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291 Prospects for understanding avirulence gene function Frank F White*, Bing Yang† and Lowell B Johnson‡ Avirulence genes are originally defined by their negative impact on the ability of a pathogen to infect their host plant. Many avirulence genes are now known to represent a subset of virulence factors involved in the mediation of the host–pathogen interaction. Characterization of avirulence genes has revealed that they encode an amazing assortment of proteins and belong to several gene families. Although the biochemical functions of the avirulence gene products are unknown, studies are beginning to reveal the features and interesting relationships between the avirulence and virulence activities of the proteins. Identification of critical virulence factors and elucidation of their functions promises to provide insight into plant defense mechanisms, and new and improved strategies for the control of plant disease. type-III secretory pathways have been reviewed extensively [5], and are remarkable for their capacity to deliver virulence and avirulence proteins into the host cells (reviewed in [6]). Although not all avirulence and virulence gene products are necessarily delivered via the Hrp pathway, this review will emphasize the intrinsic value of Hrp-associated bacterial avirulence gene products and the features of these proteins that might be relevant to their functions within the host cell. Avirulence genes as virulence genes Introduction The number of known dual-acting bacterial avirulence genes has grown steadily since the identification of avrBs2 from Xanthomonas campestris pv. vesicatoria (Table 1) [7]. The virulence effects of these genes usually affect the population of the bacteria in the infected tissue and are often discernible as causing changes in lesion size, number, or appearance (Figure 1). Mutations in avrBs2 resulted in impaired ability of Xanthomonas to grow on pepper cultivars that lacked the Bs2 gene for resistance [7]. The avrBs2 gene is found in many species of Xanthomonas and the polypeptide that it encodes has a sequence related to those of agrocinopine synthase and glycerol phosphodiesterase, suggesting that this polypeptide has an enzymatic role in the pathogenicity of many members of the genus [8]. The fact that the AvrBs2 functioned upon expression in the plant host cells suggests that the protein is secreted from the bacterium and, therefore, likely to function within the host cell [9••]. A variety of phosphorylatedlipid-derived signaling molecules in plants are potential substrates for AvrBs2 [10]. Avirulence genes cause a plant pathogen or pest to elicit a resistance response in a host plant. The genes occur in viruses, bacteria, fungi, nematodes and insects. Related genes have also been identified in species of Rhizobium suggesting their involvement in symbiotic interactions [1]. Avirulence genes are defined by corresponding resistance (R) genes of which a relatively large number have now been cloned (reviewed in [2]). The resistance response is typically accompanied by a hypersensitive reaction (HR), which is a form of programmed cell death; a burst of superoxide production; and the expression of defense genes (reviewed in [3,4]). The lack of a clear biological function attributable to many avirulence genes has been puzzling. This puzzlement has steadily given way to the view that many avirulence genes have dual functions, having a role in virulence as well as avirulence, and thus function as important mediators of the interaction between pathogen and host. This concept of avirulence gene products as virulence factors, although not new, was greatly advanced by studies of the Hrp (HR and pathogenicity) pathway, a type-III secretory pathway, and by comparisons to the closely related type-III secretion pathways of animal pathosystems. Both plant and animal One of the difficulties in assessing the intrinsic properties of some bacterial avirulence genes is that they have a pronounced effect on virulence only when present in the appropriate species, strain or pathovar, or in a pathovar on a particular host species or variety. The virulence effects of avrA and avrE have been observed in only one strain of Pseudomonas syringae pv. tomato, PT23 [11]. The avrE locus consists of two coding sequences and both polypeptides are required for virulence and avirulence activity. AvrF, the product of the open reading frame that is downstream of AvrE, has the characteristics