This induction is inhibited more than 90% by addition of a combination of auxin and cytokinin to the culture medium (Shinshi et aI., 1987). Tobacco mesophyll protoplasts synthesize P-l ,3-glucanase, chitinases, and TL proteins during in vitro culture. Their syntheses are reduced by the presence of auxin (Grosset et aI., 1990). Similar results are observed in soybean protoplasts (Jouanneau et aI., 1991). Thus, the expression of PR proteins may be under a negative control by auxin.

10.4 ROLES OF PR PROTEINS IN DISEASE RESISTANCE Several lines of evidence indicate that PR proteins are involved in disease resistance. First, PR proteins accumulate rapidly and mainly in tissues that develop necrotic local lesions rather than systemic symptoms, indicating a correlation between PR proteins and the resistance reaction (Daugrois et aI., 1990; Joosten et aI., 1989; Liao et aI., 1994). Secondly, tobacco leaves previously inoculated with TMV that exhibit a HR and are accumulating mRNAs for PR proteins develop smaller and fewer lesions than plants that are not previously infected when challenged with the same virus (Cornelissen et aI., 1986a; Hooft van Huijsduijnen et aI., 1986). Finally, the discovery that PR proteins possess activity of hydrolytic enzymes further indicates the role ofPR proteins in defense of pathogenic infection.

10.4.1 Lysis of Cell Walls ofInvading Pathogens Enzymes with the ability to attack the structure ofmicroorganisms are potential defense arsenals against pathogenic invasion. Many fungal pathogens contain P-l ,3-glucan, chitin, and chitosan as cell wall components. It is conceivable that enzymes hydrolyzing these fungal cell wall components are able to inhibit fungal growth and cause death of mycelia. Increase in activity of these hydro lases in plant tissues has, therefore, been considered an attribute of resistance. For example, inoculation of the first leaves of young cucumber

654 - Plant Pathogenesis and Resistance plants with Pseudoperonospora cubensis. Pseudomonas lachrymans, or TNV induced up to a 100-fold increase in chitinase activity in the un infected second leaves of the plants. The increase in chitinase activity in the second leaves correlates well with an enhanced resistance to challenge infection of Colletotrichum lagenarium (Metraux and Boller, 1986). Several reports have demonstrated that chitinase breaks down fungal cell walls and inhibits mycelial growth. Chitinase purified from Stylosanthes guianensis, a tropical forage legume, is able to cause death of Colletotrichum gloeosporioides hyphae (Brown and Davis, 1992). Purified bean chitinase inhibits growth of Trichoderma viride, a saprophytic fungus, at the concentration of2 ｾｧＯｭｌＮ＠ The effect is attributed to enzymatic attack on newly formed chitin in the growing hypha I tips, disturbing the balance between synthesis and hydrolysis required for tip growth (Schlumbaum et aI., 1986). Wheat germ agglutinin is a lectin that binds specifically to N-acetylglucosamine. The gold-labeled agglutinin has been seen in the vicinity of chitinasetreated Rhizoctonia solani mycelial cells, indicating the release of chitin oligosaccharides from fungal cell walls (Benhamou et aI., 1993). Not all isoforms of chitinases and P-l,3-glucanases are equal in their antifungal activities. The class I (vacuolar) chitinase and P-l,3-glucanase of tobacco (Nicotiana tabacum cv. Samsun NN) are the most active against Fusarium solani germ lings, resulting in lysis of the hyphal tips and growth inhibition. The class II isoforms of these hydrolases exhibit no antifungal activity (Sela-Buurlage et aI., 1993). Inhibition of certain plant-pathogenic fungi, however, requires a combination of chitinase and P-l ,3-glucanase. For example, purified chitinase and P-l ,3-glucanase, tested individually, do not inhibit growth of most fungi tested. Combinations of purified chitinase and P-l,3-glucanase, however, inhibited all 18 fungi tested by Mauch et al. (I 988b). Chitosanases have been evaluated for their lytic activity towards fungal spores impregnated in polyacrylamide gels. Lyses of spores of Fusarium oxysporum f. sp. radicislycopersici, Verticillium albo-atrum, and Ophiostoma ulmi. indicated by clear bands or spots against the opaque spore-containing matrix, have been observed with basic chitosanases of24 kD from stressed tomato and 19 kD from stressed barley (Grenier and Asselin, 1990).

10.4.2 Disruption of Membrane Structure and Function oflnvading Pathogens The biological function of TL proteins has not been fully elucidated. At low concentrations, zeamatin causes the rapid release of cytoplasmic materials from Candida albicans and Neurospora crassa, indicating that the protein permeabilizesthefungal plasma membranes (Roberts and Selitrennikoff, 1990). Linusitin is a 25-kD TL protein isolated from flax seeds (Borgmeyer et aI.,

Pathogenesis-Related Proteins - 655 1992). Using a fluorescent method, the amount ofthe fluorescent dye calcein released from the inside of unilamellar lipid vesicles induced by linusitin has been studied. The results indicate that calcein released is a result of the tetrameric protein aggregation. It is possible that this aggregation perturbs the membrane and forms transmembrane pores (Anzlovar et aI., 1998). Since thaumatin-like PR proteins have amino acids sequences similar to those of zeamatin and linusitin, it is conceivable that TL PR proteins have a similar membrane permeabilizing activity. In fact, the TL protein from TMVinoculated tobacco plants has been shown to lyse sporangial apex of Phytophthora infestans and inhibit hyphal growth of the fungus. The observed lysis indicates that the TL protein has a cell hydrolysis activity. Alternatively, the hydrophobic nature of the TL may interact with the plasma membrane causing membrane disruption (Woloshuk et aI., 1991). Similar observations have been made on TL protein-treated Cercospora beticola (Vigers et aI., 1992). Thionins are rich in cysteine and are positively charged. They may interact with negatively charged membrane phospholipids, causing pore formation on the membrane and disrupting membrane function (Florack and Stiekema, 1994). Plant defensins have a biphasic effect on the membrane permeabilization of Neurospora crassa: a cation-sensitive permeabilization at high defensin levels (10 to 40 11M) and a cation-resistant permeabilization at low defensin levels (0.1 to 111M). The former is resulted from a direct peptide-phospholipid interaction and the latter is via binding-site mediated membrane insertion of plant defensins (Thevissen et aI., 1999).

10.4.3 Fortification of Plant Cell Walls Anionic peroxidase (E 1.11.1.7) is located in the cell wall and catalyzes the polymerization of cinnamyl alcohols into lignin. Tobacco plants transformed with a chimeric tobacco anionic peroxidase gene synthesize high levels of peroxidase in all tissues throughout the plant. The percentage of lignin and lignin-related polymers is nearly twofold greater in cell walls of pith tissue isolated from peroxidase-overproducing plants compared to the control plants. Wound-induced lignification occurred 24 to 48 hrs sooner in plants overexpressing the anionic peroxidase than in the controls (Lagrimini, 1991). 10.4.4 Liberation of Elicitors of Defense Reactions Soybean tissues contain a factor capable of releasing a highly active phytoalexin elicitor from Phytophthora megasperma f. sp. glycinea. The elicitor-releasing factor was later identified as P-I,3-glucanase (Keen and Yoshikawa, 1983) and the elicitors as glucomannans (Yoshikawa et ai, 1981). Soybean leaves treated with mercuric chloride synthesize P-l ,3-glucanase and other PR proteins. When extracts made from the treated leaves are

656 - Plant Pathogenesis and Resistance incubated with Phytophthora megasperma f. sp. glycinea, the amount of carbohydrate solubil ized from the fungal cell walls is correlated with the P-l ,3glucanase activity in the extract. Phytoalexin elicitor-releasing activity increases rapidly in treated plants and shows a similar increase relative to P-l ,3glucanase activity. These results indicate that fungal cell wall oligosaccharide fragments solubilized by a pathogenesis-related protein P-l ,3-glucanase elicit phytoalexin accumulation (Ham et aI., 1991). Erwinia carotovora subsp. carotovora produces several cell-wall-degrading enzymes extracellularly, including endo-PL (Hinton et aI., 1989), endo-PG (Saarilahti et aI., 1990), and PNL (Chatterjee et aI., 1991). These exoenzymes are virulence factors of this pathogen. Recent results indicate that some of these cell-wall-degrading enzymes are elicitors of a host defense reaction. Treatment of tobacco plants with PG and PL prepared from this bacterium elicits rapid accumulation of the transcript for P-l ,3-glucanase. A similar plant response could be elicited by the application of polygalacturonase-treated polypectate. Tobacco seedlings inoculated with weakly virulent mutants of the bacterium that still produce pectic enzymes accumulate P-l ,3-glucanase. No accumulation of P-l,3-glucanase mRNA has been detected in plants inoculated with exoenzyme-negative mutants (PaIva et aI., 1993).

