MECHANISMS OF RESISTANCE TO PLANT DISEASES
to Plant Diseases
Edited by
A.J. Slusarenko
RWTH, Aachen, Germany
R.S.S. Fraser
Society for General Microbiology, Reading, United Kingdom, Honorary Visiting Professor, University of Manchester, U.K.
and
L.C. van Loon
Utrecht University, The Netherlands
SPRINGER-SCIENCE BUSINESS MEDIA, B.V.
A C L P . Catalogue record for this book is available from the Library of Congress.
ISBN 978-1-4020-0399-8 ISBN 978-94-011-3937-3 (eBook) DOI 10.1007/978-94-011-3937-3
Transferred to Digital Print 2001
Printed on acid-free paper
Cover illustration:
Part B , the dispersal of pigment throughout a dead cell, of Figure 22 from the article by J.W. Mansfield (p.325-370).
Provided by Ralph Nicholson.
A l l Rights Reserved
© 2000 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 2000
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.
1 CASE STUDIES
Resistance to Tobacco Mosaic Virus in Tobacco Plants R.S.S. Fraser
2 Black Rot of Crucifers 21
Anne M. Alvarez
3 The Cladosporium fulvum-Tomato Interaction 53
A Model System to Study Gene-For-Gene Relationships Pierre J.G.M. De Wit
4 The BarleylBlumeria (Syn. Erysiphe) graminis Interaction 77 Hans Thordal-Christensen, Per L. Gregersen & David B. Collinge
2 GENETICS OF DISEASE RESISTANCE 101 Basic Concepts and Application in Resistance Breeding
Beat Keller, Catherine Feuillet & Monika Messmer
3 RESISTANCE IN POPULATIONS 161
J. Frantzen
4 RESISTANCE GENES AND THE PERCEPTION AND 189 TRANSDUCTION OF ELICITOR SIGNALS IN HOST·
PATHOGEN INTERACTIONS Thomas Boller & Noel T. Keen
5 STRUCTURAL ASPECTS OF DEFENSE 231 Bruno Moerschbacher & Kurt Mendgen
6 THE HYPERSENSITIVE RESPONSE 279
Thorsten Jabs & Alan J. Slusarenko
7 ANTIMICROBIAL COMPOUNDS AND RESISTANCE 325 The Role of Phytoalexins and Phytoanticipins
J.w. Mansfield
VI
8 INDUCED AND PREFORMED ANTIMICROBIAL PROTEINS 371 w.F. Broekaert, F.R.G. Terras & B.P.A. Cammue
9 SPECIAL ASPECTS OF RESISTANCE TO VIRUSES R.S.S. Fraser
10 SYSTEMIC INDUCED RESISTANCE
L.e.
Van Loon479
521
11 TRANSGENIC APPROACHES TO CONTROL EPIDEMIC 575 SPREAD OF DISEASES
Ben
J.e.
Cornelissen & Andre SchramSUBJECT INDEX SPECIES INDEX
601 615
CASE STUDIES
RESIST ANCE TO TOBACCO MOSAIC VIRUS IN TOBACCO PLANTS
Summary
R.S.S. FRASER
Society for General Microbiology, Marlborough House, Basingstoke Road, Spencers Wood, Reading RG7 lAE, UK ([email protected])
Tobacco mosaic virus (TMV) was first studied scientifically as a plant pathogen 100 years ago. A fonn of hypersensitive resistance transferred from the wild species Nicotiana glutinosa to cultivated tobacco was shown 60 years ago to be due to a single dominant gene, named N. This causes the virus to be localised to necrotic lesions which fonn around each site of infection: the hypersensitive response (HR). N-gene resistance has proved extremely durable: only one virulent (resistance-breaking) TMV isolate has been reported to date. Another resistance gene, N', thought to be allelomorphic with N, causes a hypersensitive reaction to avirulent isolates of TMV, but numerous virulent isolates are also known. These do not induce necrosis but spread systemically and cause nonnal mosaic symptoms. The single known example of virulence against N has been mapped on TMV RNA to the replicase gene, whereas virulence against N' in different TMV isolates has been mapped to a number of locations, all within the coat protein gene. The N gene has been isolated and sequenced: it shows structural and possibly functional features in common with certain other genes for resistance to bacterial and fungal pathogens, and to other genes with known functions in control of development or response to hormones in animals. These similarities give some clues about how the N-gene product might be involved in TMV recognition and in signalling the cascade of resistance and other responses which follows. The actual mechanism which inhibits TMV spread or multiplication after resistance is induced is not yet fully clear, but may involve an inhibition of multiplication or blocking of cell-to-cell spread of the infection front.
Abbreviations
HR hypersensitive response TMV tobacco mosaic virus PR pathogenesis-related protein
A. Slusarenko, R.S.S. Fraser. and K. van Loon (eds), Mechanisms of Resistance to Plant Diseases. 1-19.
© 2000 KlulVer Academic Publishers.