of a chaperone and may be involved in the secretion of AvrE [12]. Homologs of avrE were originally identified as the critical virulence factors DspE (also known as DspA) and DspF (also known as DspB) in Erwinia amylovora; yet these homologs were dispensable for the elicitation of a HR on nonhost species, hence their identification as disease specific (Dsp) loci [12,13]. The DspEF locus also functions as an avirulence locus when transferred to P. syringae pv. glycinea [12]. The polypeptide responsible for avirulence activity has yet to be characterized. The virulence effect of avrPto was observed after its transfer to strain T1 of P. syringae pv. Addresses Department of Plant Pathology, Kansas State University, 4024 Throckmorton Hall, Manhattan, Kansas 66506, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] Current Opinion in Plant Biology 2000, 3:291–298 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations HR hypersensitive reaction Hrp HR and pathogenicity NLS nuclear localization signals R resistance 292 Biotic interactions Figure 1 (a) (b) Current Opinion in Plant Biology tomato (Figure 1) [14•,15•]. AvrRpm1 (also called avrPmaA1.RPM1) is required for full virulence of P. syringae pv. maculicola on Arabidopsis [16], yet loss of a virtually identical allele had no apparent effect in P. syringae pv. pisi on pea [17]. AvrRpt2 has a virulence effect in strain DC3000 of P. syringae pv. tomato on the No-0 land race of Arabidopsis but not on land race Col-0 (which does not have a functional RPS2) (B Kunkel, personal communication). Interestingly, avrRpt2 has also been found to have deleterious effects when expressed in Col-0, indicating that the encoded protein may affect both Arabidopsis land races, but its effects in enhancing the virulence of the bacterium are observable only on No-0 ([18]; B Kunkel, personal communication). Interestingly, avrRpt2 increased the size of the bacterial population of strain ES4326 of P. syringae pv. maculicola on the rps2 mutant of Col-0 (J Greenberg, personal communication). The intrinsic value in virulence of an avirulence gene may also require environmental conditions or epidemiological factors that are not included in the virulence assay. The avrb6 from X. campestris pv. malvacearum does not alter the size of the bacterial population within inoculated leaves but results in the release of greater numbers of bacteria to the leaf surface [19]. Thus, the release and spread, or invasiveness, of the pathogen, which are often not assayed, may be as important as the size of the pathogen population [20]. Avrb6 is a member of the avrBs3 avirulence gene family of which there are many members, including several dual-acting genes (reviewed in [21]). For example, the retention of multiple avrBs3 family members within strains of the cotton pathogen X. campestris pv. malvacearum and the rice pathogen X. oryzae pv. oryzae may facilitate the generation of additional virulence alleles by recombination [22–24]. Members without apparent virulence activity may represent defeated virulence factors or virulence factors with specificity for other host species. Additional alleles may allow a strain to have a broader host range or more rapid adaptation to the deployment of R genes in intensively Symptoms of the dual-acting avirulence genes avrPto and pthXO1 in compatible interactions. (a) Tomato leaves infected with P. syringae pv. tomato T1 with (right) and without (left) avrPto. (b) Rice leaves infected with X. oryzae pv. oryzae. Inoculations from left to right: strain PXO99A; PXO99AhrpC– (nonpathogenic); PXO99Amx2 (containing a mutation in pthXO1, an uncharacterized dualacting avrBs3 family member); and PXO99Amx2 with pthXO1. Dark areas on leaves represent water-soaked lesions. Watersoaked appearance, caused by the release of water from the infected cells, is a common symptom of foliar necrotizing bacterial pathogens. The tomato leaves were dipinoculated. Rice leaves were inoculated using needle-less syringes. managed crop species such as cotton and rice, which are grown in environments conducive to bacterial disease. Avirulence genes and general host defense responses A working model of the role of some avirulence gene products can be derived from the events in the invasion process of Yersinia spp. (and related bacteria) that use type-III secretory systems to enhance virulence and invasiveness. Bacterial lipoproteins and lipopolysaccharides of Yersinia, among other compounds, trigger host proinflammatory