10.4.5 Inactivation ofProteinases Secreted by Plant Pathogens Proteinases have been shown to be virulence factors of certain plant pathogens. Xanthomonas campestris pv. campestris produces two extra-cellular serine proteases. A protease-deficient mutant lacking both enzymes showed considerable loss of virulence in pathogenicity on mature turnip leaves (Dow et aI., 1990). Extracellular proteinases aid the pathogen in nutrient uptake by digesting foods and spreading by destructing structural integrity of host tissues. Inhibition of proteinase activity renders plants resistant to proteolytic attack by these pathogens. For example, proteinase inhibitor activity increases rapidly and remains at a high level in tomato plants following infection with an incompatible but not compatible race of P. infestans (Peng and Black, 1976). Addition of an elicitor obtained from P. parasitica var. nicotianae to tobacco cell suspension culture resulted in accumulation of a proteinase inhibitor. The de novo synthesized proteinase inhibitor has a M, of 10,500 (Rickauer et aI., 1989). Tobacco leaves that respond hypersensitively to TMV produce a proteinase inhibitor highly active against fungal and bacterial endo-proteinases. The inhibitor is a small polypeptide with a M. of 6,000 (Geoffroy et aI., 1990). Proteinase inhibitor activity increased sharply in melon seedlings infected by Colletotrichum lagenarium three days after inoculation. The activity was associated with heat stable proteins and was effective against the protease produced by the fungus as well as against trypsin. Treatment of healthy melon leaves with an elicitor of ethylene isolated from the fungus also resulted in

Pathogenesis-Related Proteins - 657 increase in proteinase inhibitor activity. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis, both ethylene and proteinase inhibitor activity were inhibited (Roby et aI., 1987).

10.4.6 Degradation of Cellular RNAs and Induction of Hypersensitive Cell Death Betv 1 proteins, a group of birch pollen allergens, have been shown to possess ribonuclease activity. They digest different RNA substrates, but not ssDNA or dsDNA, in vitro (Swoboda et aI., 1996). A parsley PR protein homologous to Betv 1 proteins has been shown to accumulate rapidly and massively in leaves inoculated with incompatible Phytophthora megasperma f. sp. glycinea (Somssich et aI., 1988). It is possible that the PR protein may possess ribonuclease activity and degrade cellular RNAs and thus contribute to hypersensitive cell death, blocking the spread of the pathogen to other parts of the infected plant (Swoboda et aI., 1996). 10.5 CONCLUSIONS The identification of some of the PR proteins as p-glucanases, chitinases, or chitosanases indicates that PR proteins may playa protective role against invasion by insects and plant pathogens. Further evidence that PR proteins are involved in disease resistance comes from the reports that plant parts containing PR proteins exhibit an enhanced resistance to subsequent inoculation of fungi, bacteria, or viruses. The contention that accumulation ofPR proteins constitutes a resistance mechanism, however, is not unequivocally supported. The PR protein concentrations have been measured at various times after primary inoculation of'Xanthi-nc' tobacco with TMV on one-half of two lower leaves. At various time intervals, plants were challenge-inoculated on the two upper leaves and on the previously uninoculated halves of the lower leaves. The amount of resistance (measured by reduction in size and number of lesions formed in the challenge inoculation) was measured. The results indicate no quantitative or temporal relationship between amounts of resistance to the challenge inoculation and the accumulation of PR proteins. Furthermore, spraying abscisic acid on plants induces an apparent resistance without accumulating PR proteins. Low doses of methyl benzimidazole-2yl-carbamate causes accumulation of PR proteins but not resistance. Nicotiana glutinosa accumulates large amounts ofa PR protein after primary inoculation ofTMV but becomes more susceptible to a challenge inoculation (Fraser, 1982). These results indicate that PR proteins are not involved in acquired systemic resistance. Leaves of 'White Burley' tobacco form systemic symptoms and necrotic lesions in response to inoculation ofTMV and TNV, respectively. PR proteins accumulate to similar extent in both leaves, indicating a lack of correlation

658 - Plant Pathogenesis and Resistance between symptom expression and PR protein accumulation (Pennazio et aI., 1983). Leaves of N sylvestris with a mosaic from earlier inoculation with a systemic strain ofTMV (TMV -C) and control plants were challenge-inoculated with a necrotizing strain ofTMV-P, RNA ofTMV-P, or turnip mosaic virus (TuMV). TMV-P produced localized necrotic lesions only in the dark green areas of the mosaic leaves. 80th RNA of TMV-P and TuMV produced localized necrotic lesions in both light and dark green areas of the mosaic leaves. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. PR proteins were not found in either light or dark green areas of mosaic leaves resulting from TMV-C inoculation. No quantitative or temporal relationship between the onset of resistance and PR protein concentration was found in light green areas challenge-inoculated with RNA ofTMV-P or TuM V or dark green areas inoculated with TMV-P. These results indicate that PR proteins may not be involved in the mechanisms of viral induced resistance (Sherwood, 1985). Transgenic plants have been used to determine the role of PR proteins in disease resistance (Table 10-11). The results are conflicting. Transgenic tobacco seedlings constitutively expressing a bean chitinase gene under the CaMV 35S promoter have shown an increased ability to survive in soil infested with fungal pathogen Rhizoctonia solani (8roglie et aI., 1991). Expression of the gene encoding a-thionin from barley endosperm conferred the transgenic tobacco enhanced resistance to Pseudomonas syringae pv. tabaci and pv. syringae (Carmona et aI., 1993). On the other hand, transgenic plants that constitutively expressed the PR-l a or PR-l b genes under the transcriptional control of the CaMV 35S promoter did not exhibit increased resistance to TMV or alfalfa mosaic virus (Cutt et aI., 1989; Linhorst et aI., 1989). Overexpression of a basic tobacco chitinase in N sylvestris did not substantially increase resistance to Cercospora nicotianae (Neuhaus et aI., 1991 b). N sylvestris plants transformed with antisense constructions ofthe coding sequence ofthe vacuolar P-l,3-glucanase effectively blocked constitutive and induced expression of class I P-l ,3-glucanase. These plants did not exhibit increased susceptibility to C. nicotianae (Neuhaus et aI., 1992). These results indicate that the expression of a single PR gene may be insufficient to provide protection against pathogenic infection. In light of the synergistic antifungal activity of chitinases and P-l ,3glucanases reported by Mauch et aI. (I 988b), it may be necessary for plants to overexpress several PR proteins to achieve enhanced resistance to fungal pathogens. Alternatively, protection against plant pathogens may require the mobilization of a complex network of different resistance mechanisms.

Pathogenesis-Related Proteins - 659 Table 10-11. Transgenic plants overexpressing pathogenesis-related proteins and their resistance to plant diseases. Plant Potato

PR proteins overexpressed STH-2, a potato PR protein with amino acid sequence similar to ribonuclease Leaf peroxidase (Prx8) of barley

Tobacco (Nicotiana benthamiana) Tobacco Tobacco chitinase I (Nicotiana sylvestris) Tobacco (Nicotiana tabacum) Tobacco (Nicotiana tabacum) cv. SamsunNN Tobacco (Nicotiana tabacum) cv. Xanthi nc Tomato cv. Moneymaker

Rice RCH I 0 chitinase I and alfalfa AGLUI acidic ｾＭＱ＠ ,3-glucanase Barley a-thionin

PR-Ia

Chitinase I or ｾＭｉ＠ ,3-glucanase I Chitinase I and ｾＭｉ＠ ,3-glucanase I

Resistance to diseases

Reference

No change in susceptibility to PVX and Phytophthora infestans No change in resistance to £rysiphe cichoracearum

Constabel et al. (1993)

No correlation between high levels of chitinase expressed and resistance to Cercospora beticola Increase in resistance to Cercospora beticola

Neuhaus et al. (1991b)

Enhances resistance to Pseudomonas syringae pv. tabaci

Carmona et al. (1993)

Increase in tolerance to infection by Peronospora tabacina and Phytophthora parasitica var. nicotianae No increase in resistance to Fusarium oxysporum f. sp. Iycopersici Concurrent expression of both PR proteins enhances resistance to F. oxysporum f. sp. Iycopersici

Alexander et al. (1993)

Kristensen et al. (1997)

Zhu et al. (1994)

Van den Elzen (1993)

Plants have an array of defense mechanisms, preformed or induced, structural or biochemical. Production of PR proteins may be an important defense mechanism in certain host-parasite interactions and may play no role in other host-parasite interactions.