2 R.S.S. Fraser
I. Introduction
A. HISTORY
It is highly fitting that the case study of virus resistance chosen for this book is not only one of the best understood examples of a mechanism of resistance to a plant-virus, but also the subject of a notable centenary in 1998, the year of writing. Tobacco mosaic virus (TMV) had been identified in the 1880-1890 period (Matthews, 1991) as a sap-transmissible disease-causing agent that could pass though bacterial filters, but it was not until 1898 that the phenomenon became fully accepted (Beijerinck, 1898). This is now generally recognised as the birth of the study of plant virology.
Forty years later, Holmes (1938) demonstrated that resistance to TMV, transferred from the wild species Nicotiana glutinosa to the cultivated tobacco N. tabacum by Clausen and Goodspeed (1925), was controlled by a single dominant gene which he named N. These publications may be regarded as the start of the scientific study of resistance to plant viruses and of its exploitation in practical crop protection. The N gene has also played a central role in the development of the classical technology of plant virus studies, such as infectivity assay (Holmes, 1929; Kleczkowski, 1950), and was the first gene for resistance to a plant virus to be isolated and sequenced (Dinesh-Kumar et aI., 1995).
B. THE VIRUS AND ITS HOSTS 1. TMVas a model virus
Before describing the phenomenon of TMV resistance, and the components of plant-virus interactions involved, it may be useful to describe TMV in more general terms as a structure and pathogen, especially for the reader not familiar with plant virology. In the context of this book, there are massive differences between viruses on the one hand, and bacteria and fungi on the other, both in terms of genetic and structural complexity, and in mechanisms of pathogenesis. TMV is taken in this section as a model virus which exemplifies a number of features of the viral 'lifestyle', an understanding of which is an essential foundation for studying the resistance mechanisms and other types of plant-virus interactions described here and in chapter 9.
A more detailed description of the diversity of viruses as plant pathogens is given in the introduction to chapter 9. It must be stressed here that plant viruses have evolved many different ways of solving their 'lifestyle issues'; TMV is one well-understood example, but should not be considered in any way typical.
TMV infects host plants through natural or experimentally-caused wounds: its only biotic transmission agents are man or animals which may transfer it casually between infected and healthy plants by surface contact. This contrasts with the highly-specific interactions other viruses have with particular vectors such as leaf-feeding insects (see chapter 9).
In susceptible tobacco plants, TMV causes the characteristic light-greenldark-green mosaic pattern from which it derives its name, together with stunting and distortion of those leaves which become infected at an early stage of development (Fig. 1).
The virus can accumulate to concentrations as high as 5-10 mg g -I fresh weight of leaf (Fraser, 1987).
Figure 1. Different types of response to 1MV infection in tobacco leaves. Centre: uninoculated, healthy leaf. Right: leaf of a susceptible variety showing the characteristic mosaic of light green/dark green tissue, after systemic spread of the virus. Left: inoculated leaf of a resistant variety carrying the N gene, showing the local lesions (arrowed) which form around each site of infection (HR).
TMV is a rigid, rod-shaped particle, 300 nm long and 18 nm in diameter, with a central hollow core 4 nm in diameter (Fig. 2). The rod contains a single RNA molecule of molecular mass 2.1 mDa, assembled with about 2100 coat protein molecules of molecular mass 17.6 kDa. In the particle structure, the RNA forms a helix, embedded within a helix of assembled coat protein molecules. Good illustrations of particle architecture are to be found in Matthews (1991), and excellent three-dimensional rotating depictions in Sgro (1995).
Figure 2. Electron micrograph ofTMV particles. 1MV in sap from an infected tobacco plant was negatively stained with methylamine tungstate. The hollow core of the particle is clearly visible in short lengths of broken particle seen end on. The scale bar indicates 50 nm. Photomicrograph by courtesy of Colin Clay.
4 R.S.S. Fraser
The TMV RNA contains the information required to specify virus multiplication and pathogenesis, although both processes clearly involve interactions with the host and participation of host-coded components. TMV RNA from the virus particle is messenger sense (positive), in that it can be translated into protein in vitro or in vivo, although the actual mechanism of expression of all the proteins encoded by the RNA is rather complex. TMV RNA was the first plant virus RNA to be fully sequenced (Goelet et at., 1982). Examination of the sequence for open reading frames (ORFs:
regions between start and stop codons coding for a recognisable sequence of amino acids) has allowed the compilation of the genetic map shown in Fig. 3. From an AUG start codon after an untranslated leader sequence of 69 nucleotides, two proteins of molecular masses 126 and 183 kDa are produced, the larger by occasional read-through of a leaky termination codon (UAG, amber) at the end of the 126 kDa sequence.
No other significant polypeptides are produced when the full length TMV RNA is translated: eukaryotic 80S ribosomes will only translate ORFs beginning at the 5'-proximal start codon.
The 126 and 183 kDa proteins are components of the TMV replicase (Osman and Buck, 1996). By comparisons with the amino acid sequences of replicase proteins from other putatively related single-stranded RNA plant and animal viruses forming what is known as the Sindbisvirus supergroup, three functional domains have been postulated. Dl is a methyltransferase activity thought to be involved in capping of mRNA with 7-methylguanosine triphosphate, which enhances translation. The full length TMV RNA and sub-genomic coat protein mRNA (see below) are capped; the other sub-genomic mRNAs are not. D2 is a helicase, and D3 is an RNA polymerase.
