Targeting the targets of Type III effector proteins secreted by phytopathogenic bacteria

Many Gram-negative bacterial pathogens of both plants and animals depend on a type III secretion system to infect their host organism (Hueck, 1998). Type III secretion systems have been most intensively studied in species of Yersinia and Salmonella, where it has been established that they function to inject multiple effector proteins directly into host cells (Hueck, 1998). In mammalian cells, these effector proteins interact physically with specific signal transduction proteins, leading to multiple changes in host cell physiology, including major alterations in the actin cytoskeleton, suppression of immune responses, and activation of apoptosis (programmed cell death) (Cornelis, 2000; Galan and Zhou, 2000). By comparison, very little is known about the function of effector proteins secreted by plant pathogens. However, several recent papers are beginning to shed new light. I will discuss the implications of these new papers and discuss some promising new directions for elucidating the function of effector proteins secreted by plant pathogens. Although the function of the type III secretion system was first elucidated in animal pathogens (Cornelis, 2000), genes encoding the components of type III systems were first identified in the plant pathogen Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986). In this early work, several P. s. pv. phaseolicola mutants were identified that failed to induce a hypersensitive resistance response on tobacco, a non-host. Importantly, these mutants also failed to cause disease on their normal host, bean. Mutations in several different genes, which were clustered in a small interval of DNA, gave rise to this phenotype. This cluster was designated the hrp cluster for hypersensitive resistance and pathogenicity. The mechanistic basis of this phenotype did not become clear until 6 years later, when the first gene sequences from the Yersinia type III secretion apparatus were published and recognized to be homologous to hrp genes from P. syringae, Xanthomonas campestris pv. vesicatoria and Pseudomonas solanacearum (now known as Ralstonia solanacearum) (Fenselau et al., 1992; Gough et al., 1992). This observation indicated that protein secretion by phytopathogenic bacteria is an essential aspect of both pathogenesis and the induction of host defence responses. Establishing the mechanistic links between type III effectors (proteins secreted by the type III system), pathogenesis, and induction of the HR in plants has been difficult. Most putative type III effectors from phytopathogenic bacteria were first identified genetically as the products of classical avirulence (avr) genes. Avirulence genes in bacteria are defined as genes that can convert a normally virulent strain to avirulence in a host-specific manner. Avirulence is usually manifested as an induction of an HR on resistant host plants. Because induction of the HR is dependent on hrp genes, it was hypothesized that the products of avr genes are secreted by the type III secretion system directly into the cytoplasm of host cells. Compelling data supporting this hypothesis have been provided by expressing avr genes directly inside plant cells using transient delivery methods and/or expression under an inducible promoter (Bonas and Van den Ackervaken, 1997). In all cases, the expression of an avr gene inside a plant cell leads to induction of an HR-like response (i.e. cell death) that is dependent on a cognate disease resistance (R) gene in the plant. It is thus assumed that the hrp secretion system’s only role relative to HR induction is the delivery of Avr proteins into plant cells; however, direct proof of such protein translocation is yet to be obtained. The existence of specific type III effectors in phytopathogenic bacteria that induce strong defence responses in plants is paradoxical, unless they also play a role in pathogenesis. Surprisingly, the first avr genes identified, which were in the soybean pathogen P. syringae pv. glycinea, appeared to make no significant contribution to pathogenesis; mutation of these genes did not affect the virulence of these strains on normally susceptible hosts, and many P. syringae strains completely lacked these avr genes (Staskawicz et al., 1984; Staskawicz et al., 1987). Many other avr genes have been identified subsequently, and several do indeed contribute to virulence as measured by symptom production, bacterial growth inside the leaf, and bacterial escape to the leaf surface (Alfano et al., 2000; Bogdanove et al., 1998; Chang et al., 