responses. The bacteria, for their part, secrete a variety of factors via a type-III pathway that interrupt the signaling processes. Some of the affected host pathway components have been identified including MAP (i.e. mitogen-activated protein) kinases, GTP-binding proteins and actin filaments, which play a role in cell organization and molecular trafficking (reviewed in [25•]). A variety of compounds produced by microorganisms also elicit defense gene expression in plants (reviewed in [26]). The elicitors are produced by a broad range of bacteria including virulent strains. The response is therefore considered nonspecific or general resistance in contrast to the specific avirulence-gene-dependent responses. MAP kinases that are activated by general elicitors have been identified in plants [27,28•], and at least one GTP-binding factor is associated with programmed cell death [29]. The virulence function of some avirulence proteins may be analogous to that of the type-III-dependent factors of pathogenic bacteria, that is, to interfere with signal and structural pathways of the general defense responses. Hrp-dependent interference with the defense gene expression of the host has been shown to occur, although the individual factors involved remain to be identified (reviewed in [30]). Genetic approaches are underway to identify the genes involved in general defense responses to virulent pathogens. Three genes, EDS1, PAD4, and NDR1, that Prospects for understanding avirulence gene function White, Yang and Johnson 293 Table 1 Bacterial avirulence genes with virulence or virulence-associated properties. Gene or protein Organism or virus Features, possible function and localization in host* Related proteins or alleles† avrBs2 X. c pv. vesicatoria Cytoplasmic avrXa7 X. o. pv. oryzae avrb6 pthN pthA avrRxv X. c. pv. malvacearum X. c. pv. malvacearum X. citri X. c. pv. malvacearum NLS, AD DNA binding, nucleus Nucleus Nucleus Cell hyperplasia, nucleus MAP kinase binding? Agrocinopine synthase, glycerol-phosphodiesterase avrBs3 family avrRpm1 avrPto avrE MYM, inner membrane? MYM, kinase binding, inner membrane Unknown avrA avrRpt2 P. s. pv. maculicula P. s. pv. tomato (T1) P. s. pv. tomato (PT23) E. amylovora P. s. pv. syringae E. amylovora P. s. pv. tomato (PT23) P. s. pv. tomato avrPphF virPphA P. s. pv. phaseolicola P. s. pv. phaseolicola Unknown Unknown, inhibits HR avrF Reference(s) [8] DspA (DspE) (Ea) [21,23] [55,56•] [22,63] [64] [65•] [35,36,66] [38,39,41] [16,67] [14•,15•] [12,13,68] Chaperone DspB (DspF) (Ea) [12,13] Cytoplasmic Cytoplasmic AvrA (Psg), AvrBs1 [68,69] [70] (a) avrBs3 family avrBs3 family avrBs3 family AvrBsT, Orf5 (Pss), YopJ (Yps), YopP (Ye), AvrA (St), y410 (Rl) AvrPpiA1 (Psp) [71•] [71•] *AD, transcription activation domain; MYM, myristoylation motif; NLS nuclear localization signal motif. †Species indicated in parentheses. Ea, E. amylovora; Psg, P. syringae pv. glycinea; Psph, Psp, P. syringae pv. pisi; Pss, P. syringae pv. syringae; St, Salmonella typhimurium; Ye, Y. enterocolitica; and Yps, Y. pseudotuberculosis. (a) B Kunkel, J Greenberg, personal communications. allow the enhanced growth of virulent pathogens have been cloned and characterized [31,32••,33]. EDS1 and PAD4 have sequence similarity to lipases, whereas NDR1 is predicted to be a membrane protein and no potential biochemical function has been assigned to it. PAD4 and EDS1 could well begin a phospholipid signaling cascade. It would be interesting to determine whether the activity of some avirulence genes, particularly avrBs2, is affected on plants in which these genes have been mutated. Biochemical approaches to characterizing defense responses to pathogens are also underway. A conserved segment of bacterial flagellin was recently demonstrated to be a potent elicitor of defense gene expression. The active peptides are present in a variety of pathogenic bacteria and perceived at the nanomolar level by a broad range of plant species [34••]. Biochemical analyses should help to identify the signaling complexes that may be targeted by the individual virulence factors from bacterial pathogens. X. campestris pv. vesicatoria (avrBsT) [36], P. syringae pv. syringae [37•], Erwinia amylovora [12] and Rhizobium leguminosarum [38]. Although avrRxv has not been shown to be required for virulence, the protein product of avrRxv has sequence relatedness to the virulence factor YopJ from Yersinia pseudotuberculosis (Figure 2). YopJ has additional homologs in Y. pestis (YopP) and