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62 Agrawal, A.A. and Karban, R. (1999) Why induced defenses may be favored over constitutive strategies in plants. In The Ecology and Evolution of Inducible Defenses (Tollrian, R. and Harvell, C.D., eds), pp. 45–61, Princeton University Press 63 Grandbastien, M.A. (1998) Activation of plant retrotransposons under stress conditions. Trends Plant Sci. 3, 181–187 64 van Dam, N. et al. (2001) Instar-specific sensitivity of specialist Manduca sexta larvae to induced defences in their host plant Nicotiana attenuata. Environ. Entomol. 26, 578–586 65 Felton, G.F. and Korth, K.L. (2000) Trade-offs between pathogen and herbivore resistance. Curr. Opin. Plant Biol. 3, 309–314 66 Thaler, J.S. (1999) Jasmonate-inducible plant

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defences cause increased parasitism of herbivores. Nature 399, 686–688 Peng, J. et al. (1999) ‘Green revolution’ genes encode mutant gibbereline response. Nature 400, 256–261 Bowling, S.A. et al. (1997) The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 9, 1573–1584 Clarke, J.D. et al. (1998) Uncoupling PR gene expression from NPR1 and bacterial resistance: characterization of the dominant Arabidopsis cpr6-1 mutant. Plant Cell 10, 557–569 Yoshioka, K. et al. (2001) Environmentally sensitive, SA-dependent defense responses in the cpr22 mutant of Arabidopsis. Plant J. 26, 447–459

Balancing selection favors guarding resistance proteins Renier A.L. Van der Hoorn, Pierre J.G.M. De Wit and Matthieu H.A.J. Joosten The co-evolutionary arms race model for plant–pathogen interactions implies that resistance (R ) genes are relatively young and monomorphic. However, recent reports show R gene longevity and co-existence of multiple R genes in natural populations. This indicates that R genes are maintained by balancing selection, which occurs when loss of the matching avirulence (Avr ) gene in the pathogen is associated with reduced virulence. We reason that balancing selection favors R proteins that function as guards, monitoring changes in the virulence target mediated by the Avr factor, rather than recognizing the Avr factor itself. Indeed, the available experimental data support the notion that guarding is prevalent in gene-for-gene interactions.

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71 Petersen, M. et al. (2000) Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103, 1111–1120 72 Hilpert, B. et al. (2001) Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions. Plant J. 26, 435–446 73 Cipollini, D. et al. Costs of induced responses. Basic Appl. Ecol. (in press) Note added in proof Recently, Don Cipollini and co-workers [73] have presented an alternative point of view on the costs of induced responses, which might be an interesting addition to what is presented here.

matching gene pairs control the outcome of the interaction for many other pathosystems [3]. A logical prediction of the gene-for-gene model is that R genes encode receptors that interact physically with products of matching Avr genes, enabling recognition of the pathogen and subsequent elicitation of an array of plant defense responses that eventually lead to resistance [4]. The structure and predicted location of R and Avr proteins are usually consistent with this model [5]. For example, most R proteins carry leucine-rich repeats (LRRs), which are thought to form a versatile binding domain that could fulfill the receptor role of the R protein. In addition, membrane-anchored R proteins mediate the perception of extracellular Avr factors, whereas cytoplasmic R proteins mediate the perception of Avr factors that are produced in or injected into the host cytoplasm by the pathogen. Although these observations agree with the ligand–receptor model, a direct physical interaction between Avr and R proteins has only been shown for the AvrPto–Pto and AvrPita–Pi-ta pairs [6–8]. In most other cases (e.g. AVR9–Cf-9 [9]), in spite of extensive and detailed studies, no evidence for a direct interaction between the two gene products has been found. Guard model

Renier A.L. Van der Hoorn Pierre J.G.M. De Wit Matthieu H.A.J. Joosten* Laboratory of Phytopathology, Wageningen University, The Netherlands. *e-mail: matthieu.joosten@ fyto.dpw.wag-ur.nl