2000; Chen et al., 2000; Gassmann et al., 2000; Guttman and Greenberg, 2001; Huguet et al., 1998; Jackson et al., 1999; Kearney and Staskawicz, 1990; Lorang et al., 1994; Ritter and Dangl, 1995; Shan et al., 2000a; Swarup et al., 1991, 1992; Tsiamis et al., 2000; Vera Cruz et al., 2000; Yang et al., 2000, 1994). In the majority of cases the contribution of a given avr gene to virulence is small. However, we know that the secretion of effectors is essential to pathogenicity, and one must therefore conclude that phytopathogenic bacteria secrete multiple effector proteins, which contribute to virulence in a quantitative or partially redundant fashion. Before one can speculate productively about the functions of type III effectors in phytopathogenic bacteria, it is necessary to review the pathology of these bacteria. Both Xanthomonas and Pseudomonas species colonize the intercellular spaces of the leaf mesophyll (the apoplast). Estimates of the amounts of sugars and minerals available in the leaf apoplast argue that there is sufficient nutrition to support high density growth of these bacteria without active transport of sugars from host cells (the symplast) (Hancock and Huisman, 1981). Thus, the key susceptibility response induced by these bacteria is likely to be leakage of water into the apoplast, and a key defence response might be deprivation of water to the site of infection, for example by cell wall thickening. For these bacteria to grow to high densities, they must either avoid inducing host defence responses, actively suppress defence responses, or both. Thus we can imagine type III effectors that specifically affect the flow of water through the plasma membrane, and other effectors that interfere with host defence signalling. The first protein shown to be secreted in a hrp-dependent manner by a phytopathogenic bacterium was the Harpin protein from P. syringae pv. syringae (He et al., 1993). Harpin differs from most Avr proteins in that it can induce a hypersensitive-like response (i.e. cell death) when injected in the leaf apoplast, implying that it might function on the outside of the plant cell. This HR-like response differs from Avr-induced HRs in that it does not appear to be genotype-specific (i.e. dependent on a cognate R gene). Recently it has been shown that purified recombinant Harpin can associate with liposomes and synthetic bilayer membranes and stimulate strong ion currents, suggesting that Harpin forms pores in these synthetic membranes (Fig. 1) (Lee et al., 2001). The role of such pores in pathogenicity is not yet clear, however, as mutations in P. syringae Harpin genes have little to no effect on HR-induction or virulence (Lee et al., 2001). It is plausible that Harpin promotes the efflux of water and nutrients from host cells, but that this represents only one of several mechanisms that P. syringae uses to induce host-cell leakage. Alternatively, Harpin-induced pores may facilitate the translocation of other type III effectors into host cells. The latter view is supported by studies that have shown Harpin to be essential for delivery of the type III effector HrmA (now called HopPsyA) to plant cells by E. coli expressing the hrp regulon from P. syringae pv. syringae (Alfano et al., 1996). This latter observation suggests that P. syringae produces proteins that are not found in E. coli, and which function redundantly with Harpin to facilitate translocation of effectors. Diverse targets of bacterial type III effectors in plant cells. Gram-negative phytopathogenic bacteria secrete multiple effector proteins into host plant cells. The Harpin protein of P. syringae is secreted into the extracellular space and can form pores in membranes, suggesting that it may facilitate entry of other type III effectors into host cells (Lee et al., 2001). P. syringae Avr proteins such as AvrRpm1 and AvrPto localize to the plasma membrane where they interact with unknown targets (Nimchuk et al., 2000; Shan et al., 2000b). This localization is dependent on an N-terminal myristylation motif, indicating that the Avr proteins are modified after entry into the host cell. X. campestris and X. oryzae Avr proteins belonging to the AvrBs3 family are nuclear localized, where they likely bind DNA and alter gene expression (Yang et al. 2000). X. campestris Avr proteins belonging to the avrRxv/avrBsT/YopJ family are believed to have SUMO protease activity, suggesting that they function to remove the SUMO peptide from SUMO modified proteins (Orth et al., 2000). This may in turn cause these target proteins to be ubiquitinated and then degraded by the 26S proteosome (Desterro et al., 1998). Several recent papers have implicated specific avr genes in the suppression of host defences. For example, the avrPphF gene can convert P. syringae pv. phaseolicola strain RW60 from avirulent to virulent on bean cultivar Tendergreen, suppressing the HR (Tsiamis et al., 2000). However, this HR suppression is host-specific, as avrPphF enhances the HR induced by strain RW60 on a second bean cultivar, Canadian Wonder. Fascinatingly, this HR-enhancing activity of avrPphF can itself be suppressed by a physically linked avr gene, avrPphC. As with avrPphF, the HR-suppressing activity of avrPphC is host-specific; avrPphC fails to suppress the avrPphF-associated HR on the bean cultivar Red Mexican. Because the HR-suppressing activities of AvrPphC and AvrPphF are host-specific, the targets of these effector proteins must vary between bean cultivars. This suggests that there has been a molecular ‘arms race’ between plants and phytopathogenic bacteria at the level of effectors and their targets, and not just between effectors and plant disease resistance gene products. In fact, the above data are consistent with a model in which the target of one effector (e.g. AvrPphC) is the R gene product responsible for detecting a second effector (e.g. AvrPphF). If AvrPhC could inactivate the protein responsible for detecting AvrPphF in cultivar Tendergreen, this would suppress the AvrPphF-induced HR. In such a scenario, AvrPphF and AvrPphC might both interact with the same R gene product. A second example of defence response suppression by a P. syringae avr gene is provided by avrRpt2. Transgenic Arabidopsis plants expressing avrRpt2 are hypersusceptible to the virulent P. syringae strain DC3000, and strikingly, are also susceptible to the avirulent strain DC3000(avrRpm1) (Chen et al., 2000). Resistance to the latter strain is mediated by the R gene RPM1, thus transgenic expression of avrRpt2 completely suppresses RPM1-mediated resistance, including the HR. Significantly, this suppression is R gene-specific, as resistance mediated by two other R genes, RPS4 and RPS5, is only slightly affected. These data are consistent with the model proposed above, in which Avr proteins can inactivate specific R gene products. If such a model were correct, avrRpt2 and avrRpm1 may have a reciprocal relationship, as co-infecting Arabidopsis plants with isogenic P. syringae strains expressing avrRpt2 and avrRpm1 suppresses defence responses by both of the cognate resistance genes RPM1 and RPS2 (Reuber and Ausubel, 1996; Ritter and Dangl, 1996). Molecular support for this model has recently been provided by co-immunoprecipitation data, which suggest that the RPS2 protein physically interacts with both AvrRpt2 and AvrB (Leister and Katagiri, 2000) (note: AvrB and AvrRpm1 both activate an RPM1-dependent HR in Arabidopsis (Bisgrove et al., 1994)). In this example, AvrB would be blocking the activation of RPS2 by AvrRpt2. Perhaps inconsistent with this simple model is the observation that avrB enhances the growth of P. syringae in soybean in the absence of avrRpt2, which indicates that AvrB is doing more than blocking an AvrRpt2-specific R gene product (Ashfield et al., 1995). Similarly, AvrRpt2 and AvrRpm1 enhance the growth of P. syringae strains in normally susceptible Arabidopsis lines (Chen et al., 2000; Guttman and Greenberg, 2001; Ritter and Dangl, 1995). These latter observations suggest that AvrRpt2, AvrRpm1 and AvrB may interfere with induction of defence responses at a level common to infection by both virulent and avirulent pathogens. In general, type III effector proteins are diverse in structure and size. Little similarity is observed among the various effectors present in a single bacterial species, or among the effectors observed in different species. A notable exception to this observation is the YopJ family of Type III effectors. Members of the YopJ family have now been reported in species of Xanthomonas, Pseudomonas, Rhizobium, Yersinia and Salmonella (Alfano et al., 2000; Ciesiolka et al., 1999). The first member of this family to be described was AvrRxv from Xanthomonas campestris pv. vesicatoria. AvrRxv is particularly noteworthy among Avr proteins because it can induce avirulence on a wide range of plant species, including both monocots and dicots (Whalen et al., 1988). Significant progress toward identifying the function of AvrRxv has recently been made, based on its similarity to YopJ. The YopJ protein is a type III effector secreted by Yersinia pseudotuberculosis, and has been shown to suppress mammalian inflammatory responses via inhibition