Genes known or predicted to be involved in virulence
Approximately 40% of the ipx fusions that we analysed in detail identify genes known or predicted to be involved in pathogenesis (Table 1). It is not surprising that we identified a large number of fusions to the hrp/hrc region, as these genes are known to be induced upon inoculation of bacteria into plant tissue (Rahme et al., 1991). Likewise, we anticipated identifying genes that encode known or predicted effectors of the type III secretion system, such as avr and vir genes and members of the conserved effector locus (CEL), as these genes are known or expected to be co-regulated with the hrp/hrc locus (Xiao et al., 1994; He, 1998; Alfano et al., 2000). The fact that our IVET strategy selected efficiently for several known genes of this class both validates the approach and suggests that many of the other genes identified in this study are also involved in pathogenesis.
Type III-dependent factors. The two avr genes identified in this screen, avrPpiB (ipx8) and avrPphD (ipx9) have been identified previously in Pst DC3000 (Cournoyer et al., 1995; Arnold et al., 2001a,b). Although avr genes confer the ability to elicit defence responses on plants carrying the corresponding resistance gene (White et al., 2000), it is hypothesized that they provide a selective advantage to the pathogen, perhaps by promoting virulence on susceptible hosts. However, only a few avr genes have been demonstrated to contribute significantly to pathogen virulence (White et al., 2000). We constructed an avrPpiB mutant Pst DC3000 strain (JB206), but were unable to detect a significant alteration in virulence towards A. thaliana or tomato in this strain (Fig. 3). The absence of a virulence phenotype for this mutant is not surprising, as many type III effector proteins are predicted to play redundant or overlapping roles in parasitism (Alfano and Collmer, 1996).
The identification of a gene with similarity to virPphA of P. syringae pv. phaseolicola (ipx7) in Pst DC3000 is exciting. virPphA was initially identified based on its virulence activity in P. syringae pv. phaseolicola and was then subsequently shown to function as an avr factor in soybeans (Jackson et al., 1999). Several groups have also recently reported the discovery of this virPphA-like gene in Pst DC3000 (G. Martin, personal communication, S.-Y. He, personal communication), and its contribution to virulence is currently under investigation.
Alfano et al. (2000) have proposed that many of the genes in the CEL encode type III effectors, as (i) most of the ORFs in the CEL are preceded by a hrp box; and (ii) the deletion of several of these ORFs (including avrE, hrpW and CEL orfs 2–5, but not CEL orf1) from the Pst DC3000 genome resulted in a strain with significantly reduced virulence on tomato. Our identification of ipx fusions to four different genes in the CEL, and demonstration that expression of these genes (with the exception of CEL orf1; see below) is regulated by HrpL (Table 1), further strengthens this hypothesis. Interestingly, we also identified an ipx fusion to a gene with similarity to CEL orf5 (ipx32) that does not map to the CEL and does not contain a conserved ‘hrp box’ in its regulatory region (Table 1). It is unclear whether this gene encodes a new virulence factor.
Our finding that CEL orf1 is not expressed in E. coli carrying the P. syringae hrpL gene (Table 1) is unexpected, given that the ORF is preceded by a well-conserved ‘hrp box’ (Table 1; Alfano et al., 2000), and that genetic studies indicate that expression of CEL orf1 in P. syringae is dependent on hrpL (Lorang and Keen, 1995). We have verified that the pIPET clones carrying the ipx10 and ipx11 fusions contain DNA extending more than 1 kb upstream of the ‘hrp box’, and thus should carry sufficient upstream regulatory sequences to direct normal expression of CEL orf1. This is the only example out of the 21 ipx genes with an ‘hrp box’ that was not induced in the E. coli HrpL expression assay (Table 1). These findings suggest that additional regulatory factors not present in E. coli may be required for the expression of CEL orf1.