The purified, functional replicase also contains a plant-coded protein (56 kDa) which is required for activity (see chapter 9), two components (54 and 50 kDa) not found in healthy plants and of unknown origin, and a 32 kDa component also occurring in healthy plants (Osman and Buck, 1997).
The TMV replicase is involved in the synthesis of a complementary full length (negative sense) RNA (Fig. 3). From this, three 3'-co-terminal sub-genomic messenger- sense RNAs are produced: all are detectable in infected plants. These contain at or close to their 5' ends the ORFs for the 54, 30 and 17.6 kDa proteins respectively. The 30 and 17.6 kDa proteins have been detected in infected plants but the 54 kDa protein has not: it is not yet established whether it is the 54 kDa component found in the purified replicase (Osman and Buck, 1997). The 17.6 kDa protein is the virus coat protein;
the function of the 30 kDa protein will be described later.
The negative sense full length TMV RNA also functions as the template for synthesis of full-length progeny virus RNA molecules (Fig. 3). These then assemble with coat protein to form the progeny virus particles. The process of assembly requires no other participating macromolecular components, and can indeed be carried out in vitro (Matthews, 1991).
This description of the molecular biology of TMV multiplication covers the events within an individual infected cell, based largely on an understanding of how the genetic information in the TMV RNA is expressed. It leaves unanswered the events before and after that process: how the infection commences after the virus particle penetrates the wound, and how infection spreads through the plant from the initially infected cell.
(a)
(b)
(c)
o 2 3 4 5
N t
1)9~H IMP
fin 6 N'
11::::Qi::::l Hti~tttl •
kilo base scale
5'
7mG _____________ _
3' ORFs inTMVRNA (messenger sense)
126kDa 183 kDa
3'
II::: :Qi:::: 1 !tJitt/lJ[JJ
Ii;'; i;';';'; i; i
1 >;.;
i; i; i;';' ;.\I';'; ;.;.;.;.;.;.;.;.; ;.;.;.;.;.;.;.;.;.;.]
Replicase sub-units
"
I~
CPI I
" "
1 ''j"i
5'
Gene expression and virus multiplication
complementary negative strand 3' co-terminal subgenomic mRNAs
?
I
I ;. ;. ;. ;.>]
54 kDa replicase subunit?.. I
~30 kDa movement protein
sa ..
17.6 kDa coat protein5'
7mG---
3' Progeny TMVRNA t ...• Self-assembly ofprogeny virus particles ... i
Figure 3. Organization and expression of the TMV genome. (a) Scale bar. (b) The various possible open reading frames (ORFs) on the genomic RNA are shown, together with putative functional domains in the replicase, as described in the text and in Lewandowski and Dawson (1995). (c) Replication and expression of the genome. Transcription to RNA copies is shown by broken arrows; translation to protein by solid arrows, and assembly of progeny virus particles by dotted arrows. The 54 kDa replicase subunit protein has not yet been shown to exist in infected plants, although its sub-genomic messenger RNA has been detected. This protein may equate to a 54 kDa protein found in the purified replicase (Osman and Buck. 1996. 1997) but not yet fully characterised. The kilobase scale (a) also shows the location of the detenninants of virulence/avirulence against the Nand N' genes (Padgett and Beachy, 1993; Taraporewala and Culver, 1996, 1997).
6 R.S.S. Fraser
The TMV particle is extremely robust: infectivity is retained for decades in dead infected plant material, soil and groundwater. Infectivity will survive heating to 80°C, and treatment of particles with high levels of ribonuclease, or mutagens such as HN02
and ultraviolet light (Matthews, 1991). How then does such a 'survivor' expose its RNA to initiate infection? The answer is that it exploits the molecular biology of the multiplication process.
The coat protein subunits covering the 80 or so nucleotides at the 5' end of the genomic RNA appear to be comparatively loosely bound to the RNA and the other coat proteins in the particle. It is thought that in a region of the cytoplasm with comparatively high pH (8.0) and low Ca2+ concentration, these subunits may disassemble, leaving the 5' end of the TMV RNA exposed. This allows a ribosome to attach to the first AUG initiation codon, and commence translation of the 1261183 kDa replicase proteins.
As it moves along the TMV RNA, the ribosome displaces further coat protein subunits, a process known as co-translational disassembly. The process has been demonstrated in vitro, and TMV particles partially stripped of coat proteins and expressing replicase proteins have been found in vivo (Shaw et ai., 1986; Wilson et ai., 1990). Co-translational disassembly effectively exposes the first three quarters of the genome from the 5' end, but translation is unable to proceed beyond the stop codon at the end of the 183 kDa protein. It appears that the 3' end of the genome is uncoated by removal of the subunits in a 3'-75' direction (i.e. the opposite to co-translational disassembly) by the newly expressed replicase, and concomitant with the synthesis of the negative strand viral RNA (Wu and Shaw, 1997). Presumably the nucleotide sequence at the 3' end of the genomic RNA where the replicase attaches and initiates transcription is accessible in the 5'-73' partially stripped particle.