Classical resistance breeding has long been used to suppress plant diseases. Many resistance genes have been introduced into crop plants, resulting in new pathogen-resistant cultivars that quickly became popular and were grown as homogeneous crops. However, in many cases, pathogens were eventually able to overcome resistance, resulting in outbreaks of large epidemics. The plant cultivar that was once ‘booming’ but now ‘busts’, forced breeders to introduce a cultivar with a new resistance trait. Repeated boomand-bust cycles in agriculture have provided material for extensive studies on various plant–pathogen interactions [1]. Genetic studies of the flax–flax-rust pathosystem led to the development of the gene-forgene model, which states that, for every dominant resistance (R) gene in the plant, there is a matching dominant avirulence (Avr) gene in the pathogen [2]. After this classic work, it became evident that http://plants.trends.com

Lack of evidence for direct Avr–R interactions stimulated scientists to propose new models for Avr perception by resistant plants. One interesting model is that R proteins confer recognition of Avr factors only when these Avr factors are complexed with their host virulence targets. This model was initially proposed [10] to explain the role of Prf in AvrPto–Pto signaling and was later referred to as the guard model [11]. In this model, Pto is considered to be the virulence target of AvrPto, which is guarded by the ‘real’R protein, Prf [10]. Although the guard model needs to be proved experimentally, it has gained increasing support from experimental data obtained for most of the intensively studied gene-for-gene pairs [12]. Table 1 shows nine examples in which the R protein seems to guard the virulence target and monitors changes of this target mediated by the Avr factor. In general, three observations support the guard model. First, no

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Table 1. Nine examples of putative guarding R proteins R protein

Putative virulence targets

Avr protein

Virulence target mutantsa

Correlationb

Cf-2 Cf-9 HRT Prf RPG4d RPM1 RPP5 RPS2 RPS5

Rcr3c

AVR2 AVR9 CP AvrPto Syringolide (AvrD) AvrB/AvrRpm1 AvrRPP5d AvrRpt2 AvrPphB

+ – – + – + – – +

ND + + + + ND ND ND ND

aExistence

HABSd,e TIPf Ptog P34h RIN4i AtRSH1j 75kDd,k PBS1l

of a loss-of-function mutant for the putative virulence target is indicated by +.

bA correlation between elicitor activity of Avr mutants and their binding affinity for the putative virulence target is indicated by +. ND, not determined. cRcr3

is specifically required for Cf-2 function but not for function of the highly homologous Cf-5 protein [44]. gene not yet cloned. eHABS is a high-affinity binding site for AVR9 that was detected in solanaceous plants [45]. The absence of a detectable interaction between AVR9 and Cf-9 [9] and the positive correlation between the affinity of AVR9 mutants for the HABS and their elicitor activity in Cf-9 plants [46] suggest that the HABS is required for Cf-9-mediated recognition of AVR9. f TIP is a putative transcription factor that interacts with the coat protein (CP) of Turnip Crinkle Virus, which acts as an elicitor for the HRT resistance gene. A positive correlation between the binding of CP mutants to TIP and their elicitor activity on HRT-containing plants suggests that TIP is required for HRT-mediated recognition of CP [47]. gPto is a kinase that probably regulates basal defense responses [48,49] and interacts directly with AvrPto [6,7]. Mutants of either AvrPto and Pto that no longer interact fail to induce defense responses [50–52]. Prf does not act upstream of Pto [53] and probably guards Pto to monitor changes induced upon binding of Pto to AvrPto [10]. hP34 is a vegetative storage protein with putative thiol-protease activity [54] that interacts with syringolides, the elicitors generated through AvrD. A positive correlation between the affinity of syringolide derivatives for P34 and their elicitor activity in Rpg4-containing plants [54] suggests that P34 is required for Rpg4-mediated recognition of AvrD. iRIN4 probably regulates basal defense responses and is modified upon binding to AvrB and AvrRpm1. It is specifically required for RPM1 function, interacting physically with the RPM1 protein and enhancing its stability (D. Mackey et al., pers. commun.). jAtRSH1 is a protein that probably mediates (p)pGpp signaling, which might regulate defense responses. It interacts physically with RPP5 but not with other R proteins [55]. k75kD is an uncharacterized, cytoplasmic protein that immunoprecipitates with both RPS2 and AvrRpt2 [56]. lPBS1 is a kinase that belongs to different a subfamily from Pto. The function of RPS5 depends on the presence of PBS1 [57]. dCorresponding

direct interaction is found between Avr factors and R proteins. Second, recognition of the Avr factor requires an additional host protein that is specific for each Avr–R pair. Third, the structure and predicted function of this host protein, or its general occurrence, suggest that it might be a virulence target for the pathogen. The available data suggest that resistance based on guarding is prevalent in gene-for-gene interactions. This raises the intriguing question of why guarding R proteins have become prevalent in nature. We propose a model that could explain this, based on recent progress in understanding the dynamics of gene-for-gene interactions in nature and their implications at the molecular level. Behavior of R genes in natural plant populations

