Molecular secrets of bacterial type III effector proteins

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Molecular secrets of bacterial type III effector proteins Thomas Lahaye and Ulla Bonas Most Gram-negative phytopathogenic bacteria are thought to inject effector proteins into the plant cell via a type III secretion system that is essential for pathogenicity. Plant targets and the mode of action of type III effector proteins, which include avirulence (Avr) proteins, are largely unknown. However, recent findings have shed light on the molecular mechanisms of Avr action. Here, we focus on two classes of Avr proteins (the AvrBs3 and AvrRxv/YopJ families) that have been suggested to act as transcription factors and proteases, respectively.

Thomas Lahaye* Ulla Bonas Institute of Genetics, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle (Saale), Germany. *e-mail: lahaye@ genetik.uni-halle.de

Phytopathogenic bacteria are equipped with a wide range of effector molecules that collectively favor growth in their natural habitat. For many Gramnegative pathogenic bacteria, colonization of the host’s tissue depends on a molecular syringe, the type III protein secretion system (TTSS), which secretes and probably translocates effector proteins into the host cell1,2. To combat infection, plants have evolved sophisticated defense systems, targeted against individual type III-secreted proteins and associated with a rapid, localized programmed cell death, the HYPERSENSITIVE RESPONSE3 (HR; see Glossary). Bacterial effector proteins that betray the parasite to the plant surveillance system encoded by resistance (R) genes have been termed avirulence (Avr) proteins. All bacterial Avr proteins analyzed to date are secreted by the type III system encoded by a group of HYPERSENSITIVE RESPONSE AND PATHOGENICITY (hrp) GENES. The evidence that Avr proteins are translocated into plant cells is still indirect and is based on the fact that in planta expression of avr genes via Agrobacterium or biolistic delivery induces an R-gene-dependent HR (Ref. 4). This avr-triggered cell death has been helpful for both the analysis of the recognition process and the investigation of type III effectors and their secretion. The idea that host–pathogen cross-talk is shaped by evolutionary forces implies that avr genes should provide a selective advantage to the bacterium in susceptible plants. Indeed, many avr genes have been shown to play a role in virulence5,6. Detailed analysis of individual avr genes [e.g. avrBs2, avrRpm1, avrE and pthA (Refs 5–8)] has provided clues about their importance for the bacterial parasite in compatible interactions. However, the biochemical function of most is still unknown. The pathogenicity function of Avr proteins is an important question, not only for geneticists and microbiologists but also for plant breeders, because the assessment of the fitness penalty associated with http://plants.trends.com

the loss of a given effector protein can be a direct measure of the durability of the matching R gene used in crop protection strategies9. Here, we summarize recent findings on two particular avr gene families, providing insights into the molecular mechanisms of the host–parasite cross-talk. Bacterial type III secretion and type III effectors are reviewed in Refs 1,5–8,10,11. Revisiting the receptor–ligand model for pathogen recognition

The genetic model of gene-for-gene resistance is based on the observation of complementary genes in the host (R genes) and in the pathogen (avr genes) that are required for the recognition to occur. Generally, this genetic model has been interpreted as a receptor–ligand interaction in which a pathogenderived Avr ligand binds directly to a corresponding R protein to initiate a resistance reaction12 (Fig. 1). An alternative interpretation of the genetic model is the more recently proposed ‘guard model’, which assumes an indirect interaction between Avr and R proteins13 (Fig. 1). This model predicts that Avr Glossary Hypersensitive response (HR) The HR involves rapid, spatially confined cell suicide (programmed cell death) concomitant with halt of pathogen spread. Hypersensitive response and pathogenicity (hrp) genes The hrp genes are required to elicit the HR in resistant hosts and non-hosts, and to be pathogenic in hosts. The hrp genes are thought to encode a type-III protein-secretion system that injects proteins into plant cells. Nuclear localization signals (NLSs) NLSs are short amino acid motifs that are sufficient and necessary for nuclear targeting of a given protein. NLS motifs are recognized by importin α, which mediates nuclear targeting in complex with importin β. Acidic transcriptional activation domain (AAD) In contrast with the DNA-binding domains, transcriptional activation domains are not well conserved. AADs are rich in acidic amino acids. κB) NF-κB is a transcription factor that plays Nuclear factor κB (NF-κ a central role in cellular responses to environmental stimuli. κB (Iκ κB): IκB interacts with NF-κB and masks the Inhibitor of NF-κ NLSs of NF-κB. NF-κB is activated by dissociation of IκB, allowing NF-κB dimers to enter the nucleus and induce gene expression. Ubiquitin Small, reusable polypeptide tag that is highly conserved in all eukaryotes. Post-translational attachment of ubiquitin to acceptor proteins targets them to the proteasome for degradation. Small, ubiquitin-related modifier (SUMO) The SUMO modification pathway is analogous to ubiquitin conjugation but requires distinct catalytic enzymes to attach or to remove the SUMO peptide from target proteins. Unlike ubiquitinylation, SUMOylation seems to enhance protein stability or to modulate their subcellular compartmentalization.