TMV particles are much too large to pass through the plasmodesmata which provide cytoplasmic continuity between adjacent plant cells (Lucas and Gilbertson, 1994).
Experiments with micro-injected dyes indicate that the size exclusion limit is of the order of 1.5-2.0 nm molecular diameter, equivalent to a molecular mass of around 1 kDa (Terry and Robards, 1987). The TMV 30 kDa protein has been shown to be tightly bound to the cell wall fraction and associated with plasmodesmata; it increases the plasmodesmatal size exclusion limit markedly to about 5-9 nm, although this is still not enough to allow the passage of intact virions (Wolf et ai., 1989). It appears instead that the infectious entity that moves from cell to cell is the viral RNA.
The 30 kDa protein has a single-stranded RNA-binding function, which opens up the free-folded TMV RNA (mean diameter 10 nm) to an extended, thinner, transferable form with a diameter around 2.5 nm (Lartey et ai., 1997). An association of the movement protein-TMV RNA complex with elements of the cytoskeleton may also facilitate delivery of the complexes to the plasmodesmata.
Cell-to-cell movement of infection does not require the TMV coat protein, as coat protein-less mutants move with the same efficiency as the wild type (Dawson et ai., 1988).
Coat protein is, however, required for long-distance transport of infection in the phloem:
it is possible that the movement of the infectious entity from mesophyll cell to sieve tube element is a different process from that involved in local cell-to-cell spread.
2. Host Range
TMV is a member of the tobamovirus genus which contains 12 members infecting diverse plant groups, including tobacco, tomato, cucumber, orchids, frangipani and pepper (Capsicum) (Lewandowski and Dawson, 1995; Brunt et ai., 1996). Although tobamoviruses from different host groups tend to have a high degree of sequence similarity, with a minimum in the range 60-80%, the evidence is that in nature each is adapted to its particular host species or related group of species, with a comparatively narrow host range (Bald, 1960). In contrast, TMV and certain other tobamoviruses have been shown to have very wide experimental host ranges in laboratory tests of hundreds of species in numerous families (Horvath, 1978).
TMV causes systemic mosaic on numerous Nicotiana species, including N. sylvestris, N. tomentosa and N. tomentosiformis. An N-gene type of HR is also found in numerous species, including N. glutinosa, N. rustica, N. gosseii, N. suaveolens and N. repanda (Valleau, 1952). This may suggest that the association of TMV with the genus Nictotiana is one of long standing, and that resistance to the virus evolved at an early stage of speciation (Holmes, 1951). An alternati ve theory (Valleau, 1952), that the virus first spread from the wild host plantago to cultivated tobacco crops which had evolved susceptibility, appears untenable.
II. The Genetics and Phenotype of Resistance
A. THENGENE
In N. glutinosa and N. tabacum cultivars contammg the N gene, TMV does not spread systemically, but is localised to small areas of several hundred infected cells around each point of infection. After a few days, the infected cells become necrotic - the hypersensitive response (HR). Virus multiplication ceases (Fig. 4), although infectious virus can still be isolated from the lesions (Siegel, 1960). As described elsewhere in this book, HR is a common resistance response to invading bacteria, fungi and viruses of many species or types, although for viruses in particular there are numerous other types of resistance response (chapter 9).
The N gene was transferred from N. glutinosa to N. tabacum via an interspecific synthetic hybrid, N. digluta (Clausen and Goodspeed, 1925). It was thought that there had been a substitution of the entire N. glutinosa chromosome carrying the N gene (chromosome Hg) for the N. tabacum chromosome H, and that the N. glutinosa chromosome has been physiologically stable within the N. tabacum genetic background. This was thought to explain the ease with which the resistance gene was introgressed into commercial cultivars. Later, Gerstel (1948) did observe some exchange of segments between the Hand Hg chromosomes. A useful review of the complex literature on the genetic and breeding history of the N gene is given by Dunigan et al. (1987).
Since its introduction, N-gene resistance has been widely incorporated into commercial tobacco cultivars; cigar-smoking readers may have noticed its presence as betrayed by the occasional lesion on the cigar outer leaf. However, TMV still
8 R.S.S. Fraser
causes severe crop losses in important tobacco-growing areas such as North and South Carolina, USA. This is because it has not yet been possible to breed flue-cured tobacco cultivars carrying the N-gene with the same quality and yield characteristics as susceptible cultivars (Barnett, 1995), despite the early discovery of the gene and elucidation of the inheritance of resistance. The problem may stem from other genes affecting yield and quality in the persisting Hg chromosomes or segments of it.
Growers clearly prefer to risk losing a portion of their crop each year to TMV, in return for the economic benefits of higher quality and hoped for higher yield of the susceptible cultivars.
The N gene, where used in tobacco cultivars, has been remarkably durable, in that the resistance has not been overcome by TMV types occurring in tobacco cultivation.
A single resistance-breaking (virulent) strain has been isolated from pepper (CsiIlery et a!., 1983). This was initially characterised as an isolate of the related tobamovirus tomato mosaic virus, but later as another related tobamovirus, Solanum dulcamare yellow fleck virus (Sanfa~on et a!., 1993).