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674 - Plant Pathogenesis and Resistance Vigers AJ, Roberts WK, SelitrennikoffCP (1991) A new family of plant antifungal proteins. Mol Plant-Microbe Interact 4:315-323 Vigers AJ, Wiedemann S, Roberts WK, Legrand M, Selitrennikoff CP, Fritig B (1992) Thaumatin-like pathogenesis-related proteins are antifungal. Plant Sci 83: 155-161 Wadsworth SA, Zikakis JP (1984) Chitinase from soybean seeds: purification and some properties of the enzyme system. J. Agric Food Chern 32:1284-1288 Walter MH, Liu J-W, Grand C, Lamb CJ, Hess D (1990) Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members ofa ubiquitous new class of conserved PR proteins including pollen allergens. Mol Gen Genet 222:353-360 Walter MH, Liu JW, WOnn J, Hess D (1996) Bean ribonuclease-like pathogenesis-related protein genes (YprJO) display complex patterns of developmental, dark-induced and exogenousstimulus-dependent expression. Eur J Biochem 239: 281-293 Wasternack C, Atzorn R, Jarosch B, Kogel KH (1994) Induction of a thionin, the jasmonateinduced 6 kDa protein of barley by 2,6-dichloroisonicotinic acid. J Phytopathol 140:280-284 White RF (1979) Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco. Virology 99:410-412 White RF, Dumas E, Shaw P, Antoniw JF (1986) The chemical induction ofPR (b) proteins and resistance to TMV infection in tobacco. Antiviral Res 6: 177-185 Woloshuk CP, Meulenhoff JS, Sela-Buurlage M, van den Elzen PJM, Cornelissen BJC (1991) Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant Cell 3:619-628 Xu Y, Chang PFL, Liu D, Narasimhan ML, Raghothama KG, Hasegawa PM, Bressan RA (1994) Plant defense genes are synergistically induced by ethylene and methyljasmonate. Plant Cell 6:1077-1085 Yalpani N, Enyedi AJ, Le6n J, Raskin I (1994) Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco. Planta 193:372-376 Yamagami T, Funatsu G (1994) The complete amino acid sequence ofchitinase-a from the seeds of rye (Secale cereal). Biosci Biotech Biochem 58:322-329 Yoshikawa M, Matama M, Masago H (1981) Release of a soluble phytoalexin elicitor from mycelial walls of Phytophthora megasperma var. sojae by soybean tissues. Plant Physiol 67:1032-1035 Zhu B, Chen THH, Li PH (1995) Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato. Plant Mol BioI 28: 17-26 Zhu Q, Lamb CJ (1991) Isolation and characterization of a rice gene encoding a basic chitinase. Mol Gen Genet 226:289-296 Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ (1994) Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Bioffechnology 12:807-812

Index Note: page numbers in italics refer to tables; those in bold refer to figures

A AAL-toxins, 312 as herbicides, 382 sites of action of, 301, 314,314 Aceric acid, 56 ACRL-toxins, 301, 368 ACT-toxins, 354 biosynthesis of, 355 site of action of, 301 Acyl carrier protein (ACP), 7, 191,195 Acyl-ACP desaturase, 9, 11 Acyl-ACP thioesterase, 7, 9, 11 ADP/O ratio, 149 in diseased plants, 165 AF-toxins,354 biosynthesis of, 355 site of action of, 301 Agrobacterium rhizogenes genes on Ri plasm ids, 265 genetic maps of Ri plasm ids, 262 Agrobacterium tumefaciens iaaH, 243, 246 iaaM, 243, 246 in plant genetic engineering, 260 interaction with plant cells, 254 ipt, 243, 246 production of polygalacturonases by biovar 3 of, 80 T-DNA,245 Ti plasmids, 241 vir region. 245 Agropine, 245 AK-toxins, 354 as resistance suppressors, 434 biosynthesis of, 355 site of action of, 301 Albugo candida effect on chlorophyll content, 154; photosynthesis, 151; Rubisco activity, 159 Alternaria alternata genetics of melanin production, 31 production of altertoxins, 366;

cutinase, 15; maculosin, 316; melanin, 33; polygalacturonases, 81; tenuazonic acid, 303 Alternaria alternata f sp. lycopersici production of AAL-toxins, 312; fumonisins, 311 Alternaria brassicae production of destruxins, 334 Alternaria brassicicola elicitation of plant defensin production in Arabidopsis and radish, 643 production of brassicicenes. 294; cutinase, 15 Alternaria cassiae production of alterperylenol, altertoxin, stemphyltoxins, and stemphyperylenol, 366 Alternaria cinerariae production of curvularin, ap-dehydrocurvularin, 358 Alternaria citri production of ACRL-toxins, 368; ACT-toxins, 355; tentoxin, 323; tenuazonic acid, 303 Alternaria fragariae production of AF-toxins. 354 Alternaria kikuchiana production of AK-toxins. 354; tenuazonic acid, 303 Alternaria mali production of AM-toxins, 326; tentoxin, 323; tenuazonic acid, 303 Alternaria solani production of alternaric acid, 368; solanapyrones, 368; tentoxin, 323; zinniol, 356; Alternaria tenuis production oftentoxin, 323; tenuazonic acid. 303 Alternaria zinniae production of zinniol, 356 Alternaric acid, 368 biosynthesis of, 369 Altertoxins, 365

676 -

Plant Pathogenesis and Resistance

AM-toxins, 301, 326 trans-4-Aminoproline, 382 Angelicin, 550, 550 Anhydrofusarubin, 360, 361 Anthranilamides, 574, 575 Anthraquinones, 361 Apigenin, 182, 184, 186 Apigeninidin, 536 Apiose,56 Apium graveolens production of angelicin, bergapten, columbianetin, isopimpinellin, psoralen, xanthotoxin, 550 Arabidopsis thaliana production of camalexin, 572; plant defensins, 643; thionins, 643 Arabinanases, 84 Arabinans,55 Arabinofuranosidase, 84 Arachidonic acid, 426, 581 as an elicitor, 418, 577 Arachis hypogaea production of resveratrol, 546 Arbutin, 339, 433 Ascochitine,359 as a herbicide, 382 Ascochyta fabae production of as co chi tine, 359 Ascochyta hyalospora production of ascochitine, 359; hyalopyrone,372 Ascochyta pisi elicitation of chitinase production in pea, 631 production of ascochitine, 359 Ascochyta rabiei degradation of maackiain and medicarpin by, 541 Aspartate carbamoy Itransferase effect of AAL-toxins on, 313, 314 Aspergillus aculeatus production of arabinanase, 84; rhamnogalacturonanases, 83 Aspergillus niger production of arabinanases and arabinosidase; 84; polygalacturonases,81 Aspergillus oryzae production of pectin methylesterase, 83; 13-1,4-xylanase, 85 Aucuparin,551 Auxins (see IAA)

Avena sativa production of avenanthramides, 574; thaumatin-like proteins, 638 Avenanthramides, 574 Ayapin, 550, 550 Azorhizobium caulinodans host specificity, 177

B Bacterial canker of almond, 271 Bacteroids, 179 Barley stripe mosaic virus effect on chlorophyll synthesis, 156 Bean pot mottle virus effect on nitrogen-fixing activity, 218; nodulation, 215 Bean yellow mosaic virus effect on nitrogen-fixing activity, 218; nodulation, 215 Benzothiadiazole, 459 Bergapten, 550, 550 Beta vulgaris production of betagarin, 535; betavulgarin, 536; 13-1,3glucanases, 628; lipid-transfer proteins, 644 Betagarin, 535 Beticolins, 301, 362 Bipolaris cynodontis production of bipolarox in, 343 Bipolaris sorokiniana production of helminthosporol and prehelminthosporol, 346; sorokinianin and victoxinine, 347; 13-1,4-xylanase, 85 Bipolaris zeicola production of BZR-cotoxins, 335 Bipolaroxin, 343 as a herbicide, 382 Bjerkandera adusta production of MnP, 92 Bjerkandera sp. strain BOS55 production of LiP and MnP, 92 Blumeriellajaapii effect on photosynthesis, 151 Botryosphaeria obtusa production of p-hydroxybenzaldehyde and tyrosol, 357 Botrytis cinerea elicitation of production of plant