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avrBs3 family Structural motifs and their potential function

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Fig. 1. Models describing compatible and incompatible plant–bacteria interactions. The bacterial type III secretion system is thought to deliver bacterial avirulence (Avr) proteins into the plant cell. Interaction of the Avr protein with a matching host target protein (the so-called pathogenicity target, P) modulates P function to the benefit of the bacterial parasite (left-hand side). The receptor–ligand model predicts that a direct interaction between Avr and a matching resistance (R) protein initiates the resistance reaction. In the guard model, the R protein safeguards a matching pathogenicity target P. In resistant plants, the complex of Avr and pathogenicity target is recognized by the R protein, which, in turn, initiates the resistance reaction.

effectors interact with certain host proteins (referred to as ‘pathogenicity targets’) to promote disease. R proteins ‘guard’ the pathogenicity target and, upon recognition of an Avr–pathogenicity-target complex, initiate plant defense responses14. The guard model is appealing because it provides an explanation for the dual recognition capacity of some R proteins, such as RPM1 (Ref. 15) and Mi-1 (Ref. 16), because they could guard the same host component targeted by unrelated Avr products. Furthermore, the model agrees with the fact that a direct physical interaction has not been observed for most matching Avr–Rprotein pairs, in spite of substantial experimental efforts (e.g. Ref. 17). Gene-for-gene interaction, in the context of the guard model, requires not only corresponding Avr and R determinants but also pathogenicity targets that act as a ‘molecular glue’ between complementary Avr and R proteins. Absence of matching targets might explain why the majority of R genes are only functional in plants that are closely related to the R-gene donor species, a phenomenon designated ‘restricted taxonomic functionality’18. Recently, the Arabidopsis R protein RPS2 has been found in a protein complex together with its matching bacterial Avr protein, AvrRpt2, and additional host proteins, which is consistent with the guard model19. Yet, with the exception of matching Avr and R proteins, the host components of the protein complex that triggers the defense response (which has the proposed designation of the ‘resistosome’) and their function remain unknown. http://plants.trends.com

Members of the avrBs3 gene family [named after its first sequenced member, from Xanthomonas campestris pv. vesicatoria (Xcv)20] have been identified in Xanthomonas6,7,21 and Ralstonia solanacearum (http://sequence.toulouse.inra.fr/R.solanacearum). AvrBs3-homologous proteins share 90–97% amino acid sequence identity21 and are secreted via the Hrp type III pathway22,23. Members of the avrBs3 family were identified by (1) their ability to induce the HR in resistant hosts20,24, (2) increasing watersoaking or inducing canker-like (tumor-like) virulence symptoms25,26 and (3) sequence homology27,28. Differences between avrBs3 family members are almost exclusively confined to the central domain, composed of a variable number (13.5–25.5) of almost identical 34 amino acid repeats (Fig. 2a). Another structural hallmark of AvrBs3-like proteins is the presence of functional NUCLEAR LOCALIZATION SIGNALS29,30 (NLSs) and an ACIDIC TRANSCRIPTIONAL ACTIVATION DOMAIN (AAD) at the C-terminus31–34. Yeast two-hybrid studies using AvrBs3 revealed that the NLS motifs interact specifically with pepper (Capsicum annuum) importin α (Ref. 34), which, together with importin β, mediates the passage of eukaryotic proteins through the nuclear pore complex35. The interaction between AvrBs3 and importin α suggests that the prokaryotic effector has recruited the host’s nuclear import machinery and that the target of AvrBs3 is in the nucleus (Fig. 2b). Mutational studies of AvrXa7, a member of the AvrBs3 family, support this model because deletion of the NLSs resulted in reduced pathogenicity33. The DNAbinding activity of AvrXa7 (Ref. 33) and the fact that all AvrBs3 homologs contain an acidic C-terminal transcriptional activation domain31–34 suggest that these effectors act directly or indirectly as transcription factors, modulating the host transcriptome to the benefit of the bacterial intruder. Repeats determine the specificity of avirulence and virulence