Resistance conferred by the N gene to avirulent isolates is temperature sensitive, breaking down at 28-30°C to allow systemic spread of the virus (Takahashi, 1975;
De Laat and Van Loon, 1983), but this does not appear to have detracted from the usefulness of the gene in practical crop protection.
300 250 c 0 .~ !:1 200
c Q)
u c
0 150
<: u
~
100>
~ E-<
50 0
0 5 10 15 20
Days after inoculation
Figure 4. The effects of the hypersensitive response on multiplication of TMV. Tobacco cv. White Burley, which contains the N' resistance gene, was inoculated with virulent (+) or avirulent (_) isolates of TMV.
Virus concentration was measured by polyacrylamide gel electrophoresis of extracted nucleic acids, and TMV RNA concentration expressed as !!g g ·1 fresh weight. Necrotic lesions began to appear on leaves inoculated with the avirulent isolate at the point shown by the arrow.
B. THEN' AND EN GENES
These genes are of interest for cytogenetic reasons and for what they tell us about plant-virus interactions, rather than because of any practical value in plant breeding.
The cultivated species N. tabacum does not occur naturally in the wild, but is an amphidiploid derived from the wild species N. sylvestris and N. tomentosiformis and presumably selected by an early plant breeder on the American continent. N. sylvestris is systemically infected by many isolates of TMV, but forms the typical local lesions of HR when inoculated with other (avirulent) isolates (Weber, 1951). This response is controlled by a dominant gene, named N', thought to be allelomorphic with N (Valleau, 1952; Dunigan et al., 1987). A gene from N. sylvestris and certain N.
tabacum cultivars with similar ability to discriminate TMV strains, but showing incomplete dominance, was named nS by Weber (1951), who suggested that it might be synonymous with N'.
N. tomentosiformis plants are systemically infected by (susceptible to) all isolates of TMV. N. tabacum cultivars may exhibit HR to avirulent isolates of TMV if they have inherited the Nt gene, or are infected systemically by all isolates of the virus if they do not have N' from their natural parentage and have not had N transferred by the plant breeder. In the former case, it must be assumed that tobacco cultivars showing systemic infection by all strains of TMV have somehow lost the N genes on the chromosome pairs derived from the N. sylvestris parent, or that N has become suppressed or masked in some way.
N. tomentosiformis plants, and N. tabacum cultivars susceptible to all isolates of TMV, are referred to as containing the n gene. This may be either an inactive allelic form of Nand N', or may be a null allele of them (Fraser 1986; Dunigan et al., 1987).
The latter explanation assumes that when a resistance gene is transferred from a wild species via an introgressed chromosome or chromosomal segment, there may be no corresponding DNA sequence in the susceptible parent genome. This question is difficult to resolve by classical cytogenetic methods, but the functional status or otherwise of the putative n allele may be approachable now that the N-gene and related sequences can be studied directly (Dinesh-Kumar et al., 1995).
Melchers et al. (1966) found a spontaneous mutation of the nn variety Samsun which gave a necrotic reaction to the same isolates of TMV as N'-containing varieties, and may have represented a back mutation of n to N', or a release from a masking effect.
They named this gene EN.
Genetically, the tobacco-TMV interaction displays a limited form of the gene-for-gene interaction between resistance/susceptibility in the host and virulence/avirulence in the pathogen, which is set out in Table 1. The TMV -N' gene interaction has an interesting historical significance in the development of genetic knowledge: the ability to create local-lesion-forming isolates by nitrous acid mutagenesis of systemic mosaic-inducing strains provided some of the first experimental evidence that mutation resulted from a single base alteration (Gierer and Mundry, 1958).
More complex gene-for-gene interactions between plants and viruses are discussed in chapter 9.
10 R.S.S. Fraser
Table 1. The gene-for-gene interaction between TMV and tobacco
TMV isolate
Host resistance Virulent Avirulent
N systemic mosaic local lesion
(very rare) hypersensitive response
N' systemic mosaic local lesion
(common) hypersensitive response
n systemic mosaic systemic mosaic!
! In a virus isolate, virulence/avirulence is assigned in the light of interaction with a specific resistance gene. Strictly speaking, the term avirulence has no meaning in the context of a host with no known resistance gene against that virus, but this is a matter of semantics, . and does not compromise the integrity of the gene-for-gene interaction.
III. The Biochemistry of the Resistance Response
A. RECOGNITION
The widely supported model for HR, for bacterial and fungal pathogens as well as viruses, is that a 'recognition event' occurs between some product of the resistance gene, and an avirulence determinant in the pathogen. This recognition then induces a series of responses, possibly by complex signalling pathways, which give effect to the resistance mechanism(s) that block the pathogenic process, and a cascade of associated changes. The challenges have been to identify the critical recognition event and the molecular participants in it, the nature of the resistance mechanism(s) induced, and those changes which are secondary. The comparatively simple gene-for-gene interaction between tobacco and TMV has lent itself to analysis of some of these questions.