Index - 677 defensins, 643; ribonuclease-like protein, 641 production of cutinase, 15; p-I ,3glucanases, 86; pectate Iyases, 74; pectin methylesterases, 83; polygalacturonases,81; rhamnogalacturonanases, 83 Bradyrhizobium elkanii host specificity, 177 Bradyrhizobium japonicum host specificity, 177 production ofrhizobitoxine, 304 Brassica campestris c1ubroots caused by Plasmodiophora brassicae, 275 production of brass in in, cyclobrassinin, and methoxybrassinin, 571; P-I ,3glucanases, 628 Brassica juncea production ofbrassilexin, 571 Brassica oleracea production of methoxybrassinin, 571 Brassilexin, 571,572 Brassinin,571 biosynthesis of, 572 metabolism by Leptosphaeria maculans, 571,593 Brefeldin A, 358 as a resistance suppressor, 378, 434 site of action of, 301 BZR-cotoxins, 335 structures of, 336

c CJC 1 ratio, 167 in diseased plants, J67 Ca2+ as a second messenger, 441 Calvin cycle, 140 Camalexin, 572 biosynthesis of, 573 metabolism by Rhizoctonia solani, 593 site of action of, 588 Camelina sativa production of camalexin and 6methoxycamalexin, 573 Canavalia ensiformis production of maackiain and medicarpin, 538 Capsicein characteristics of, 422

structure of, 423 Capsicum annum production of capsidiol, 560; P-I,3glucanases, 628; lipid-transfer proteins, 644 Capsidiol, 560 biosynthesis and metabolism of, 561 Carotenoids biosynthesis of, 136 in diseased plants, 157 Casbene, 565 biosynthesis of, 567 Casbene synthase, 566, 567 Cebetins, 362 Cell wall chemical components of, 54, 72 functions of, 52 structure of, 52, 72 Cell-wall degrading enzymes arabinanases, 84 arabinosidases, 84 biosynthesis of, 93 cellobiohydrolases, 86, 88 cellulases, 86, 88, 98 correlation with virulence, 104 galactanases, 86 glucanases, 85 P-I,3-glucanases, 86, 86 in disease resistance, 106 in infected tissues, 102 in plant pathogenesis, 101 laccases, 91, 92 lignin peroxidases, 91, 92 manganese-dependent peroxidases, 91,92 pectate Iyases, 72, 74 pectin Iyases, 78, 79 pectin methylesterases, 82, 83 polygalacturonases, 79, 81, 97 proteases, 89, 90 reproduction of disease symptoms with, 103 rhamnogalacturonases, 83 secretion of, 98 xylanases, 84. 85 Cellobiohydrolases, 86, 88 Cellulases, 86, 88 Cellulose biosynthesis of, 67 structure of, 61 Ceratocystis fimbriata f. sp. plalani

678 - Plant Pathogenesis and Resistance production of cerato-platanin, 341 Ceratocystis ulmi production of cerato-ulmin, 341 Cerato-platanin, 341 Cerato-ulmin, 341 Cercidiphyllum japonicum production of mag nolo I, 551 Cercospora beticola elicitation of production of chitinase, 632; P-I,3-glucanases, 628 production ofbeticolins, cebetins, Cercospora beticola toxin (CBT), and cercosporin, 361 Cercospora kikuchii production of cercosporin, 363 Cercospora personata production of cercosporin, 363 Cercosporidium personatum effect on photosynthesis, 150 Cercosporin, 301,363 biosynthesis of, 364 Cerebrosides as elicitors, 427 Ceriporiopsis subvermispora production oflaccase and MnP, 92 Chitin, 420 as an elicitor, 420, 576 Chitinases as PR proteins, 629 classes of, 630 Chitooligosaccharides as elicitors, 648 Chitosan, 420 as an elicitor of phytoalexin production, 576 Chitosanases as PR Proteins, 635 Chlorophylls biosynthesis of, 133, 135 effect of pathogens on, 154 Chloroplasts, 132, 134 Chrysin, 184, 186 Chrysoeriol, 184, 187 Cicer arietinum production of maackiain and medicarpin, 538 Cichoralexin, 563, 563 Cichorium intybus production of cichoralexin, 563 Citrus exocortis viroid elicitation ofthaumatin-Iike protein production in tomato, 638

Cladosporium cucumerinum production of pectate Iyases, 74; polygaIacturonases,81 Cladosporium fulvum effect on respiration, 163 elicitation of production of chitinase, 632; P-I,3-glucanases, 628 Clavibacter michiganensis subsp. michiganesis production of cellulases, 88 Clavibacter michiganensis subsp. sepedonicus production of cellulases, 88 Claviceps purpurea production ofP-I,3-glucanases, 86; P1.4-xylanase, 85; Clubroots of crucifers, 275 Cochliobolus carbonum production of arabinosidase, 84; P-I,3-glucanases, 86; P-I,4glucanases, 88; polygaIacturonases, 81; proteases, 90; P-I,4-xylanase, 84,85 Cochliobolus heterostrophus elicitation of ribonuclease-like protein production in sorghum, 641 production of cutinase, 15; melanin, 32; HMT toxins, 352 genetic of melanin production, 3 1 Cochliobolus miyabeanus production of melanin, 30 genetics of melanin production, 31 Cochliobolus spicijer, 372 Cochlioquinones, 360 biosynthesis of, 360 Coleostephus myconis production of mycosinol, 569 Colletotrichin, 301, 350 Colletotrichum capsici production of colletotrichin, 350; cutinase, 15 Colletotrichum gloeosporioides elicitation of ribonuclease-like protein production, 641 production of cutinase, 15; pectate Iyases, 74; pectin Iyases, 79; polygalacturonases,81 Colletotrichum gloeosporioides f. sp. malvae production of pectate Iyases, pectin lyase and polygalacturonases, 78 Colletotrichum lagenarium

Index - 679 effect on Rubisco activity, 159 elicitation of chitinase production in cucumber, 63/ genetics of melanin production, 31 inhibition of Rubisco synthesis, 158 production ofcutinase, /5; melanin, 30 Colletotrichum lindemuthianum elicitation of ribonuclease-like protein production in bean, 64/ production of melanin, 30; pectin Iyases, 79; polygalacturonases, 8/ Colletotrichum musae production ofpolygalacturonases, 8/ Colletotrichum tabacum production of colletotrichin, 350 Columbianetin, 550, 550 Coniophora puteana production of cellulases, 88 Convolvulanic acid, 373 Convolvulopyrone, 367 Coronafacic acid, 306 structural and functional similarity to methyljasmonate, 310 Coronamic acid, 306 structural and functional similarity to I-aminocyclopropane-I-carboxy lic acid (ACC), 310 Coronatine, 306 as a resistance suppressor, 378 biosynthesis of, 307 effect on photosynthesis, /5/ Cotonefurans, 552 Coumarins, 549 biosynthesis of, 550 Coumestrol, 184, /86, 545 Crown and root galls of Gypsophila, 272 Cryphonectria parasitica production of cellulases, 88; cutinase, /5; polygalacturonases, 8/ Cryptogeins as elicitors ofPR-protein induction, 648 characteristics of, 422 Cucumber mosaic virus effect on chlorophyllase activity, 155; fructose 1,6-bisphosphatase activity, /60; photosynthesis, /5/; respiration, /63; Rubisco activity, /59 elicitation of ｾ＠ -I ,3-glucanase production in pepper, 628 Cucumis sativus

production of chitinases, 63/ Cupressotropolones, 557 Curvularin, 358 Curvulin, 357 as a herbicide, 382 Cuticle biological functions of, 4 chemical composition of, 6 structure of, 5, 6 Cuticular wax as an inducer, 431 Cutin, 6 biosynthesis of monomers, 7, 11, 11, 13 polymerization of monomers, 12, 14 Cutinase biosynthesis of, 15 catalytic properties of, 21 catalytic triad of, 21, 11 correlation with virulence, 25 CUT genes, 18 in penetration of cuticle, 23 induction of biosynthesis in fungal spores, 17 molecular and structural properties of, 20,11 production by plant pathogens, 14, /5 Cyclic AMP as a second messenger, 451 Cyclic photophosphorylation, 138, 139 Cyclobrassinin, 571, 571 Cyclobrassinin sulphoxide, 571, 571 Cyclopaldic acid, 373 Cyperine, 295, 30/ p-Cystathionase effect of rhizobitoxine on, 306 Cytochalasins, 315 Cytokinins, 240 biosynthesis of, 143 in Agrobacterium tumefaciensinoculated tissues, 258 in vitro production by Agrobacterium tumefaciens, 24/; Pseudomonas syringae pv. savastanoi, 268, 268 role in crown gall formation, 260; hairy root formation, 265