In nature, the ongoing battle between plants that develop novel resistance specificities and pathogens that try to circumvent recognition by these plants can be seen as an arms race. Such an arms race implies a transient polymorphism of R genes, which means that high disease pressure causes the replacement of old R genes by new ones, resulting in relatively young R genes and monomorphic R gene loci [13]. However, recent studies of functional RPM1 and Pto genes in species of Arabidopsis and Lycopersicon, respectively, revealed that these genes already existed before speciation [14,15]. In addition, the extensive polymorphism in RPP1, RPP13 and RPS2 alleles, suggests that these genes are also ancient [13,16]. Slow R gene evolution also agrees with the suppressed recombination observed at many R gene http://plants.trends.com

loci [17–20]. In studies on AvrRpm1 recognition in Arabidopsis and AVR9 recognition in Lycopersicon pimpinellifolium, it was also observed that, in natural populations, plants recognizing an Avr factor co-exist with plants that lack this trait [14,21]. Other studies have shown that the greatest diversity of resistance traits in natural plant populations is found in areas with the highest disease pressure [22]. Together, these data indicate that the frequencies of R genes in natural plant populations are balanced by frequency-dependent selection. Thus, an R gene becomes prevalent as a result of its selective advantage, whereas the frequency of such an R gene is reduced when the corresponding pathogen causes less disease pressure. This balancing selection implies the existence of two counteracting forces: a cost of virulence for the pathogen (explained below) and a cost of resistance for the host, which is poorly understood [23]. Balancing selection results in a balanced polymorphism of R genes and can maintain Avr–R gene pairs over an indefinite period of time. To illustrate balancing selection of Avr–R gene pairs, the metaphor of ‘trench warfare’ was introduced [14]. Similarly, the term ‘recycling polymorphism’ has been used to describe the dynamics of R genes under balancing selection [24]. Balancing selection and the cost of virulence

The virulence role of the Avr factor is crucial for balancing selection. Many Avr factors contribute to virulence of the pathogen [25] but their relative contributions are often difficult to assess in

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R gene frequency (%)

100

R2

R4

R5

R6

R3

R3 R4 R1

R1 0 Plant generations

TRENDS in Plant Science

Fig. 1. Model for selection of R genes in natural plant populations showing the frequency of various R genes (R1–R6) in the plant population over a long period of time. The marks on the x axis represent several dozen plant generations. Circles mark the point at which new R genes are generated (filled circles) or when pathogens circumvent recognition by a mutation in the matching Avr gene (open circles). Selection pressure after these events results in the rise and fall, respectively, of the R gene frequency. Incidentally, non-functional R genes become extinct (crosses). Recognition of the Avr gene product by R1, R3 and R4 gene products cannot be circumvented without a reduction in virulence of the pathogen, resulting in maintenance of these genes by balancing selection (horizontal lines). By contrast, recognition of Avr factors that match R2, R5 and R6 can be circumvented by mutations in the Avr gene without causing reduced virulence of the pathogen. Consequently, the modified Avr genes will replace the original Avr gene in the pathogen population, and the matching R genes are likely to become rare (R5) or extinct (R2 and R6 ).

laboratory experiments [26–28]. In addition, most pathogens lacking Avr genes were isolated from crops grown as monocultures. These pathogens might have been able to compensate for their reduced virulence during prolonged maintenance on susceptible crops. In nature, this compensation is less likely to occur because the pathogen continuously encounters different plant genotypes. Balanced polymorphism of R genes in nature will only occur when the matching Avr gene contributes to virulence of the pathogen. Without any virulence function of the Avr factor, the selection pressure on the pathogen, imposed by the plant population that acquires the matching R gene, will result in selection for loss or mutated versions of the Avr gene. As a result, the original Avr gene will become rare or even extinct. However, Avr factors with a virulence role will be maintained in the pathogen population even though this will result in avirulence on a subpopulation of the host. For example, selection against strains lacking an Avr gene has been observed for the avrXa7 gene of the rice blast fungus [29]. Only some of the Avr–R gene pairs are maintained by balancing selection