1. Mapping of the Determinants of Virulence and Avirulence on TMV
TMV is a comparatively simple plant virus. The genetic map (Fig. 3) shows that almost the entire genome is taken up by the replicase, movement protein and coat protein functions, essential to the full replicative cycle in the susceptible plant. There is no 'spare capacity' that could be devoted purely and solely to determinants of virulence or avirulence, if these determinants operated at the protein level. Even if the viral determinant in the recognition event were to operate at the RNA level, or as a product of a virus-coded protein, this would have to be in the overall context of another function for that gene in the pathogenicity of the virus.
The ability to make cDNA copies of plant viruses with RNA genomes, and the fact that these copies can themselves be infectious (Weber et aI., 1992) or can be used to
produce infectious RNA transcripts (Meshi et aI., 1986), revolutionised the approach to genetic mapping and analysis of virulence. Knowledge of sequence data and appropriate restriction enzymes have been used to make artificial recombinants between virulent and avirulent isolates, which could then be tested for biological activity in resistant and susceptible plants. This allowed mapping of determinants first to particular functional regions of the viral genome, and ultimately with the aid of site-directed mutagenesis to individual nucleotide residues and consequent changes in single amino acids in the derived proteins. Intriguingly, given the proposed allelomorphic nature of Nand N', the determinants of virulence/avirulence mapped to different viral functions. For N' the determinant is in the coat protein gene (Culver and Dawson, 1989; Pfitzner and Pfitzner, 1992). Later work demonstrated that a number of amino acid residues at non-contiguous positions in the coat protein sequence are essential for triggering the HR response, but in the three-dimensional folded structure of the protein these come together to form a surface which was proposed as the binding site with the host receptor (Taraporewala and Culver, 1996; 1997). For N, the rare example of a virulent isolate allowed mapping of the determinant to the 126 kDa replicase gene (Ikeda et aI., 1993;
Padgett and Beachy, 1993) (Fig. 3).
Examples of virulence/avirulence determinants for other resistance genes which map to other viral functions are given in chapter 9.
2. Isolation of the N Gene
Early attempts to isolate the N gene utilised differential hybridization procedures to compare messenger RNA or translated protein populations from HR-expressing and non-expressing hosts, and while several additional proteins were found to be expressed during HR, none could be specifically associated with N-gene activity (Smart et aI., 1987, Dunigan et aI., 1987).
Later work involved the use of transposon tagging to disable N-gene function, and selection procedures to detect mutant plants with the tagged N gene (Whitham et aI., 1994; Dinesh-Kumar et aI., 1995). The maize transposon Ac integrates itself into the plant genome and can disable gene function at the point of integration. Tobacco plants with the NN genotype were treated with Ac, and crossed with nn plants. The Nn progeny seedlings were then inoculated with TMV and maintained at 30°C, to allow systemic multiplication of the virus. The seedlings were then shifted to 21°C, when those with a functional N gene suffered lethal systemic necrosis, whereas those with the N gene disabled by transposon insertion survived. One unstable mutant line, giving rise to sec,tored progeny, was shown to contain an Ac element insertion in a large open reading frame. This was used to recover a homologous full length gene from a genomic library of N. glutinosa. This gene was shown to be the N gene by transformation into susceptible tobacco, which subsequently exhibited HR when inoculated with TMV. Further confirmation that this single gene was enough to switch on the HR response after infection came from the demonstration that it could also confer HR in transformed tomato plants (Whitham et aI., 1996). This also demonstrated that all the genetic elements downstream from the initial recognition event between the N gene product and TMV, and necessary for the full expression of HR, are present in tomato as well as in tobacco.
12 R.S.S. Fraser
3. Sequence oj the N Gene and Similarities to Other Genes
Sequence analysis of the N gene cDNA and genomic clones indicated that the gent:
contains five exons which could be spliced to give a protein with 1144 amino acids and a molecular mass of 131 kDa. An alternative splicing route might give rise to a truncated protein of 75 kDa, containing the N-terminal region and an additional C-terminal region of 36 amino acids (Whitham et al., 1994, Dinesh-Kumar et al., 1995) (Fig. 5).
Full-length and truncated forms have been reported for other receptor proteins such as mammalian cytokine and growth factor receptors, although the possible significance of the two forms in vivo and in regulation of N-gene activity is not known.
Structures of pathogen resistance genes in plants
HostJPathogen Gene Resistance gene structure 'r!J
~ ... 00000000
Tobacco/TMV N
N---C
Also
Arabidopsisl RPS21RPMI Tomatol Pseudomo/U1s
'r!J
~
...
0+TobaccolTMV N N C possible truncated
fonn
Flax/flax rust L6 N
- ...
00000000C
Tomato/Cladosporium
-
00000000 !ffulvum Cfl2 N C
Figure 5. Structural features of the N gene for TMV resistance. in full length and possible truncated form. based on data from Whitham el al. (1994). and comparison with other known genes for resistance to bacterial and fungal pathogens. (0000) leucine-rich repeats; (T) nucleotide binding sites; ("11") cytoplasmic signalling domains which may affect nuclear transcription factors; (0) regions of conserved nucleotide sequence; (+) additional sequence of 30 amino acid residues in the putative truncated form of the N-gene protein; (~) leucine zipper or Toll-like domain; ( .. ) signal peptide for membrane transport;
(n) transmembrane domain. Based on data in Bent (1996) and Gebhardt (1997).