CEL ORF1 exhibits similarity to murein lytic transglycosylases (38% amino acid identity to E. coli MltD; Alfano et al., 2000), proteins predicted to encode peptidoglycan hydrolases. It has been hypothesized that this protein may contribute to the virulence of P. syringae by facilitating insertion of the type III secretion apparatus through the peptidoglycan layer of the bacterial envelope (Alfano et al., 2000). Our finding that the CEL orf1 mutant strain (JB205) has reduced virulence (Fig. 3) is consistent with this hypothesis. However, we observed only a small decrease in virulence for this strain, suggesting that there may be one or more additional genes in Pst DC3000 encoding a similar function that compensates, in part, for the loss of CEL ORF1 activity. This subtle reduction in virulence may also account for the fact that, in a previous study, no significant change in virulence was observed for a Pst DC3000 CEL orf1 mutant (referred to as avrE transcription unit II by Lorang and Keen, 1995).
Pectin lyase. One of the intriguing genes identified in this screen encodes a putative pectin lyase (PlyD, ipx31). Pectin, a polysaccharide, is a major structural component of primary plant cell walls. Cell walls serve not only to provide shape and strength to plant cells, but also function as a barrier to pathogen entry, and presumably also to the establishment of direct cell–cell contact between pathogen and plant. The induction of pectolytic enzymes could facilitate the assembly of functional type III secretion complexes that allow transport of bacterial proteins through the plant cell wall and cytoplasmic membrane (Alfano and Collmer, 1997; Hu et al., 2001). Alternatively, these enzymes may contribute to degradation or softening of plant cell walls, increased osmotic fragility and eventual plant cell death (Gross and Cody, 1985). Although several P. syringae pathovars are reported to exhibit pectolytic activity (Gross and Cody, 1985), the role of pectolytic enzymes in the pathogenesis of P. syringae is not understood. Our finding that the plyD-like gene defined by ipx31 is induced during growth in plant tissue and is regulated by HrpL (Table 1) suggests that this gene is involved in pathogenesis.
The Pst DC3000 plyD mutant (KP391) did not exhibit reduced virulence when inoculated onto tomato or A. thaliana plants (Fig. 3; data not shown). It is possible that, like many other plant pathogenic bacteria, Pst DC3000 possesses multiple pectolytic enzymes and that a subtle reduction in virulence resulting from mutation of one of these was not detectable in these experiments (Alfano and Collmer, 1996). To date, only one other Pst DC3000 protein with similarity to a pectolytic enzyme has been reported. HrpW, one of the proteins encoded in the CEL, is a type III-secreted protein that contains a domain with similarity to pectate lyases (Charkowski et al., 1998). However, although HrpW specifically binds to pectate, the protein lacks pectolytic activity. Mutation of the Pst DC3000 hrpW gene did not result in a detectable reduction in virulence on tomato (Charkowski et al., 1998). We have carried out a preliminary search of the available PstDC3000 unfinished genome sequence (www.TIGR.org) for additional pectolytic genes and found one additional gene encoding a putative pectolytic enzyme with similarity to PlyA from Glomerella cingulata (Q00374, 38% amino acid identity/51% amino acid similarity; J. Boch and B. Kunkel, unpublished).
Coronatine biosynthetic genes. Thirteen of the ipx fusions are to genes in the cfl/cfa operon, which encodes enzymes that catalyse synthesis of coronafacic acid, a component of the P. syringae phytotoxin coronatine (COR). Given that the cfl/cfa locus in Pst DC3000 is very large (spanning ≈19 kb; F. Alarcón-Chaidez and C. Bender, personal communication), it is not surprising that we isolated multiple fusions to this region. COR is believed to function as a molecular mimic of the endogenous plant hormone methyl jasmonate (Bender et al., 1999) and may promote colonization of host tissue by altering plant defence signalling pathways (Mittal and Davis, 1995; Kloek et al., 2001). Our observation that the cfl/cfa operon is induced within 6 h after infiltration into plant tissue (Table 2; data not shown) is consistent with previous findings (Li et al., 1998), and supports the hypothesis that COR is important at an early stage of infection.