Some xanthomonads, such as X. campestris pv. malvacearum (Xcm) and Xanthomonas oryzae pv. oryzae (Xoo), contain multiple copies of avrBs3 homologs21. Sequential inactivation of avrBs3-homologous genes in Xcm has shown that some gene family members increase the ability to cause watersoaking symptoms on cotton, and that this effect is additive36. Analysis of AvrBs3 homologs in Xoo revealed that their virulence function is not merely additive but specific to each family member37. This finding raises the question of how these highly conserved effector molecules exert their specific virulence function at the molecular level. Deletion of repeat units and domain swaps between different AvrBs3 homologs provided experimental evidence that both avirulence and virulence specificity is governed by the repeats25,31,33,38. Based on the assumption that the proteins act as transcription factors, it is tempting to

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Fig. 2. (a) Structure of AvrBs3 from Xcv, the prototype of the AvrBs3 protein family. The central domain is composed of 17.5 nearly identical 34-amino-acid repeats. The C-terminus contains two functional nuclear localization signals (NLS) and an acidic transcriptional activation domain (AAD). (b) Proposed model for molecular mechanisms determining virulence and avirulence of AvrBs3-like proteins. AvrBs3-like proteins are translocated into the plant cell via the bacterial type III secretion system. NLSs interact with importin α, which, together with importin β, catalyzes the transport of AvrBs3-like proteins into the nucleus. Modulation of the host’s transcriptome is achieved by direct or indirect interaction of AvrBs3 with DNA. Recognition of AvrBs3 and its homolog often depends on NLSs, suggesting that corresponding R proteins [e.g. pepper (Capsicum annuum) Bs3] are located in the nucleus. However, some R proteins probably recognize AvrBs3-like proteins in the cytoplasm (e.g. tomato Bs4). Abbreviation: HR, hypersensitive response.

speculate that the repeat domain determines, directly or indirectly, the recognition of specific promoters. Analysis of host transcripts induced by AvrBs3 and AvrBs4 from Xcv (Refs 23,27), a close homolog of AvrBs3, indeed revealed gene activation spectra that are specific for each Avr protein, supporting the hypothesis of repeat-specific gene induction (E. Marois and U. Bonas, unpublished). R-gene-mediated recognition

Deletion of the NLSs, which are present in all AvrBs3 family members, in most cases abolished Avr recognition by the plant23,31–33, suggesting that the cognate R proteins are in the nucleus. In addition, detection by the host’s defense system requires the presence of the C-terminal AAD (Refs 30,31). http://plants.trends.com

Mutations in the NLSs or the AAD might cause conformational changes that abolish interaction with the R protein, yet the endogenous NLS and AADs can be functionally replaced by the heterologous viral SV40 NLS (Ref. 30) and the herpes simplex VP16 AAD (Refs 32,34), respectively. This suggests that R-proteinmediated induction of cell death occurs downstream of nuclear localization and gene activation. Does the current knowledge on avrBs3 family members agree with the conceptual framework of the guard model (Fig. 1)? According to this model the AvrBs3 interactor importin α (Ref. 34) might be the pathogenicity target of AvrBs3. In compliance with the proposed pathogenicity function of Avr proteins, binding of AvrBs3 to importin α would promote disease. Yeast two-hybrid studies with importin α revealed that it binds equally well to AvrBs3 and the homologous protein AvrBs4 (B. Szurek and U. Bonas, unpublished). However, the AvrBs3-triggered HR, mediated by the pepper Bs3 gene, is not inducible by AvrBs4. This indicates that importin α is probably not the pathogenicity target that is guarded by the R protein Bs3. AvrRxv/YopJ family Sequence-related type III effector proteins in plant and mammalian pathogens

The isolation of R genes14,39 has revealed common structural motifs in their encoded proteins, such as