Balancing selection can explain many recent observations but does not provide an explanation for the generation of new R genes with novel specificities. Therefore, R gene dynamics in a natural plant population probably reflect a combination of balancing selection and an arms race, the latter perhaps being relatively slow (Fig. 1). As has been suggested previously, R gene analogs (RGAs) are randomly generated, most likely through a birth-and-death process [30]. Most of these new RGAs have no direct function and probably exist as rare genes for relatively short periods of time. Some of them might confer recognition of host factors, http://plants.trends.com

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and those triggering defense autonomously would have suicidal effects and would quickly disappear from the plant population. Incidentally, a novel RGA confers recognition of a pathogen and becomes an R gene when it confers resistance. Its selective advantage will soon result in an increase in its frequency in the plant population (Fig. 1). A similar model for RGA dynamics, entitled ‘evolved recycling polymorphism’, has been proposed recently [24]. It is important in our model that only some of the newly generated R genes will be maintained by balancing selection. As explained above, balancing selection will only occur for R genes when the corresponding Avr gene of the pathogen contributes to virulence. If this is not the case, the corresponding R gene will become rare or even extinct. Importantly, this selection will eventually result in maintenance of only those R genes that cause reduced virulence when the pathogen tries to circumvent recognition (Fig. 1). Three additional comments can be added to this model. First, there is little epidemiological evidence supporting this model because only a few studies have been performed, and in these studies, population dynamics have been studied for only a dozen generations of both host and pathogen [31]. Therefore, studying pathosystems with short life cycles or exploiting fossils or herbarium samples [32] might be useful. Second, the average frequency of an R gene that is maintained in the plant population might reflect the relative contribution of the matching Avr gene to the virulence of the pathogen. For example, the Avr gene matching R3 (Fig. 1) would be more important for virulence of the pathogen than the Avr gene that matches R1. Third, R gene dynamics in natural plant populations are different from those in crop plants. The extreme resistance that is desired in monocultures of crop plants results in boom-and-bust cycles. Strains of pathogens that overcome R genes present in monocultures can cause epidemics, even if they have a reduced virulence through loss of Avr factors. These strains have no real competitors. To protect crops better in modern agriculture, it would be useful to employ multilines, in which many plant varieties that differ only in a single R gene are mixed, thereby simulating the polymorphism of natural plant populations [11]. The recent success of growing mixed rice cultivars illustrates the potential of this strategy, resulting in a 94% reduction in the occurrence of rice blast [33]. Selection for guarding R proteins

The proposed model for the behavior of gene-for-gene interactions in natural populations of plants and pathogens (Fig. 1) provides an explanation for the putative prevalence of guarding R proteins. To illustrate the selection process at the molecular level, imagine a particular virulence target in a susceptible host (Fig. 2a) that is modified by an Avr factor of the pathogen (Fig. 2b). This virulence target can represent a complex of multiple cellular components and its Avr-induced modification might, for example, represent a conformational change,

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(a) Before infection VT

(b) During infection Avr

VT Susceptibility (VT modification)

(c) New R proteins R2

Avr

Avr

R3 VT Resistance (by guarding)

Resistance (direct interaction)

(d) Selected R protein Avr′

R3

Implications for future research

VT Resistance (by guarding) TRENDS in Plant Science

Fig. 2. Simplified model to illustrate natural selection for guarding R proteins. (a) Before infection, a virulence target (VT) exists in the host plant. (b) Infection of a susceptible plant results in binding of the avirulence factor (Avr) to its VT, resulting in a modification of the VT (altered shape) and leading to virulence (susceptibility of the host). (c) In a resistant plant population, different R proteins (e.g. R2 and R3) have evolved that recognize the Avr factor itself (R2, blue) or an Avr-induced modification of the VT (R3, red). These two R proteins correspond to the R genes used in Fig. 1. The recognition event activates the R protein, triggering defense responses that render the plant resistant. Direct recognition (as mediated by R2) can be circumvented by alterations of the Avr factor without affecting its virulence function. By contrast, the recognition mediated by R3 cannot be circumvented by alteration of the Avr factor without losing its virulence function. (d) During selection for mutations in the Avr gene that result in a novel Avr factor (Avr′), R3 is likely to be maintained as a common R protein in the plant population by balancing selection, whereas R2 becomes rare or even extinct. Thus, only guarding R proteins are maintained as a result of this selection.