of the nuclear factor kappa B (NF-κB) pathway. This inhibition appears to be accomplished in part by preventing phosphorylation of several different mitogen activated protein kinase kinases (MAPKKs) (Orth et al., 1999). Recently, YopJ was shown to have homology to cysteine proteases (Orth et al., 2000). The substrate of YopJ appears to be the SUMO-1 protein, which is a small ubiquitin-like protein that is post-translationally added to lysine groups on multiple targets in mammalian cells (Hochstrasser, 2000). YopJ appears to remove SUMO-1 from these proteins (Orth et al., 2000). Mutation of the catalytic amino acids in YopJ abolishes SUMO-1 protease activity, and abolishes the ability of YopJ to block inflammatory responses in macrophages (Orth et al., 2000); thus, SUMO-1 protease activity correlates with YopJ virulence function. Removal of SUMO-1 from target proteins by YopJ likely affects their function and/or stability. For example, SUMO-1 can compete with ubiquitin for the same lysine groups on target proteins, thus blocking ubiquitination (Desterro et al., 1998). Blocking ubiquitination will prevent proteolysis of targets by the ubiquitin-dependent protein degradation pathway. It is therefore plausible that YopJ could enhance the degradation of specific targets by enabling ubiquitination of these targets. It has also been shown that SUMO-1 modification can affect the intracellular localization of target proteins (Matunis et al., 1998). How SUMO-1 protease activity relates to the inhibition of MAPKK phosphorylation by YopJ is not yet clear. To apply this information to phytobacteria, Orth et al. (2000), created site-specific mutations in the conserved catalytic residues of the YopJ homologue AvrBsT from X. campestris pv. vesicatoria. AvrBsT induces an HR in pepper plants and in Nicotiana benthamiana. The catalytic site mutants of AvrBsT failed to induce an HR, whereas a mutation away from the catalytic core had no effect. These data suggest that protease activity is required for HR induction by AvrBsT. This in turn suggests that recognition of AvrBsT by a cognate R gene product may not be by a direct protein : protein interaction, as is often assumed. Instead, AvrBsT may be recognized indirectly by the changes it induces on specific host-cell proteins (Fig. 1). Although protease activity appears to be necessary for the HR-inducing capacity of YopJ homologues, we do not yet have any information on how this activity relates to pathogenesis of plants. Unfortunately, mutations in such have failed to any on it to their role in plant in a diverse of phytopathogenic bacteria that they do make an A second family of type III effectors that is at among Xanthomonas is the AvrBs3 Members of this family are by the of direct and 1996). The amino sequences by and the of the of both the Avr and virulence functions of these proteins (Yang et al., 2000). The of these proteins are often and nuclear localization and an activation both of which are required for virulence and avirulence activity (Yang et al., 2000). A recent in how of this family function was the that the protein has activity (Yang et al., 2000). a was shown to bind and this is by but only by The for DNA suggests that the activity may have The DNA activity, with the and activation suggests that this family of effectors functions by host cell gene (Fig. however, direct activation of specific plant genes by an AvrBs3 family member has not been The of an activation and nuclear localization for avirulence activity suggests that these Avr proteins may be indirectly recognized by plant R gene as proposed above for YopJ family or that they are recognized as part of a with their targets. A more direct of identifying targets of type III effectors is to plant using a with the Avr protein as the Although several groups are this with various avr genes, only one has been published thus and 2000). In this latter work, the AvrPto protein from P. syringae pv. was as a to a AvrPto proteins were The most common gene identified in the was than which a protein with similarity to a protein from was found to be induced in both resistant and susceptible isogenic lines infection with P. syringae pv. and proteins that are homologous to a small a protein with a strong similarity to The AvrPto protein a myristylation at its which is required for plasma membrane localization and avirulence function et al., thus, the with might a that is necessary for activation of AvrPto inside the plant cell. of proteins that interact with AvrPto in the is However, the is to the of these which may be difficult. the most to will be to a in This