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leucine-rich repeats, Toll–Interleukin-1 receptor-like domains and serine/threonine kinases, which are also found in multidomain proteins of insect and mammalian host defense pathways2,14,40. Therefore, it has been speculated that innate immunity in both plants and animals evolved from common ancestral modules41. Interestingly, one class of type III-secreted effectors is conserved in mammalian and plant pathogens42. AvrRxv, from the plant pathogen Xcv, and YopJ, from the mammalian pathogen Yersinia pseudotuberculosis, were the first members of this family to be isolated43,44. Additional members of the AvrRxv/YopJ family have been identified in the mammalian pathogens Yersinia enterocolitica [YopP (Ref. 45)] and Salmonella [AvrA (Ref. 46)], the plant pathogens Xcv [AvrBsT (Ref. 47), AvrXv4 (Ref. 48)], Pseudomonas syringae [ORF5 (Ref. 49), AvrPpiG (Ref. 50)] and Erwinia amylovora (Ref. 51), and the plant symbiont Rhizobium spp. [y410 (Ref. 52)]. AvrRxv/YopJ family members in mammalian pathogens

The nearly identical proteins YopJ and YopP from Yersinia spp. are the best-studied members of the AvrRxv/YopJ family. They induce apoptosis of macrophages and downregulate proinflammatory cytokines53, thus directly affecting innate immunity and indirectly affecting acquired immunity (Fig. 3). Although Yersinia spp. can invade several cell types, the induction of apoptosis by YopJ or YopP is restricted to macrophages45,54,55. Hence, macrophage cell death can be considered to be a pathogenicity function of YopJ and YopP, promoting bacterial growth in the host56,57. In vitro studies showed that YopJ and YopP interfere with mitogen-activated protein kinases (MAPKs) and the NUCLEAR FACTOR κB (NF-κB)58,59, both key regulatory elements of the host’s immune system60–64. MAPKs play essential roles in the transduction of extracellular signals to cytoplasmic and nuclear effectors65. In mammals, three of the five subgroups of the MAPK family respond to stress (the c-Jun N-terminal kinase, the p38 kinase and extracellular-signal-regulated kinase66), all being suppressed by Yersinia spp.67 NF-κB is a transcriptional activator of proinflammatory cytokines63 but also counteracts pathogen-induced apoptosis68. Activation of NF-κB requires its release from a complex containing INHIBITOR OF NF-ΚB (IκB) by the phosphorylation of IκB. This, in turn, initiates ubiquitinylation and subsequent proteolytic degradation of IκB. Liberation of NF-κB is inhibited by YopJ and YopP, which promotes macrophage apoptosis59,69. Recently, YopJ and YopP were found to bind IKKβ and MAPK kinases (MAPKKs), the upstream regulators of NF-κB and MAPKs, thereby inhibiting their kinase function58,69. Computer-aided analyses of the YopJ sequence added another twist to this complex model and revealed homologies to adenovirus protease59. http://plants.trends.com

Significantly, the invariant residues determining the protease catalytic site (catalytic triad) were conserved among all AvrRxv/YopJ family members, and YopJ mutants altered in the catalytic triad failed to interfere with the MAPK and NF-κB pathways. However, in vitro, the protease activity of YopJ was not traceable59. Hints about potential YopJ substrates were provided by the observation that adenovirus protease shows sequence homology to a group of cysteine proteases, the UBIQUITIN-like protein-processing enzymes59. Interestingly, previous in vitro studies of the yeast ubiquitin-like protein-processing enzyme Ulp1 revealed that is has a dual activity: (1) proteolytic activation of SMALL UBIQUITIN-RELATED MODIFIER (SUMO), which is ligated to a target protein; and (2) deSUMOylation of tagged targets70. SUMO resembles ubiquitin71–73 but probably acts antagonistically to it74. Given that YopJ induces macrophage cell death, proteolytic activation of SUMO by YopJ might trigger SUMO-dependent IκB stabilization and thereby inhibit the anti-apoptotic activity of NF-κB (Fig. 3). Co-transfection experiments with YopJ and SUMO did not result in a decrease of free or bound SUMO but in a general decrease of SUMO protein59. Therefore, the proposed function of YopJ as a SUMO-processing protease remains questionable. Do other AvrRxv/YopJ homologs share functional similarities with YopJ and YopP? The Salmonella AvrA protein shares ~60% identity with the YopJ and YopP proteins from Yersinia and is their closest known relative46,75. However, a Salmonella AvrA mutant strain did not reveal any changes in cytokine expression or macrophage killing75. Likewise, heterologous expression of AvrA did not complement YopJ or YopP mutations in Yersinia75, suggesting that, in spite of structural conservation, AvrA and YopJ/YopP are functionally dissimilar. AvrRxv/YopJ family members in plant pathogens