D Daidzein, 183,184, /87,536,537 Datura stramonium production oflubimin and rishitin, 562

680 - Plant Pathogenesis and Resistance Daucus carota production of 6-methoxymellein, 570 Debneyol, 560 biosynthesis and metabolism of, 561 ap-Dehydrocurvularin, 358 De-O-Methyldiaporthin, 372 3-Deoxy-o-lyxo-2-heptulosaric acid, 56 Desoxyhemigossypol, 564 biosynthesis of, 565 site of action of, 588 Destruxin B, 301, 334 Dianthalexins,574 Dianthramides, 574 biosynthesis of, 575 Dianthus caryophyllus production of dianthramides and dianthalexins, 574 2,6-Dichloroisonicotinic acid activation of systemic acquired resistance, 458 as an elicitor of PR-protein induction, 650 Dihydrophenanthrene, 548, 549 Dihydrostilbenes, 548, 549 2,7-Dihydroxycadalene, 564, 565 7,4'-Dihydroxytlavan, 535 7,4'-Dihydroxytlavone, 186 Diplodia natalensis production ofpolygalacturonases, 81 Diterpenoids, 565, 567 Drechslera gigantea production of gigantenone, pestasol, phaseolinone, and phomenone, 343 Drechslera indica production of curvulin, O-methylcurvulinic acid, 357 Drechslera oryzae production of ophiobolins, 351 Drechslera siccans production of de-O-methyldiaporthin, 372

E Eicosapentaenoic acid, 418, 426, 581 Elicitins, 342, 421 as elicitors of defense responses, 418 characteristics of, 422 Elicitors, 416 abiotic, 417, 417 biotic, 417, 418,421

Embden-Meyerhofpathway, 144,144 Emenolone, 553 Endo-p-l,4-glucanases, 86, 88 Endo-pectate Iyases as elicitors of phytoalexin production, 576; PR-protein induction, 648 Endo-polygalacturonases as elicitors of phytoalexin production, 576; PR-protein induction, 648 Endoproteinases as PR proteins, 640 Energy-capture process, 132 photosynthesis, 132 in relation to energy utilization, 133 Energy-utilization process, 141 in relation to energy capture, 133 Enniatins, 301, 332 biosynthesis of, 333 Epicuticular wax, 5, 6, 6 Epidermis, 4, 5 Epoxiconazole as an elicitor of PR-protein induction, 650 Eriobofuran, 552 Eriobotrya japonica production of aucuparin, 551; eriobofuran, 552 Eriodictyol, 183,184,186 Erwinia carotovora subsp. atroseptica production ofpectate Iyases, 74. 76; pectin Iyases, 79; polygalacturonases,81 Erwinia carotovora subsp. carotovora production of cellulases, 88; pectate Iyases, 74. 75; pectin Iyases, 79; polygalacturonases, 81; proteases, 90

Erwinia chrysanthemi production of cellulases, 88; pectate lyases,73, 74; pectin Iyases, 79; pectin methylesterase, 83; polygalacturonases, 81; proteases, 90; P-l,4-xylanase, 85 Erwinia herbicola pv. gypsophilae, 272 Erysiphe graminis f. sp. avenae effect on chlorophyll content, 154; photosynthesis, 151 Erysiphe gram in is f. sp. hordei effect on chlorophyll content, 154; fructose 1,6-bisphosphatase activity, 160; photosynthesis, 151;

Index - 681 respiration, 163; Rubisco activity, 159; state 3 and state 4 respiration and ADP/O ratio, 165 elicitation of production of chitinases, 631; thaumatin-like proteins, 638; thionins, 643 Erysiphe graminis f. sp. tritici effect on carotenoid content, 157; fructose 1,6-bisphosphatase activity, 160 Erysiphe polygoni

effect on photosynthesis, 151 Erythronic acid, 185,187 Ethylene as an elicitor ofPR-protein induction, 648 Eutypine, 297, 301 effect on state 3 and state 4 respiration and ADP/O ratio, 165 Exo-G1ucanases, 87, 88 Exo-pectate Iyases, 73 Exo-polygalacturonases, 79

wheat, 632 Fusarium lateritium

production of enniatins, 332 Fusarium moniliforme

production ofpolygalacturonases, 81; fusaric acid, 303 Fusarium oxysporum f. sp. ciceri production ofpectate Iyases, 74 polygalacturonases, 81 Fusarium oxysporum f. sp. dianthi effect on respiration, 163 Fusarium oxysporum f. sp. lycopersici production ofpectate Iyases, 74,78; polygalacturonases, 81 Fusarium oxysporum f. sp. melonis production of cellulases, 88; polygalacturonases, 81; ｾＭＱＬＴ xylanase,85 Fusarium oxysporum f. sp. pisi production ｯｦｾＭＱＬＴｸｹｬ｡ｮｳ･＠ 85 Fusarium roseum culmorum

production of cutinase, 15

Exserohilum monoceras

Fusarium sambucinum

production ofmonocerin, 371 Extensins, 62

Fusarium scirpi

F

Fusarium solani f. sp. phaseoli

production of enniatins, 332 production of enniatins, 332

Falcarindiol, 569 Falcarinol, 569 Famesylquinone, 360 las (fasciation) locus, 270 Fasciation diseases, 270 Fatty acids biosynthesis of, 7, 11, 12, 13 Fi (fasciation-inducing) plasmid, 270 fIX (nitrogen fixation) genes, 180 Flavonoids, 535 Foeniculoxin, 343 Formononetin, 536, 537 Fructose-I,6-bisphosphatase, 140 activity in diseased plants, 160 Fumonisins, 301, 311 biosynthesis of, 312 Furanocoumarins, 550 Fusaric acid, 302 effect on respiration, 163,301,303 Fusarium avenaceum

production of enniatins, 332 Fusarium culmorum

elicitation of chitinase production in

elicitation of production ｯｦｾＭＱＬＳ glucanases, 628; plant defensin, 643 Fusarium solani f. sp. pisi

elicitation of plant defensin production in pea, 643 production of cutinase, 15; pectate lyases,74 Fusarium tricinctum

production of enniatins, 332 Fusarubin, 360 Fusicoccins 347 modes of action of, 302, 349 Fusicoccum amygdali

production offusicoccins, 347

G Galactanases,86 Galactans, 56 Gaeumannomyces graminis var. avenae

production of ｾＭｉ＠

,4-xylanase, 85

Gaeumannomyces graminis var. tritici

production of polygalacturonases, 82

682 - Plant Pathogenesis and Resistance Ganoderma lucidum production oflaccase, 92 Gene regulation, 463 Genetic maps nitrogen fixation (nifandfLX) genes, 183 nodulation (nod and no/) genes, 183 Ri plasmids, 262 Ti plasmids, 246 Genistein, 183, 184, 187, 536, 537 Geotrichum candidum production ofpolygalacturonases, 82 Gigantenone, 343 as a herbicide, 382 Globodera rostochiensis production of cellulases, 88 ｾＭｉ＠ ,3-Glucanases as PR proteins, 626, 628 classes of, 628 Glucomannan, 60 ｾＭｇｬｵ｣ｯｳｩ､｡･Ｌ＠ 87, 88 Glutinosone, 562, 562 Glyceollins, 543 biosynthesis of, 539 sites of action of, 588 Glycine max effect of diseases on nodulation of, 215 production ofcoumestrol, 545; daidzein, 536; formononetin, 536; glyceollins, 543 Glycine-rich proteins, 62 Glycolysis, 144, 144 Glycoproteins as elicitors of phytoalexin production, 576 Gossypium spp. production of desoxyhemigossypol, hemigossypol and lacinilene e, 564 Gossypol, 564 biosynthesis of, 565 GTP-binding proteins as second messengers, 450 Gypsophila paniculata gall formation caused by Erwinia herbicola pv. gypsophilae and pv. betae,272