Salmonella typhimurium (AvrA). YopJ, YopP and AvrA are secreted by type-III systems and required for virulence [39–41]. Recently, YopJ has been shown to bind MAPKK1 and IKKβ (an inhibitor of NFκb kinase kinase), which are involved in the activation of NFκb during an inflammatory response to infection by Yersinia [42••]. It is tempting to speculate, therefore, that AvrRxv and related proteins from plant pathogenic bacteria interact with MAP kinases that are otherwise activated by general defense signaling mechanisms. Shared virulence factors of plant and animal pathogenic bacteria Signal pathways that are conserved in both plant and animal cells may be responsible for the broad membership of the avrRxv/yopJ gene family. Members of this family are found in a diverse collection of Gram negative pathogenic and symbiotic bacteria. The original avrRxv gene was identified after its transfer from X. campestris pv. vesicatoria to X. campestris pv. phaseoli, thereby converting X. campestris pv. phaseoli into an incompatible strain on some bean cultivars [35]. Related genes have been identified in Avirulence gene products are targeted to cellular organelles The products of two groups of avirulence genes with virulence effects appear to be targeted to specific organelles within the host cell. Members of the first group are identified by the presence of a myristoylation motif, which, if functional, would localize the protein to the cytoplasmic surfaces of membranes (Figure 3). The motif in AvrPto is required for avirulence and virulence activity (X Tang, personal communication). Replacement of the glycine at the second position (G2A) destroyed all AvrPto activity, yet the modified protein was still secreted and capable of interaction with Pto, the corresponding R-gene product in the yeast two-hybrid system. Upon expression in the 294 Biotic interactions Figure 2 Yopj AvrA(St) AvrRxv AvrBsT Y4LO Consensus Figure 3 50 64 120 98 66 *** SRM DVEVMPALVIQANNKYPEMNLNLVTSPL EET DLEMMPFLVAQANKKYPELNLKFVMSVH RLMDIENLPHLVRSYDNRLNNLNLRSFDTPG TKADVENKYYLAHAYNERFPELHLSCHDSAQ LSLDIRNLPLLAASYNRRYPDLDLRHMDSPA Dve mp L n kypem L s 80 94 150 128 96 Current Opinion in Plant Biology AvrPto AvrPto AvrRpm1 AvrRpm1 AvrB AvrB AvrC AvrC Pto Pto p60Src p60Src * MGNICVGGSRMAH MGCVSSTSRSTGY MGCVSSKSTTVLS MGNVCFRPSRSHV MGSKYSKATNSIN MGSSKSKPKDPSQ Current Opinion in Plant Biology Alignment of the amino-terminal region of members of the AvrRxv family. The alignment shows that the residues of the amino-terminal portion of the protein, which are the products of five genes of the avrRxv family, are conserved. The region includes part of the putative SH2 domain of YopJ [40]. Replacement mutation of DVE (***) in YopJ causes the loss of YopJ activity. plants, wild-type AvrPto was found almost exclusively in the membrane fraction of the host cells, whereas the G2A mutant was present in the soluble fraction (X Tang, personal communication). Curiously, Pto also has a myristoylation site (Figure 2). Replacement of the glycine at position two in Pto, however, had no effect on its ability to confer resistance in tomato [43]. One possible caveat of this work was that the mutant Pto gene was expressed using the strong CaMV35S promoter, which may not result in expression patterns that are representative of those under the endogenous promoter. Tests using the wild-type promoter are in progress (G Martin, personal communication). Rpm1, the R-gene product corresponding to AvrRpm1, is associated with the inner plasma membrane [44]. AvrRpm1 has a myristoylation motif and presumably is also targeted to the inner plasma membrane surface. AvrB and AvrC, which are related to each other by sequence similarity [45], also have myristoylation motifs, although their significance is unknown [46]. AvrB is also recognized by Rpm1 [47]. The functions of AvrPto and AvrRpm1 in virulence are unknown. Pto is a serine/threonine kinase, and AvrPto is known to bind Pto in resistance signaling [48,49]. AvrPto might, therefore, be a kinase binding protein. Targeting the avirulence proteins to the inner membrane would be consistent with a possible role for them in interfering with a receptorlinked signaling pathway. Potential myristoylation motifs in avirulence proteins. The asterisk indicates the glycine at the number two position, which is the site of myristoylation. Replacement of G with A in AvrPto eliminates both virulence and avirulence activity. The amino-terminal sequences of Pto and pSrc60, a previously characterized