Several AvrRxv/YopJ homologs have been identified in plant pathogens owing to their recognition by certain plant R proteins48,76,77. Sequence conservation between YopJ/YopP and AvrBsT, a YopJ/YopP homolog from the phytopathogen Xcv, is less striking than between YopJ/YopP and AvrA from Salmonella. Nevertheless, residues that determine the catalytic triad are strictly conserved between YopJ/YopP and AvrBsT. Mutations in the catalytic triad of AvrBsT abolished its capacity to induce the HR in Nicotiana benthamiana and pepper59. These findings led the authors to speculate that AvrBsT also acts as a protease. However, it should be emphasized that, in contrast to YopJ/YopP-induced macrophage cell death, the AvrBsT-triggered HR is not beneficial to the bacterial parasite and must be considered to be an ‘unwanted’ (avirulence) activity of AvrBsT. If we assume that the catalytic triad is essential for an asyet-unknown virulence function of AvrBsT, these conserved residues are probably the targets of choice for the plant immune system. Therefore, it is not

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Acknowledgements We are grateful to anonymous reviewers for improving this manuscript during revision. We apologize to colleagues whose work was not cited or discussed because of space limitations. Helpful suggestions on the manuscript by Desmond Bradley, Eric Marois and Sebastian Schornack are especially appreciated. Work in our laboratory was supported by grants from the Deutsche Forschungsgemeinschaft to U.B. and T.L. (SFB 363 and LA 1338/1-1) and the European Community (BIO-CT97-2244).

Fig. 3. Modulation of macrophage signaling by the Yersinia type III effector YopJ/P. Pattern-recognition receptors (PRRs) on the host cell recognize pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharides, and activate defense. Upon contact between Yersinia and the macrophage, YopJ/P is injected into the cytosol and specifically binds to IKKβ and MAPK kinases (MAPKKs). Binding of YopJ/P to MAPKKs inhibits phosphorylation of MAPKs (p38, ERK1/2 and JNK) and blocks activation of targets such as the transcription factor CREB (cyclic-AMP-response-element-binding protein). Binding of YopJ/P to IKKβ inhibits activation of the NF-κB. Hence, pathogen-induced phosphorylation, ubiquitinylation and subsequent proteasomic degradation of IκB are inhibited and NF-κB remains in the cytoplasm. Proteolytic activation of a small ubiquitin-related modifier (SUMO) is possibly the trigger for SUMOylation and thereby stabilization of IκB. Abbreviations: MAPK mitogen-activated protein kinase; NIK, NF-κBinducing kinase; P, phosphorylation; Su, SUMO; Ub, ubiquitin.

unexpected that mutations in the catalytic triad of AvrBsT resulted in a loss of recognition59. What is known about the virulence function of AvrRxv/YopJ-like effector proteins in plant pathogens? Growth curves on a susceptible plant host of a Xcv mutant strain lacking avrRxv were indistinguishable from the wild type47 and did not http://plants.trends.com

provide any evidence for a virulence function. However, the lack of any phenotype that can be scored might be the result of functional redundancy, because the mutant still contained AvrBsT (Ref. 47). The presence of NLSs in AvrBsT, AvrRxv and AvrXv4 (Refs 47,48) might indicate a function in the plant nucleus, but localization studies have not been described to date and the relevance of the NLSs to virulence and avirulence is unknown. Further analysis is needed to determine the pathogenicity function of YopJ/YopP homologs in plant pathogens. Given that the highly related proteins YopJ/YopP and AvrA differ in function in their respective hosts, an unbiased approach appears to be most appropriate to unraveling the selective value of AvrRxv/YopJ homologs in plant–pathogen interactions. Future prospects

Ongoing research on bacterial model systems and upcoming bacterial genome sequence data will undoubtedly reveal additional AvrBs3- and