H Hairy roots, 261

Harpin as an elicitor, 424 He-toxins, 326 effect on chlorophyll content, 154 site of action of, 302, 329 TOX2 locus, 327 Helianthus annuus production of ayapin, scopoletin, 550 Helminthosporium carbonum production of He-toxins, 326 Helminthosporium maydis. race T production ofHMT-toxins, 352 Helminthosporium sacchari production of HS-toxins, 345 Helminthosporium sativum production of cutinase, 15 Helminthosporium turcicum production of ｾＭｉ＠ ,4-xylanase, 85 Helminthosporium victoriae production of HV-toxins, 329; victoxinine,347 Helminthosporol, 302, 346 Hemicellulases, 84 Hemicelluloses, 58 Hemigossypol, 564 biosynthesis of, 565 Heterodera cajani effect on leghemoglobin content, 216 Heterodera glycines effect on iron metabolism in soybean, 220; leghemoglobin content, 216; nitrogen-fixing activity, 218; nodulation, 215 production of cellulases, 88 Hevea brasiliensis production of scopoletin, 551 Hircinol, 548, 549 HMT-toxins, 302, 352 effect on state 3 and state 4 respiration, 165 Homogalacturonans, 54 Hordeun vulgare effect on respiration by Erysiphe graminis f. sp. hordei, 163 production of chitinases, 631; lipidtransfer proteins, 644; thaumatinlike proteins, 638; thionins, 643 HS-toxins, 302, 345 HV-toxins, 302, 329 Hyalopyrone, 372 as a herbicide, 382 3-Hydroxyacyl-Aep dehydrase, 9, 11

Index - 683 p-Hydroxybenzaldehyde, 357 4-Hydroxybenzoic acid, 357 7-Hydroxycostal, 560 7-Hydroxycostol, 560 Hydroxydianthalexins, 574, 575 7-Hydroxyflavan, 535 6-Hydroxyisosclerone, 357 6-Hydroxymellein, 371 4-Hydroxy-3-(3'-methyl-2'-butenyl)benzoic acid,357 Hydroxyproline-rich glycoproteins, 62 agglutination of microbes by, 514 in disease resistance, 514 Hymatoxins, 349 Hypoxylon mammatum production ofhymatoxins, 349

Ipomeamarone, 559, 559 Ipomoea batatas production of 7-hydroxycostal and 7hydroxycostol, 560; ipomeamarone, 559 ipt, 244 Irenolone, 553 Isocoumarins, 371 Isoflavonoids, 536 biosynthesis by elicited cells, 587 Isomarticin, 360 IsopimpineIlin, 550, 550 Isosclerone, 357 Isoseiridin, 373

I

Jasmonic acid as a second messenger, 445 biosynthesis of, 447 Javanicin, 360, 361 Junghuhnia separabilima production of laccase, 92; liP, 92 Kaempferol, 184, 187 3-Ketoacyl-ACP reductase, 9, 11 3-ketoacyl-ACP synthases, 8, 11 2-Keto-3-deoxymannooctulosonic acid, 56 Kievitone, 537 metabolism by Fusarium solani f. sp. phaseoli, 593

iaaH in A. rhizogenes, 262; Agrobacterium tume/aciens, 243, 246, 253, 255; Erwiniaherbicolapv. gypsophilae, 272; Pseudomonas syringae pv. savastanoi, 266 iaaM in A. rhizogenes, 262; Agrobacterium tume/aciens, 243, 246, 253, 255; Erwinia herbicolapv. gypsophilae, 272; Pseudomonas syringae pv. savastanoi, 266 Indole phytoalexins, 571,572 Indole-3-acetic acid (IAA) biosynthesis of, 239 in Agrobacterium rhizogenesinoculated plant tissues, 266 in Agrobacterium tume/aciensinoculated plant tissues, 257 in vitro production by Agrobacterium tume/aciens. 241; Pseudomonas syringae pv. savastanoi, 266 role in crown gall formation, 260; hairy root formation, 265; olive knot formation, 269 Induced resistance, 486 Inducers, 431 Infection threads, 178 Inositol phosphates as second messengers, 448 Inositol sphingophospholipids as elicitors, 427

J-K

L Laccase, 71, 91, 92 Lacinilene C, 564 biosynthesis of, 565 Lactuca sativa production of lettucenin A, 563 Leghemoglobins, 215 as affected by plant diseases, 216 Lettucenin A, 563 biosynthesis of, 563 Leucinopine, 245 Lignification as a resistance mechanism, 501 elicitors of, 498 impedes fungal invasion, 502 Lignin peroxidase (liP), 91 Lignins biosynthesis of, 68, 69, 498

684 - Plant Pathogenesis and Resistance monolignols of, 69 Lilium maximowiczii production ofyurinelide, 553 Loroglossol, 548, 549 Lotus corniculatus production of sativan and vestitol, 544 Lubimin, 562 biosynthesis and metabolism of, 562 Lupinus albus production of ribonuclease-like proteins, 641 Luteolin, 182, 184, /87, 188 Luteolinidin, 536 Lycopersicon esculentum production of chitinase, 632; P-1,3glucanases, 628; thaumatin-like proteins, 637, 638

M Maackiain, 538, 539 degradation by Ascochyta rabiei, 541; Nectria haematococca, 594 Macrophomina phaseolina production of phaseolinone, 343; P-l,4-xylanase,85 Maculosin, 302, 316 as a herbicide, 382 Magnaporthe grisea genetics of melanin production, 31 production of cutinase, 15; melanin, 85 30; ｾＭｉＬＴｸｹｬ｡ｮｳ･＠ Magnolol,551 Malformins, 331 Malus domestica production of ribonuclease-like proteins, 641 Malusfuran, 552 Manganese-dependent peroxidase (MnP), 91 Mannopine, 245 Mansonones, 564 Marticin, 360, 361 Medicago sativa production of medicarpin, 538 Medicarpin, 538 biosynthesis of, 539 detoxification by plant pathogens, 541, 593 Melanin biosynthesis inhibitors of, 34

biosynthesis of, 30,31 chemical and physical properties of, 29 genetics of, 3 1 in penetration of cuticle, 34 mediation of hydrostatic pressure in appressorium, 35, 36 Mellein,371 Meloidogyne incognita effect on leghemoglobin content, 216; respiration, /63 Mesorhizobium ciceri host specificity, 177 Mesorhizobium loti host specificity, /77 Methoxybrassinin, 571, 572 6-Methoxycamalexin, 572 6-Methoxymellein, 570, 588 5-Methoxyvestitol, 544, 545 O-Methylcurvulinic acid, 357 2-0-Methyl fucose, 56 2-0-Methyl xylose, 56 Mitochondria, 143, 143 Momilactones, 566 biosynthesis of, 567 Monilicolin A as an elicitor, 418, 421 Monilinia fructicola production of chitinase, 15; monilicolin A, 421 Monocerin,371 Monosaccharides as building blocks of cell wall polysaccharides, 64, 64 formation of sugar nucleotides, 65 Monoterpenoids, 557 Musa paradisiaca production of emenolone, irenolone, and musanolones, 553 Musanolones, 553 Mycosinol, 569 Mycosphaerella pinodes production of supprecins, 436

N Naphthoquinones, 360 Narcissus pseudonarcissus production of 7-hydroxyflavan, 7,4dihydroxyflavan, and 7,4'dihydroxy-8-methylflavan, 535 Naringenin, 184, 186