myristoylated proto-oncogene from chicken [72], are shown for comparison. Family members differ in the number and the order of the repeats as identified by the variable region (Figure 4). Critical, yet unknown, structural features that are involved in specificity for avirulence and virulence lie within the repeat domains [19,24,52]. Functional nuclear localization signals (NLS) are present in the carboxyl-coding portion of the prototype gene product [53], and are required for the avirulence activity of avrBs3 and avrXa10 [54,55]. The NLS-coding regions of avrXa7 are also required avirulence and virulence activities, both of which can be restored by the addition of the SV40 T-antigen motif (B Yang et al., unpublished results). The function of the avrBs3-like gene products upon reaching the nucleus is unknown. Curiously, the proteins have a transcriptional activation domain in the carboxy terminus, which is required for the avirulence activities of AvrXa10, AvrXa7 and AvrBs3 [55,56•]. AvrXa7 also requires the activation domain for virulence activity and has recently been shown to be a double-stranded DNA binding protein (B Yang et al., unpublished results). Thus, AvrXa7 and, by inference, the other members of the family have features consistent with those of transcription factors and possibly act by altering the transcription of one or more genes. The highly conserved nature of the members of the family may reflect a conserved target in the diverse host species. Conclusions and further speculations The second group of targeted bacterial avirulence proteins are encoded by the avrBs3 family members [50,51]. These proteins, which are restricted to the genus Xanthomonas, appear to be targeted to host nuclei. The family consists of a large number of near-identical genes with varying R-gene specificities in a variety of host plants, and some members are dual functional (Table 1). Within the middle third of the protein is a repeated sequence of 34 amino acids that varies in copy number from 14 copies in Avrb6 from X. campestris pv. malvacearum to 25 copies in AvrXa7 (reviewed in [51]). The repeats are highly conserved with the most consistent variation present at codons 12 and 13 of the individual repeat units. Understanding the role of avirulence genes may provide some practical benefits for crop protection. Identification of virulence factors and their functional domains should allow geneticists and genetic engineers to focus on strategies that are directed at the critical factors of pathogens. The plants themselves may already have provided some of the tools. Over time, the process of adaptation between pathogen and host may have led to recognition of critical virulence factors or critical domains within the factors by host plants, forcing the pathogen, in a sense, to decide between triggering a resistance reaction or converting to a less virulent strain. The longer the time needed for the pathogen to adapt to an R-gene product, the greater the Prospects for understanding avirulence gene function White, Yang and Johnson durability of resistance. Particularly successful adaptations by plants may even alter the host range of a pathogen species. The possibility of selecting R genes that correspond to important virulence factors of a pathogen is already in the process of being tested. Laugé et al. [57] were able to identify an R gene (Cf-ECP2), which corresponds to the virulence factor ECP2 from Cladosporium fulvum, by expressing the protein in the PVX vector. ECP2 is present in all strains of C. fulvum and, therefore, may be critical to the capacity of the fungus to cause significant disease. Whether the Cf-ECP2 gene recognizes an important domain of ECP2, and how readily ECP2 can convert to an unrecognized yet active form, remains to be shown. The Bs2 gene corresponds to the widely occurring and dual-acting avrBs2 and represents an R gene that could potentially be deployed against a variety of Xanthomonas disease complexes. The gene was recently cloned, and its effectiveness in a variety of crop species can now be tested [9••]. Some of the potential difficulties of this approach are perhaps indicated by the fact that Bs2 has only been shown to be effective in Solanaceous species. Nevertheless, only a few heterologous expressions have been performed, and, if highly restricted, the gene activity in other species may be improved after the limiting factors of Bs2 activity in heterologous species are better understood. This review has emphasized