Acknowledgements We apologize to all our colleagues whose work could not be reviewed here because of space limitations. We thank Frank Takken, Marco Kruijt, Bas Brandwagt, Ronelle Roth, Maarten de Kock and Rianne Luderer for critically reading the manuscript, and Jonathan Jones for valuable discussions. We also gratefully acknowledge Jeff Dangl for sharing unpublished data.

(de)phosphorylation, ubiquitination, or recruitment or release of additional factors. Over many generations of the host, many R proteins have been randomly generated and have been selected for detection of the Avr factor itself (R2; Fig. 2c) or of an Avr-induced modification of the virulence target (R3; Fig. 2c). As discussed above, balancing selection will select for R genes that confer recognition that cannot be circumvented without causing reduced virulence of the pathogen. Importantly, recognition based on a direct physical interaction between R protein and Avr factor can easily be circumvented by alteration of the Avr structure, without affecting its virulence role. However, recognition by R proteins that guard the virulence target cannot be circumvented by mutations in the Avr gene without affecting its virulence function. These R proteins will be maintained in the plant population http://plants.trends.com

(Fig. 2d), explaining the putative prevalence of guarding R proteins in current gene-for-gene interactions. There are at least three aspects that are not included in this simplified model. First, the mode of action can differ between different guarding R proteins. For example, the R protein might dissociate from, or bind to the virulence target upon Avr binding; remain bound to the virulence target or detect more distal Avr-induced modifications. Second, Avr mutations with intermediate effects might lead to counteradaptation of the matching R protein. Such microscale co-evolution would be similar to that observed between polygalacturonases or chitinases and their inhibitors [34]. Third, an additional possibility for a pathogen to circumvent recognition without a reduction in virulence is to rely on virulence factors that interfere with R protein guarding. For example, AvrRpt2 interferes with AvrRpm1 recognition [35]. Similarly, AvrPphC interferes with AvrPphF recognition, whereas AvrPphF itself interferes with recognition of another, uncharacterized, Avr factor [36].

From the model described above and from the available data (Table 1), we predict that guarding is the main mechanism in gene-for-gene-based resistance, although there are already exceptions. For example, AvrPita interacts directly with the R protein Pi-ta [8]. However, it is intriguing that AvrPita is a putative metalloprotease [8], which could hint to its function in virulence for the pathogen. Therefore, it is possible that Pi-ta is the virulence target of AvrPita, and that modification of Pi-ta is recognized by the actual, yet unidentified, R protein. In addition to the gene pairs presented in Table 1, there are various additional observations that might be explained by the guard model. For example, the dual specificity of RPM1 [37] and Mi [38,39] might be due to the presence of different Avr factors with the same virulence target. In addition, the restricted taxonomic functionality of some R genes, such as Bs2 [40] and Cf-9 [41], might be due to the absence of the virulence target in other plant species. Finally, digenic resistance, in which a cross between two susceptible lines results in resistant progeny [42,43], might be the result of polymorphism in both the virulence target and the R gene. Significantly, the guard model puts limits on the use of R genes in transgenic crops. Even though most R proteins belong to conserved families, their function might depend on virulence targets that are not necessarily conserved across the plant kingdom. This decreases the potential to use a simple model plant species as a source of natural or artificial R genes to introduce resistance in another plant species. Therefore, the identification of virulence targets is as crucial as the identification of R genes for transgenic approaches to improve disease resistance in crops. In conclusion, we anticipate that the guard model marks a turning point in research on gene-for-gene