Index - 685 Nematoloma frowardii production of MnP, 92 Neovasinin,370 biosynthesis of, 370 Neovasipyrones, 370, 370 Nicotiana benthamiana expressing barley leaf peroxidase, 659 Nicotiana debneyi production of debneyol, 560 Nicotiana sylvestris expressing chitinase I, 659 Nicotiana tabacum expressing alfalfa P-I ,3-glucanases, barley a-thionin, and rice chitinase 1,659 production of chitinases, 632; P-I,3glucanases, 628; PR proteins, 627; thaumatin-like proteins, 638 Nissolin, 543 Nitrogen fixation (nif, fIX) genes, 200 products and functions of, 203, 204 Nitrogenase, 201 dinitrogenase, 20 I dinitrogenase reductase, 201 Nodulation (Nod) factors biosynthesis of, 189, 200 structure of, 201 Nodulation (nod. nol. noe) genes, 180 products and functions of, 195 Nodules, 178 amide-forming, 181 determinate, 179 indeterminate, 179 ureide-forming, 182 Nodulins, 207 early, 207, 208 late, 207, 209 Non-cyclicphotophosphorylation, 138,139 Nopaline, 245 Norjavanicin, 360,361 Nuclear localization signal sequences, 252

o Octopine, 245 Olive knots, 265 Ophiobolins, 351 Opines structure of, 245 Orchinol, 548, 549 Oryza sativa

production of momilactones and oryzalexins, 566; phytocassanes, 567; sakuranetin, 535; thaumatinlike proteins, 638 Oryzalexins, 566 biosynthesis of, 567 Osmotin, 636 Oxalic acid as an elicitor of phytoalexin production, 577. 581; PR protein production, 648, 648 Oxidative burst, 454 Ozone as an elicitor ofPR-protein production, 650

p Panus tigrinus production oflaccase and MnP, 92 Papilla callose in, 487 chemical Components of, 487 elicitation of, 494 in resistance to penetration, 490 phenolics in, 489 proteins and enzymes in, 488 silicon in, 488 Pathogenesis-related (PR) proteins biosynthesis of, 647 degradation of cellular RNA by, 657 disruption of membrane structure of invading pathogens by, 654 elicitation of, 647 families of, 625, 627 fortification of plant cell walls by, 655 in disease resistance, 653 inactivation of proteinases secreted by plant pathogens by, 656 induction of hypersensitive cell death by, 657 liberation of elicitors of defense reactions by, 655 lysis of cell walls of invading pathogens by, 653 with unknown biological function, 645 Pectate Iyases, 72, 73 as elicitors of PR-protein production, 648 production by plant pathogens, 74 Pectin degradative pathway, 94

686 - Plant Pathogenesis and Resistance Pectin Iyases, 73, 78, 79 Pectin methylesterases, 73, 82, 83 Pentose phosphate pathway, 144, 145 Peroxidases as PR Proteins, 640 Perylenequinones, 362 Pestalopyrone, 367 petasol, 343 as a herbicide, 382 Phanerochaete chrysosporium production of cellulases, 88 Phaseolinone, 343 as a herbicide, 382 Phaseollin, 543 biosynthesis of, 539 metabolism by plant pathogens, 544 site of action of, 588 Phaseolotoxin, 321 site of action of, 302, 322 Phaseolus lunatus production of coumestrol, 545 Phaseolus vulgaris production of chitinases, 631; coumestrol, 545; kievitone, 537; ribonuclease-like proteins, 641 Phlebia ochraceofulva production of LiP, 92 Phlebia radiata genes encoding LiP, 92 production of LiP and MnP, 92 Phoma exigua production of phomenone, 343 Phoma Iingam elicitation of chitinase production in oilseed rape, 631 production of phomalide, 331; phomapyrones, 367; sirodesmins, 316 Phoma tracheiphila production of melle in, 371 Phomalide, 331 Phomapyrones, 367 Phomenone, 343 as a herbicide, 382 Phomopsis cucurbitae production ofpolygalacturonases, 82 Phomopsis foeniculi production offoeniculoxin, 343 Photosynthetic light reaction, 138, 139 Photosystem I, 138, 139 Photosystem II, 138,139

Phyllosticta maydis production of PM-toxins, 354 Phytoalexins accumulation in resistant tissues, 583 biosynthetic pathways, 528 definition of, 527 detoxification of, 592,593 effect on electron transport system, 590; membrane integrity and function, 589 elicitation of, 575 in disease resistance, 590 modes of action of, 588, 588 Phytocassanes, 567 Phytoferritin, 220 Phytophthora capsici effect on biosynthesis of chloroplast fatty acids, 153, 155 production of cutinase, 15 Phytophthora infestans elicitation of production of chitinases, 632; ｾＭｉ＠ ,3-glucanases, 628; ribonuclease-like proteins, 641; thaumatin-Iike proteins, 638 production of pectin methyl esterase, 83 Phytophthora megasperma f. sp. glycinea effect on respiration, 163; CJC, ratio, 167 elicitation of ribonuclease-like protein production in parsley, 641 Phytotoxins as elicitors of phytoalexin production, 381 as biofungicides in disease control, 381 as disease-resistance suppressors, 377 as pathogenicity factors, 375 as potential herbicides, 381, 382 as primary determinants of pathogenicity, 293 as probes in plant disease diagnostics, 381 as probes in plant physiology research, 380 as secondary determinants of pathogenicity,293 as tools in breeding disease resistant plants, 378 as virulence factors, 376 detoxification of, 380 host-specific (host-selective), 293

Index - 687 molecular sites of action of, 301 non-host-specific (non-host-selective), 293 produced by plant pathogenic bacteria, 300 produced by plant pathogenic fungi, 294 role in plant pathogenesis, 375 Phytuberin, 562 biosynthesis and metabolism of, 562 Phytuberol, 562, 562 Piceatannol, 548 Pinosylvin, 546 Pinus sylvestris production of pi nosy Iv in, 547 Pisatin,538 biosynthesis of, 539 detoxification by plant pathogens, 593, 594 Pisum sativum production of P-I ,3-glucanases, 628; maackiain and pisatin, 538; plant defensins, 643; ribonuclease-like proteins, 641 Plant defensins as PR Proteins, 642, 643 Plasmodiophora brassicae, 275 Plastoquinones, 137 PM-toxins, 302, 354 Podosphaera leucotricha effect on photosynthesis, 151 Polyacetylenes, 569 Polygalacturonase inhibitory proteins, 106, 107

Polygalacturonases, 79, 81 as elicitors ofPR protein production, 648 Polygalacturonic acid as an elicitor, 421 Preformed resistance, 486 Prehelminthosporol, 346 Proline-rich proteins, 62 Protease inhibitors as PR Proteins, 638 Protein phosphorylation! dephosphorylation, 453 Prunus avium production ofthaumatin-Iike proteins, 636 Prunus persica

infection with Taphrina deformans: effect on respiration, 163 Pseudomonas amygdali, 271 Pseudomonas andropogonis production of rhizobitoxine, 304 Pseudomonas cepacia production of polygalacturonases, 82 Pseudomonas fluorescens production ofpectate Iyases, 74,77 Pseudomonas lachrymans elicitation of chitinase production, 631 Pseudomonas marginalis production ofpectate Iyases, 74, 77; pectin Iyases, 79 Pseudomonas solanacearum production of cellulases, 88; polygalacturonases, 80 Pseudomonas syringae pv. atropurpurea production of coronatine, 306 Pseudomonas syringae pv. coronafaciens production of tabtoxin, 318 Pseudomonas syringae pv. glycinea production of coronatine, 306 Pseudomonas syringae pv. maculicola production of coronatine, 306 Pseudomonas syringae pv. morsprunorum production of coronatine, 306 Pseudomonas syringae pv. phaseolicola production of phaseolotoxin, 321 Pseudomonas syringae pv. savastanoi, 265 Pseudomonas syringae pv. syringae elicitation ofthaumatin-Iike protein production in rice, 638 production of syringomycins, syringopeptins, syringostatins, syringotoxins, 337 Pseudomonas syringae pv. tabaci production of tabtoxin, 318 Pseudomonas syringae pv. tagetis production oftagetitoxin, 374 Pseudomonas syringae pv. tomato production of coronatine, 306 Pseudomonas tolaasi production of proteases, 90 Pseudomonas viridiflava production ofpectate Iyases, 74,77 Psoralen, 550, 550 Pterocarpans, 538 biosynthesis of, 539 Puccinia allii effect on chlorophyll content, 154; Rubisco activity, 159; stomatal

688 - Plant Pathogenesis and Resistance conductance, 153 Puccinia gram in is f. sp. avenae elicitation ofthaumatin-like protein production, 638 Puccinia graminis f. sp. tritici effect on C,jC 1 ratio, 167; photosynthesis, 152 elicitation of production of chitinase, 628 632; ｾＭＱＬＳｧｬｵ｣｡ｮｳ･＠ Pyrenolide as a herbicide, 382 Pyrenolines, 375 Pyrichalasins, 3 15 Pyricularia oryzae effect on photosynthesis, 151 production of cytochalasins, 3 15; 6-hydroxymellein, 371; tenuazonic acid, 303 Pyrufurans, 552 Pyrus communis production ofpyrufurans, 552