the possible role of avirulence genes in altering the host’s pathogen surveillance system. The mode-of-action by which virulence is enhanced has not, however, been determined for any bacterial avirulence gene product. Some virulence factors may act on physiological and homeostatic processes other than defense mechanisms of the host cells. The secretion of auxin by bacteria, for example, has been proposed to stimulate the release of nutrients from host plant tissues and to assist in bacterial growth in the phyllosphere [58]. The behaviors of some avirulence proteins raise interesting questions regarding their function in virulence and resistance. Do the apparent requirements for specific localization motifs for virulence indicate that the proteins are targeted to specific sites, possibly defense signaling complexes, within the host? Does the simultaneous localization requirements for avirulence activity indicate that the host R-gene products are either similarly targeted or exist as components or guardians of the host complexes [59]? Other evidence fuels the speculation that the virulence and avirulence activities of some genes may be intimately linked. Recent preliminary reports indicate that the flagellin receptor, which is associated with general resistance, is a serine/threonine receptor kinase related to the product of the R gene Xa21 [60]. Mutations EDS1 and NDR1 affect both general resistance (i.e. allow the greater growth of virulent strains) and R-gene induced resistance [61,62]. Rapid progress in determining the biochemical functions of bacterial virulence factors is expected thanks to the wealth of information provided by the analysis of animal pathosystems and the development of Arabidopsis as a model system. Understanding the function of virulence factors will provide important insights toward understanding 295 Figure 4 (a) B P S S B NLS A B C AD (b) Repeat AvrXa10 AvrBs3 Avrb6 PthA PthN 1 NI HD HD NI NI 2 HG NG NI NG HD 3 NI NS NG NI HD 4 HG NG HD NI NI 5 NI NI HD NG HD 6 NI NI NI HD NI 7 NN NI HD NG NG 8 HD HD NI HD NI 9 10 11 12 13 14 15 16 17 18 NI HD NN HG NS NG HD NG HD NG NS NS HD HD HD NG HD NG NS HD HD HD NN NG NG NG NG NG NS HD HD NG NG NN HD NI NG NG NN Current Opinion in Plant Biology (a) Map of the prototype gene in the avrBs3 family. The central repeat domain is represented by series of open boxes. Black boxes designate nuclear localization motif sequences A, B, and C, and the transcriptional activation domain. The conserved restriction sites B, BamHI; P, PstI; and S, SphI are shown. (b) Organization of 34-aminoacid repeat units in selected members of the avrBs3 family. Each individual repeat unit is represented by the amino-acid residues at positions 12 and 13. the mechanisms of plant defense, and practical strategies aimed at limiting crop losses caused by plant disease. Update Evidence for the myristoylation of AvrRpm1 and its requirement for the proper functioning of the protein in virulence and avirulence has been recently published [73]. A similar requirement for the avirulence activity of AvrB and AvrPphB was also shown [74]. Evidence has also been presented that the cysteine residue adjacent to the acylated glycine residue may be subject to palmitoylation. Alterations of C3 in AvrRpm1 decreased the efficacy of the protein, although not to the extent of the alteration of G2 [73]. The AvrPphB myristoylation site is only exposed after a proteolytic cleavage at K62/G63 [74]. Expression and localization of the proteins within the plant demonstrated that the AvrRpm1 and AvrB are found in the cell plasma membrane [73]. Acknowledgements The authors would like to thank Brad Porter, Xiaoyan Tang and Jian-Min Zhou for helpful discussions, and the colleagues who supplied preprints and unpublished information. The expert assistance of Ms. Diana Pavlasko was used in the preparation of the manuscript. This is publication number 00-363-J from the Kansas Agriculture Experiment Station. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Viprey V, Del Greco A, Golinowski W, Broughton WJ, Perret X: Symbiotic implications of type III protein secretion machinery in Rhizobium. Mol Microbiol 1998, 28:1381-1389. 296 Biotic interactions 2. Martin GB: Functional analysis of plant disease resistance genes and their downstream effectors. Curr Opin Plant Biology 1999, 2:273-279. 3. Richberg MH, Aviv DH, Dangl JL: Dead cells do tell tales. Curr Opin Plant Biol 1998, 1:480-485. 20. 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