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AvrRxv/YopJ-like proteins, as well as new type III effector families (e.g. Refs 78,79). Major challenges for the future will be to extract and archive this wealth of information efficiently and to uncover the biological function of the effector proteins. Sequences that are indicative of type III effectors (e.g. homology to effectors from plant and animal pathogens, and/or regulatory sequences in their putative promoter sequences) should allow their in silico identification. However, complementary screening approaches that do not rely on computerized sequence evaluation need to be developed to identify the plethora of type III effectors. To assess the role of functionally redundant bacterial effector families, not only single, but also multiple gene knockouts need to be generated and analyzed for their effect on the plant–pathogen interaction. References 1 Cornelis, G.R. and Van Gijsegem, F. (2000) Assembly and function of type III secretory systems. Annu. Rev. Microbiol. 54, 735–774 2 Staskawicz, B.J. et al. (2001) Common and contrasting themes of plant and animal diseases. Science 292, 2285–2289 3 Klement, Z. and Goodman, R.N. (1967) The hypersensitive reaction to infection by bacterial plant pathogens. Annu. Rev. Phytopathol. 5, 17–44 4 Bonas, U. and Van den Ackerveken, G. (1997) Recognition of bacterial avirulence proteins occurs inside the plant cell: a general phenomenon in resistance to bacterial diseases? Plant J. 12, 1–7 5 Kjemtrup, S. et al. (2000) Effector proteins of phytopathogenic bacteria: bifunctional signals in virulence and host recognition. Curr. Opin. Microbiol. 3, 73–78 6 White, F.F. et al. (2000) Prospects for understanding avirulence gene function. Curr. Opin. Plant Biol. 3, 291–298 7 Vivian, A. and Arnold, D.L. (2000) Bacterial effector genes and their role in host–pathogen interactions. J. Plant Pathol. 82, 163–178 8 Innes, R. (2001) Targeting the targets of type III effector proteins secreted by phytopathogenic bacteria. Mol. Plant Pathol. 2, 109–115 9 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 10 Aldridge, P. and Hughes, K.T. (2001) How and when are substrates selected for type III secretion? Trends Microbiol. 5, 209–214 11 Romantschuk, M. et al. (2001) Hrp pilus – reaching through the plant cell wall. Eur. J. Plant Pathol. 107 12 Keen, N.T. (1990) Gene-for-gene complementarity in plant–pathogen interactions. Annu. Rev. Genet. 24, 447–463 13 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 14 Dangl, J.L. and Jones, J.D.G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411, 826–833 15 Grant, M.R. et al. (1995) Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science 269, 843–846 http://plants.trends.com

In the past, the virulence function of type III effectors was mainly studied using in planta growth curves of respective bacterial mutant and wild-type strains. However, assays performed in the laboratory are probably not sufficient to disclose subtle effects or to uncover as-yet-unknown functions of type III effector proteins. Therefore, field studies must be used in combination with new experimental assays and reporter systems to unravel type III effector function. Recent in vivo studies with epitope-tagged Avr and R proteins indicate that both components aggregate with host proteins in a large protein complex (the ‘resistosome’). Identification of these host proteins will clarify whether these are identical to signaling components of disease resistance pathways, which were previously identified by mutational screens.

16 Vos, P. et al. (1998) The tomato Mi-1 gene confers resistance to both root-knot nematode and potato aphids. Nat. Biotechnol. 16, 1365–1369 17 Luderer, R. et al. (2001) 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 18 Tai, T.H. et al. (1999) Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato. Proc. Natl. Acad. Sci. U. S. A. 96, 14153–14158 19 Leister, R.T. and Katagiri, F. (2000) A resistance gene product of the nucleotide binding site – leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo. Plant J. 22, 345–354 20 Bonas, U. et al. (1989) Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol. Gen. Genet. 218, 127–136 21 Gabriel, D.W. (1999) The Xanthomonas avr/pth gene family. In Plant–Microbe Interactions (Vol. 4) (Stacey, G. and Keen, N.T., eds), pp. 39–55, APS Press 22 Rossier, O. et al. (1999) The Xanthomonas Hrp type III system secretes proteins from plant and mammalian pathogens. Proc. Natl. Acad. Sci. U. S. A. 96, 9368–9373 23 Ballvora, A. et al. (2001) Genetic mapping and functional analysis of the tomato Bs4 locus, governing recognition of the Xanthomonas campestris pv. vesicatoria AvrBs4 protein. Mol. Plant–Microbe Interact. 14, 629–638 24 De Feyter, R. et al. (1993) Gene-for-genes interactions between cotton R-genes and Xanthomonas campestris pv. malvacearum avr genes. Mol. Plant–Microbe Interact. 6, 225–237 25 Yang, Y.N. et al. (1994) Host-specific symptoms and increased release of Xanthomonas citri and X. campestris pv. malvacearum from leaves are determined by the 102-bp tandem repeats of pthA and avrb6, respectively. Mol. Plant–Microbe Interact. 7, 345–355 26 Swarup, S. et al. (1991) A pathogenicity locus from Xanthomonas citri enables strains from several pathovars of Xanthomonas campestris to elicit cankerlike lesions on citrus. Phytopathology 81, 802–809 27 Bonas, U. et al. (1993) Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific

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