Comparisons of the amino acid sequence of the N gene with sequences of other genes of known activities revealed a number of intriguing similarities, which may provide clues to the mechanism of gene action. Firstly, the N gene shows some close structural similarities to a number of other plant genes for disease resistance, including the RPS2 and RPM1 genes for resistance to Pseudomonas syringae pathovars in Arabidopsis (Bent et al., 1994; Grant et al., 1995); Prj for resistance to P. syringae in tomato (Salmeron et al., 1994), and to a lesser extent, L6 for resistance to Melampsora lini in flax (Lawrence et al., 1995). There are therefore common structural elements in genes for resistance to quite distinct types of pathogen: viral, bacterial and fungal. As shown
in Fig. 5, the common elements include those referred to as cytoplasmic elements, three nucleotide binding sites, and a region of leucine-rich repeats. The possible functional significance of these common structural regions will be considered in the next section.
It is important to point out, however, that the product of the N gene is predicted to be cytoplasmic, on the basis of its sequence, whereas several of the genes for resistance to fungal and bacterial pathogens contain trans-membrane domains. This difference may reflect the cytoplasmic location ofTMV multiplication, and the possible involvement of membranes in the recognition of and response to microbial pathogens.
Although there is strong similarity between these genes in terms of functional regions, this is not reflected in strong conservation of nucleic acid sequence, apart from two highly conserved regions (Gebhardt, 1997). Other plant resistance genes against bacterial and fungal pathogens are also known which do not share some or all of these functional regions (Bent, 1996, Gebhardt, 1997).
B. SIGNAL TRANSDUCTION
The main structural motifs described above in N and certain other resistance genes also occur in a number of other proteins whose function is more clearly understood, for example in the activity of mammalian hormones, or control of insect development.
There are helpful indicators here of how the resistance genes may operate against pathogens, although no single clear-cut mechanism emerges. Rather, the implication is that the resistance response is likely to be multifaceted, and composed of pathogen- specific and non-specific elements.
Leucine-rich repeats have been identified in genes in a number of organisms and associated with diverse functions; they are thought to mediate protein-protein interactions (Kobe and Deisenhofer, 1995), and have been proposed as the site of pathogen-specific recognition through interaction with the avirulence gene product (Bent, 1996; Jones and Jones, 1997). The leucine-rich regions are imperfect repeats of units approximately 25 amino acids long: pathogen-specificity might be determined by the other amino acids present. It is likely that the leucine and other hydrophobic residues form the inside of a 'fist' -like structure with the other, intervening amino acids exposed (Bent, 1996). An alternative model is that the leucine-rich repeats might be involved in the interaction with other proteins involved in later stages of signal transduction and activation of the various components of the resistance response (Dixon et aI., 1996).
The nucleotide binding sites appear to be essential for the HR-inducing function, as mutations in these sites may eliminate function, although the mechanism of action is still unclear (Bent, 1996).
The large N-terminal domain of the N gene bears similarity to the so-called cytoplasmic signalling domains in the Drosophila Toll protein, which is a receptor involved in the establishment of polarity during insect development, and in the mammalian interleukin-l receptor involved in response to cytokine IL-l (Whitham et aI., 1994). Both are thought to activate transcription factors which cause changes in gene expression. It has been suggested that this region of the N gene might similarly be involved in the mechanism of activation of genes involved in the HR.
14 R.S.S. Fraser
A further proposal by analogy with the animal pathways is that this region of N might be involved in the stimulated production of activated oxygen species.
The 'oxidative burst' is one of the earliest detectable events in the onset of HR (Doke and Ohashi, 1988; see chaper 6). It may be involved in redox regulation of transcription factors affecting gene expression, as well as potentially in other disease resistance and signalling mechanisms (Tenhaken et ai., 1995; Hammond-Kosack and Jones, 1996). Specifically, it is likely that the oxidative burst is involved in the induction of salicylic acid synthesis, an important signalling molecule for further downstream changes (Klessig and Malamy, 1994; Leon et ai., 1995). The development of HR in N gene tobacco plants inoculated with TMV was inhibited when the plants were exposed to low oxygen pressure, but virus multiplication was not inhibited by low oxygen (Mittler et ai., 1996).
C. HOW DOES N-GENE RESISTANCE TO TMV WORK?
During the hypersensitive response to TMV - and indeed to numerous other pathogens in many plant species - a series of changes occurs in the host as part of programmed cell death in the lesion, in surrounding non-necrotic tissue, and systemically. The changes include accumulation of phytoalexins, synthesis of proteins not normally present in healthy plants, such as the pathogenesis-related (PR) proteins (see chapters 9 and 10), increased activities of certain enzymes such as phenylalanine ammonia lyase and chalcone synthase, alterations in plant cell wall structure, and changes in concentrations of plant growth regulators. The early literature is reviewed in Fraser (1985; 1987) and more recent developments in Hammond-Kosack and Jones (1996).