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interactions. After the cloning of a large array of R genes during the past decade, the identification of virulence targets of Avr factors is a new challenge for the future. Sensitive (bio)assays to assess the References 1 Johnson, R. (1984) A critical analysis of durable resistance. Annu. Rev. Phytopathol. 22, 309–330 2 Flor, H.H. (1942) Inheritance of pathogenicity in Melampsora lini. Phytopathology 32, 653–669 3 Thompson, J.N. and Burdon, J.J. (1992) Gene-forgene coevolution between plants and parasites. Nature 360, 121–125 4 Keen, N.T. (1990) Gene-for-gene complementarity in plant–pathogen interactions. Annu. Rev. Genet. 24, 447–463 5 Takken, F.L.W. and Joosten, M.H.A.J. (2000) Plant resistance genes: their structure, function and evolution. Eur. J. Plant Pathol. 106, 699–713 6 Tang, X. et al. (1996) Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase. Science 274, 2060–2063 7 Scofield, S.R. et al. (1996) Molecular basis of genefor-gene specificity in bacterial speck disease of tomato. Science 274, 2063–2065 8 Jia, Y. et al. (2000) Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J. 19, 4004–4014 9 Luderer, R. et al. (2000) No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum. Mol. Plant–Microbe Interact. 14, 867–876 10 Van der Biezen, E.A. and Jones, J.D.G. (1998) Plant disease-resistance proteins and the gene-forgene concept. Trends Biochem. Sci. 23, 454–456 11 Dangl, J.L. and Jones, J.D.G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411, 826–833 12 Luderer, R and Joosten, M.H.A.J. (2001) Avirulence proteins of plant pathogens: determinants of victory and defeat. Mol. Plant Pathol. 2, 355–364 13 Bergelson, J. et al. (2001) Evolutionary dynamics of plant R-genes. Science 292, 2281–2285 14 Stahl, E.A. et al. (1999) Dynamics of disease resistance polymorphism at the Rpm1 locus of Arabidopsis. Nature 400, 667–671 15 Riely, B.K. and Martin, G.B. (2001) Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto. Proc. Natl. Acad. Sci. U. S. A. 98, 2059–2064 16 Caicedo, A.L. et al. (1999) Diversity and molecular evolution of the RPS2 resistance gene in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 96, 302–306 17 Chin, D.B. et al. (2001) Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa). Genetics 157, 831–849 18 Wei, F. et al. (1999) The Mla (powdery mildew) resistance cluster is associated with three NBSLRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley. Genetics 153, 1929–1948 19 Van Daelen, R.A.J.J. et al. (1993) Long-range physical maps of two loci (Aps-1 and GP79) flanking the root-knot nematode resistance gene (Mi) near the centromere of tomato chromosome 6. Plant Mol. Biol. 23, 185–192 20 Ganal, M.W. et al. (1989) Pulsed field gel electrophoresis and physical mapping of the large DNA fragments in the Tm-2a region of chromosome 9 in tomato. Mol. Gen. Genet. 215, 395–400 21 Van der Hoorn, R.A.L. et al. (2001) Intragenic http://plants.trends.com

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virulence role of Avr factors have to be developed and a combination of various biochemical and genetic approaches should allow recognition mechanisms mediated by guarding R proteins to be unraveled.

recombination generated two distinct Cf genes that mediate AVR9 recognition in the natural population of Lycopersicon pimpinellifolium. Proc. Natl. Acad. Sci. U. S. A. 98, 10493–10498 Leonard, K.J. (1997) Modelling gene frequency dynamics. In The Gene-for-Gene Relationship in Plant–Parasite Interactions (Crute, I.R. et al., eds), pp. 211–231, CAB international, Wallingford, UK Purrington, C.B. (2000) Costs of resistance. Curr. Opin. Plant Biol. 3, 305–308 Holub, E.B. (2001) The arms race is ancient history in Arabidopsis, the wildflower. Nat. Rev. Genet. 2, 515–527 Van ’t Slot, K.A.E. and Knogge, W. Peptide products of pathogen virulence genes as elicitors. Crit. Rev. Plant Sci. (in press) Chen, Z. et al. (2000) The Pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells. Mol. Plant–Microbe Interact. 12, 1312–1321 Shan, L. et al. (2000) A cluster of mutations disrupt the avirulence but not the virulence function of AvrPto. Mol. Plant–Microbe Interact. 13, 592–598 Laugé, R. et al. (1997) The in planta-produced extracellular proteins ECP1 and ECP2 of Cladosporium fulvum are virulence factors. Mol. Plant–Microbe Interact. 10, 725–734 Vera Cruz, C.M. et al. (2000) Predicting durability of a disease resistance gene based on an assessment of the fitness loss and epidemiological consequences of avirulence gene mutation. Proc. Natl. Acad. Sci. U. S. A. 97, 13500–13505 Michelmore, R.W. and Meyers, B.C. (1998) Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res. 8, 1113–1130 Burdon, J.J. and Thrall, P.H. (1999) Spatial and temporal patterns in coevolving plant and pathogen associations. Am. Nat. 153, S15–S33 Ristaino, J.B. et al. (2001) PCR amplification of the Irish potato famine pathogen from historic specimens. Nature 411, 695–697 Zhu, Y. et al. (2000) Genetic diversity and disease control in rice. Nature 406, 718–722 Stahl, E.A. and Bishop, J.G. (2000) Plant–pathogen arms race at the molecular level. Curr. Opin. Plant Biol. 3, 299–304 Ritter, C. and Dangl, J.L. (1996) Interference between two specific pathogen recognition events mediated by distinct plant disease resistance genes. Plant Cell 8, 251–257 Tsiamis, G. et al. (2000) Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease. EMBO J. 19, 3204–3214 Grant, M.R. et al. (1995) Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science 269, 843–846 Rossi, M. et al. (1998) The nematode resistance gene Mi of tomato confers resistance against the potato aphid. Proc. Natl. Acad. Sci. U. S. A. 95, 9750–9754 Vos, P. et al. (1998) The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids. Nat. Biotechnol. 16, 1365–1369 Tai, T.H. et al. (1999) Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in