R Raphanus sativus production of lipid-transfer proteins, 644; plant defensins, 643; spirobrassinin, 571 Receptors, 437, 438, 439, 439, 440, 440 Recognition, 416 Respiration rates changed in diseased plants, 163 Respiratory control ratio, 149 Resveratrol, 546 detoxification by Botrytis cinerea, 593 Rhamnogalacturonan I, 54 Rhamnogalacturonan II, 54, 56 Rhamnogalacturonanases, 83 Rhizobitoxine, 304 biosynthesis of, 305 site of action of, 302, 306 Rhizobium etli host specificity, 177 Rhizobium galegae host specificity, 177 Rhizobium leguminosarum host specificity, 177 Rhizobium lupini host specificity, 177 Rhizobium tropici host specificity, 177

Rhizoctonia solani effect on C6!C 1 ratio, 167 production of cutinase, 15; pectin Iyases, 79; pectin methylesterase, 83; polygalacturonases, 82 Rhizopus stolonifer production of polygalacturonases, 82 Rhodococcus jascians, 270 Ri (root-inducing) plasmids, 261 Ribonuclease-Like Proteins as PR Proteins, 640 Ribulose-l ,5-bisphophate carboxylase! oxygenase (Rubisco), 139, 140 activity in diseased plants, 159 Ricinus communis production of casbene, 565 Rigidoporus lignosus production oflaccase and MnP, 92 Rishitin, 562 biosynthesis of, 562

s Saccharum officinarum production of piceatannol, 548 Safynol, 569 Sakuranetin, 535 Salicin, 339 Salicylate hydroxylase, 458 Salicylic acid, 429 as an elicitor, 648, 649 biosynthesis of, 430 Sativan, 544, 545 Sclerotinia sclerotiorum production of cellulases, 88; polygalacturonases, 82 Scoparone, 549,550 Scopoletin, 550, 550 Second messengers, 441 Secondary metabolites, 526 relation to primary metabolites, 528 Seiricardines, 346 Seiridin, 373 Seiridium cardinale production of seiricardines, 346; seiridin, 373 Septoria nodorum elicitation ofthionin production, 643 production of melle in, 371 Sesquiterpenoids, 558

Index - 689 Signal molecules activation of nod genes, 184 that elicit defense responses, 416 that induce compatible host-parasite interactions, 431 that suppress defense responses, 433 Signal transduction in resistance responses, 442 in systemic acquired resistance, 457 oxidative burst in, 454 protein phosphorylationl dephosphorylation in, 453 Sinorhizobium fredii host specificity, 177 Sinorhizobium meliloti host specificity, 177 Sirodesmins, 316 biosynthesis of, 317 Solanapyrones, 368 Solanum commersonii production ofP-I,3-glucanases, 628; thaumatin-Iike proteins, 638 Solanum tuberosum production of chitinases, 632; P-I,3glucanases, 628; ribonuclease-like proteins, 641 Solavetivone, 562 biosynthesis and metabolism of, 562 Sorghum bicolor production of apigenindin and luteolinidin, 536; ribonuclease-like proteins, 641 Sorokinianin, 347 Soybean mosaic virus effect on nitrogen-fixing activity, 218; nodulation, 215 Sphaerotheca fuliginea effect on chlorophyll content, 154; photosynthesis, 151 Sphaeropsidin A, 350 Sphaeropsis sapinae f. sp. cupressi production of sphaeropsidin A, 350 Sphinganine N-acyltransferase effect of AAL-toxins on, 314 Spiciferones,372 Stachydrine, 184, 185, 187 Starch biosynthesis and degradation, 160 affected by pathogens, 161 State 3 respiration, 149, 165 State 4 respiration, 149, 165

Stemphylium botryosum production of stemphyltoxins and stemphyperylenol, 365, 365 Stemphyltoxins, 365, 365 Stemphyperylenol, 365, 365 Stilbenoids, 545 Streptomyces scabies production of cutinase, 15; thaxtomins, 317 Suberin, 507, 508 Suberization, 507 as a resistance mechanism, 508 elicitation of, 509 in disease resistance, 507 Succinamopine, 245 Sucrose as phytoalexin elicitor, 421, 428, 582 Sugar nucleotides, 65 Supprescins as resistance suppressors, 434, 436 Suppressors, 433 Syringomycin, 337 as an elicitor, 418 Syringopeptins, 337 effect on state 3 and state 4 respiration, 165 Syringostatin, 337 Syringotoxin, 337 Systemin as an elicitor, 421, 429

T T-complex, 247 T-DNA,245 expression in transformed cells, 253 integration into plant nuclear genome, 252 nuclear targeting, 252 processing for intercellular transport, 251 T-toxins, 392 Tabtoxin, 318 site of action of, 321 Tagetitoxin, 374 effect on RNA polymerases, 374 Taphrina deformans causes witches' broom on peach, 273 effect on chlorophyll content, 154; chlorophyllase activity, 155; photosynthesis, 151; respiration, 163

690 - Plant Pathogenesis and Resistance Taphrina pruni effect on growth regulators, 273 TCA cycle, 146, 146 Tentoxin, 323 site of action of, 325 Tenuazonic acid, 303 Tetronic acid, 185, 187 Thaumatin-Iike proteins as PR Proteins, 635, 638 Thaumatococcus daniellii, 635 Thaxtomins, 317 Thionins as PR Proteins, 642, 643 Threonine-hydroxyproline-rich glycoproteins, 62 Ti (tumor-inducing) plasmids, 241 genes on, 246, 255 T-DNA, 245, 246 vir region, 245 Tobacco leaf curl virus effect on chlorophyll content, 154; Rubisco activity, 159 Tobacco mosaic virus effect on Rubisco activity, 159 elicitation of production of chitinase, 632; ; P-I ,3-glucanases, 628; thaumatin-Iike protein, 638 Tobacco necrosis virus elicitation of production of chitinases, 631; thaumatin-Iike proteins, 638 Tobacco ringspot virus effect on nitrogen-fixing activity, 218; nodulation, 215 elicitation of chitinase production, 631 a-Tocopherol, 431, 432 Trametes versicolor genes encoding LiP, 92 Trifolium spp. production of medicarpin, 538 Trigonelline, 185,187 Triterpenoids, 568, 568 Triticones, 315 as herbicides, 382 Triticum aestivum production of chitinases, 632; p-I,3glucanases, 628; thaumatin-like proteins, 637, 638 Tryptophol, 297, 382 Tubakia dryina production of isosclerone and hydroxyisosclerones, 357

Tyrosol,357

u Ulmus americana production of mansonones, 564 Ulocladium consortiale production of cutinase, 15 UmbeIliferone, 184, 550, 550 Uromyces muscari effect on carotenoid content, 157; chlorophyll content, 154 Uromyces phaseoli effect on C"IC, ratio, 167 Uromyces viciae-fabae production of pectin methylesterase, 83 Ustilago spp. gall formation on maize, 274; Zizania latifolia (=2. caduciflora), 274 UV-irradiation as an elicitor of PR-protein induction, 650

v Venturia inaequalis production of cutinase, 15; polygalacturonases, 82 Verticillium albo-atrum production of polygalacturonases, 82 Verticillium dahliae production of melanin, 30 effect on photosynthesis, 151; stomatal conductance, 153 Vestitol, 544, 545 Vicia/abae production of wyerone and wyerone acid, 569 Victorins, 329 as elicitors, 418 Victoxinine, 347 a-viniferin, 546 E-Viniferin,546 Vitis vinifera production of resveratrol, a- and Eviniferin, 546

w White clover mosaic virus effect on leghemoglobin content, 216;

691 nitrogen-fixing activity, 218; nodulation, 215 Witches' broom diseases, 273 Wound tumor virus, 276 Wyerone, 569 Wyerone acid, 569

x Xanthomonas campestris pv. campestris production of cellulases, 88; pectate Iyases, 74, 77 Xanthomonas campestris pv. malvacearum production ofpectate Iyases, 74 Xanthomonas campestris pv. phaseoli effect on photosynthesis, 151 Xanthomonas campestris pv. phormiicola production ofN-cononafacoyl-L-valine and N-cononafacoyl-L-isoleucine, 308 Xanthomonas campestris pv. vesicatoria elicitation of lipid-transfer protein production, 644 Xanthotoxin, 550, 550 site of action of, 588 Xylanases, 84, 85 as elicitors ofPR protein production, 648 Xylans,58 Xylella fastidiosa production of proteases, 90 Xyloglucans, 59

Y-z Yurinelide, 553 Zeamays gall formation caused by Ustilago maydis, 274 production of chitinases, 631; ｾＭｉＬＳ glucanases, 628 Zeamatin, 636 Zinniol, 356 Zizania lati/olia gall formation caused by Ustilago esculenta, 274