has been accomplished by protein from in now make it to protein : protein in cells et al., 2000). Although such have not yet been to effector : target in plant cells, this appears very one would also the function of the genes, either by gene or by and then the with and without pathogen it is not clear would support a role as an effector For example, the target is the of a defence a mutation of such a target could to either defence response expression it were a or defence response expression it were a If the target water and ion flow the plasma membrane, one might to of However, mutations in many different genes can rise to such thus an observation of such may not support for the of an As described above, it appears that the majority of type III effector proteins from phytopathogenic bacteria contribute to virulence in a small quantitative or are partially redundant in function. This observation suggests that it will be to mutants that have become resistant to infection because of an in a target of a type III to this is to a phenotype by a specific effector in the absence of the This can be accomplished by directly expressing the type III effector gene inside a susceptible plant cell. For example, when avrB is in Arabidopsis using an delivery it induces a response in several that the cognate resistance gene RPM1 (Nimchuk et al., 2000). this phenotype is host-specific, as the no response to avrB expression (Nimchuk et al., 2000). of this indicates that a single mutation in the is responsible for the of response to In work, and have for Arabidopsis mutants that to AvrB and identified all of which were to the mutation present in Dangl, The gene identified by these mutations represents a likely target of the AvrB and is by of effector proteins in plant cells has also to effector : target For example, the AvrB and AvrRpm1 effector proteins have been shown to localize to the plasma membrane of plant cells (Nimchuk et al., 2000; Shan et al., 2000b). In all cases, this localization is dependent on a putative myristylation at the of these proteins, suggesting that these Avr proteins are by host-cell after translocation into the plant cell. with this AvrB and AvrRpm1 become when plant cells (Nimchuk et al., 2000). Significantly, mutations that block myristylation in any of these proteins block both virulence and avirulence implying that these effectors function at the host-cell plasma membrane (Fig. and that there may be a mechanistic between effector function and induction of As with the AvrBs3 and YopJ this observation suggests that recognition of these Avr proteins in resistant may only when with their targets, or that these Avr proteins are recognized inconsistent with the however, is the recent of several amino in AvrPto that its avirulence but not its virulence function et al., If type III effectors and their targets form a plant cells, it be to the or the an is added to the could then proteins using The latter will be by the recent of the and Arabidopsis for of these in the As above, several lines of suggest that Avr proteins may be recognized in resistant only when with their targets. If this is then of the target protein recognition of the Avr protein in resistant This first proposed by and and has been the of this are by the protein of and the protein of is required for recognition of AvrPto and is required for recognition of et al., 1994; et al., 1999). Both and protein but these kinases appear to be only and 2001). In both cases, specific recognition of the Avr protein a second gene encoding a protein and a site of plant R gene products. Thus recognition of both AvrPto and two proteins, an protein and a second putative target with the and AvrPto interact with other in a et al., 1996). However, is the target of it be the only AvrPto enhances the virulence of P. syringae pv. on lines that et al., 2000; Shan et al., It will be to proteins can interact with effector : target protein The targets of type III effectors from phytopathogenic bacteria This is in part to a of how these pathogens cause which it to likely targets. However, direct expression of effector proteins in plant cells is new and to identifying these targets, as has of the The to be sequences for several phytopathogenic bacteria, including Xanthomonas Pseudomonas syringae, and Ralstonia solanacearum will several new type III effectors. expression of these effectors in plants may new into how these bacteria cause and how these effector proteins are recognized in resistant host plants. I and the of for in was supported by from the of and

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