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tomato. Proc. Natl. Acad. Sci. U. S. A. 96, 14153–14158 Van der Hoorn, R.A.L. et al. (2000) Agroinfiltration is a versatile tool that facilitates comparative analysis of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol. Plant–Microbe Interact. 13, 439–446 Buell, C.R. and Somerville, S.C. (1997) Use of Arabidopsis recombinant inbred lines reveals a monogenic and a novel digenic resistance mechanism to Xanthomonas campestris pv campestris. Plant J. 12, 21–29 Singh, R.P. and McIntosh, R.A. (1984) Complementary genes for resistance to Puccinia recondita tritici in Triticum aestivum. I. Genetic and linkage studies. Can. J. Genet. Cytol. 26, 723–735 Dixon, M.S. et al. (2000) Genetic complexity of pathogen perception in plants: the example of Rcr3, a tomato gene required specifically by Cf-2. Proc. Natl. Acad. Sci. U. S. A. 97, 8807–8814 Kooman-Gersmann, M. et al. (1996) A highaffinity binding site for the AVR9 peptide elicitor of Cladosporium fulvum is present on plasma membranes of tomato and other solanaceous plants. Plant Cell 8, 929–938 Kooman-Gersmann, M. et al. (1998) Correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptide elicitors. Plant Physiol. 117, 609–618 Ren, T. et al. (2000) HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to Turnip Crinkle Virus. Plant Cell 12, 1917–1925 Tang, X. et al. (1999) Overexpression of Pto activates defense responses and confers broad resistance. Plant Cell 11, 15–29 Xiao, F. et al. (2001) Expression of 35S::Pto globally activates defense-related genes in tomato plants. Plant Physiol. 126, 1637–1645 Chang, J.H. et al. (2001) Functional studies of the bacterial avirulence protein AvrPto by mutational analysis. Mol. Plant–Microbe Interact. 14, 451–459 Frederick, R.D. et al. (1998) Recognition specificity for the bacterial avirulence protein AvrPto is determined by Thr-204 in the activation loop of the tomato Pto kinase. Mol. Cell 2, 241–245 Sessa, G. et al. (2000) Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto–Pto-mediated hypersensitive response. EMBO J. 19, 2257–2269 Rathjen, J.P. et al. (1999) Constitutively expressed Pto induces a Prf-dependent hypersensitive response in the absence of avrPto. EMBO J. 18, 3232–3240 Ji, C. et al. (1998) Characterisation of a 34-kDa soybean binding protein for the syringolide elicitors. Proc. Natl. Acad. Sci. U. S. A. 95, 3306–3311 Van der Biezen, E.A. et al. (2000) Arabidopsis RelA/ SpoT homologs implicate (p)ppGpp in plant signaling. Proc. Natl. Acad. Sci. U. S. A. 97, 3747–3752 Leister, R.T. and Katagiri, F. (2000) A resistance gene product of the nucleotide binding site – leucine rich repeat class can form a complex with bacterial avirulence proteins in vivo. Plant J. 22, 345–354 Swiderski, M. and Innes, R.W. (2001) The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily. Plant J. 26, 101–112