It does seem that many of the observed changes, although induced by TMV infection and occurring as part of the local lesion response, are not directly involved in virus resistance. Thus there is convincing evidence that some PR proteins are involved in resistance to fungal and bacterial pathogens (Zhu et ai., 1994) but no firm evidence that they have any direct role in virus resistance (Fraser, 1982), although contrary statements do appear in the literature (e.g. Chivasa et ai., 1997).
The early pattern of multiplication of TMV in resistant plants, such as shown in Fig. 4, and the fact that HR resistance does not operate when isolated protoplasts are infected (Otsuki et ai., 1972) suggest that the restriction of virus multiplication and spread requires induction of the host mechanism; it is not constitutive in the initially infected cells. The fact that virus particles can be detected outside the necrotic region (Da Graca and Martin 1976; Konate et ai., 1983) suggests that cell necrosis in the core of the lesion is not the active defence mechanism, but a secondary response.
This leaves two likely mechanisms. The tissue around the focus of infected cells may have induced in it some inhibitor of virus multiplication, which increasingly restricts multiplication. Alternatively or additionally, the spread of virus from cell to cell might be inhibited.
Possible inhibitors of virus replication (IVR) have been isolated from TMV -infected N-gene tobacco plants (Gera et ai., 1990, 1993), and these might be involved in the resistance response. The extent of inhibition of virus multiplication in the assays used appeared too low to account for the complete effectiveness of N in stopping
infection, and non-specific inhibitory effects on plant metabolism were not excluded.
Another group of putative antiviral factors (A VF) was reported by Sela et al. (1987) and extensive comparisons were drawn with the human interferon system, but the relationship to N-gene resistance in vivo is not clear. Some of the plant proteins purified using monoclonal antibodies to human interferon have now been shown to have no sequence similarity to interferon (Edelbaum et aI., 1990), and to correspond to the TMV-induced PR proteins t3-1,3-glucanase and another PR of unknown function (Edelbaum et aI., 1991).
Inhibition of cell-to-cell spread appears an attractive possible explanation of localizing resistance, and for other examples of mechanisms of resistance to plant viruses the evidence that it is the main mode of action is quite compelling (chapter 9).
What is known is that creating the conditions required for cell-to-cell spread of infection is an active process, involving a virus-coded movement protein and modification of the plasmodesmata (Lucas and Gilbertson, 1994). In the case of the HR response to TMV infection of tobacco, it may be notable that the amount of 30 kDa movement protein in the cell wall fraction decreases sharply as virus localization commences and necrosis appears (Moser et al., 1988).
It is also possible that restriction of virus spread involves the creation of physical barriers by alterations in wall structure or plasmodesmatal properties. There are several reports of increased lignification and deposition of callose at the lesion edge (reviewed by Fraser, 1985; Hammond-Kosack and Jones, 1996). Interestingly, N-gene tobacco plants transformed to express an antisense construct of the basic tobacco class 1 t3-1,3-glucanase, and as a result showing reduced glucanase activity, were shown to have decreased spread of TMV when inoculated (Beffa et aI., 1996). This was attributed to an increased deposition of callose on the plasmodesmatal pores, which may have hindered cell-to-cell spread of the virus. While this is an attractive possible explanation of enhanced localisation, it is difficult to reconcile with the increased glucanase activity stimulated by necrotic infection in the form of PR proteins, and the smaller lesions formed on inoculation of leaves with acquired systemic resistance (chapters 9 and 10). Clearly, there are many intriguing clues to how N-gene resistance to TMV may operate: the elucidation of the mechanism, or the relative importance of different components of the mechanism, is eagerly awaited.
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BLACK ROT OF CRUCIFERS
ANNE M. ALVAREZ University of Hawaii,
Department of Plant Pathology
3190 Maile Way, Honolulu, H196822 ([email protected])
"In July, 1889, cabbage in the vicinity of Lexington was badly affected with a rot, which bore marks of being caused by the bacteria in the tissue. In some gardens two-thirds of the heads were affected, and of these more than half were completely invaded and rendered worthless ... The invaded leaves became brown and watery at first; later, to become black as the decay had reached an advanced stage. The heads ... gave forth a peculiarly noxious odor such as cabbage alone among vegetables is capable of producing. This final rotting was doubtless ordinary decomposition brought about by septic bacteria ... it seems to me we have here a well-marked disease of cabbage ... attributable, perhaps, to several causes working together, and at least encouraged by the vital activities of bacteria ... it is only during periods of high temperature and excessive rainfall that the organisms are able to invade and break down the tissues of plants." - and thus proceeds the first description of black rot as observed in Lexington, Kentucky (Garman, 1890).
Summary
In just over 100 years, the focus on host-pathogen interactions in the black rot disease has shifted gradually from basic aspects of the disease cycle to enzyme production and gene regulation at the molecular level. The wealth of information provided through a long history of research makes it an interesting case study. Yet, one first asks whether anyone pathosystem can provide a well-rounded view of plant-microbe interactions. Although far from complete, a thorough examination of a single bacterial disease provides a framework for deciphering the language of host-pathogen signalling mechanisms.
To understand this system in depth, we take a broad overview of the disease, the pathogen and its natural variability. Then beginning with initial inoculum and epiphytic coloniz