The regulation of COR gene expression in Pst DC3000 is not fully understood. Recent studies indicate that the expression of Pst DC3000 cfa genes is induced by plant signals (Li et al., 1998) and possibly also by temperature (Rohde et al., 1998), a factor that has a major effect on COR production in P. syringae pv. glycinea (Bender et al., 1999). Our data suggest that cfa gene expression is not directly regulated by HrpL (Table 1). Consistent with this is the finding that there are no sequences resembling an ‘hrp box’ in the regulatory region for the cfl and cfa genes (F. Alarcon-Chaidez and C. Bender, unpublished). However, the observation that an element resembling an ‘hrp box’ is present upstream of corR (V. Joardar and B. Kunkel, unpublished), a regulatory gene that governs COR gene expression in P. syringae pv. glycinea, suggests that COR biosynthesis in Pst DC3000 is coordinated with the expression of the hrp/hrc system.
Genes involved in adaptation for growth in plant tissue
Little is known about the environment faced by P. syringae upon colonization of plant tissue. The identification of ipx fusions to genes likely to be involved in amino acid biosynthesis (ipx42–43) suggests that the apoplastic concentration for some amino acids may be low. This finding is consistent with previous genetic studies demonstrating that many P. syringae mutants impaired for amino acid biosynthesis are unable to grow or cause disease on plant tissue (Cuppels, 1986; Kloek et al., 2000; D. Brooks, A. Kloek and B. Kunkel, submitted). These results may also reflect the fact that some amino acids, such as isoleucine (ipx43), are precursors for phytotoxins (e.g. coronatine; Bender et al., 1999).
Maintenance and/or modification of the bacterial cell envelope may be important for adaptation to changing environmental conditions encountered during pathogenesis (Heithoff et al., 1997; Young and Miller, 1997; Graham and Clark-Curtiss, 1999). In this study, we identified two fusions that define genes that may be involved in bacterial cell wall metabolism. The ipx36 gene encodes a protein with similarity to the muropeptide transporter AmpG, which is involved in the uptake of peptidoglycan turnover products (Holtje, 1998). The ipx35 fusion defines a cicA-like gene, which has been proposed to be involved in regulating cell wall synthesis and morphology in Caulobacter crescentus (Fuchs et al., 2001). The recent finding that a mutation in the N-acetylmuramyl-L-alanine amidase (ampD) gene of Ralstonia solanacearum results in decreased virulence indicates that functional peptidoglycan recycling is important for the virulence of plant pathogens (Tans-Kersten et al., 2000).
The possibility that our screen may predominantly have enriched for genes that are expressed relatively early during infection may account for the fact that we did not identify more genes obviously involved in nutrient acquisition or stress responses. Future studies aimed at identifying genes that are induced at later times during disease interactions may provide additional insight into the metabolic and biosynthetic pathways required for adaptation and growth in plant tissue.
Identification of potential new virulence genes
The largest class of ipx fusions identifies genes that have similarity to proteins with unknown functions (ipx52–65) or show no homology to entries in the databases (ipx51, 66–79). We are excited by the possibility that several of these genes may encode novel virulence factors. One class of fusions appears to be dependent on HrpL. Many genes whose expression is directed by HrpL are believed to be involved with type III secretion, by encoding either structural components of the apparatus or substrates that are secreted via the system (Xiao et al., 1994; Xiao and Hutcheson, 1994). Thus, some of the HrpL-dependent novel genes may encode new effector proteins of the hrp/hrc secretion system. As the hrp/hrc system plays a pivotal role in pathogenesis, it is of prime interest to identify the substrates of this system and to gain a better understanding of the function of these proteins in promoting parasitism and disease.
The regulatory pathways governing induction of P. syringae genes upon inoculation into host tissue are not well understood. Presumably, expression of the genes involved in pathogenesis is activated upon perception of cues (e.g. pH, nutritional cues, specific plant signals) present in the host environment. Based on our analysis of 79 ipx fusions, we have found little evidence in support of the hypothesis that P. syringae pathogenesis genes are induced in response to plant-specific signals. Only two of these fusions (ipx46 and ipx59) appeared to be expressed only in planta, as they did not exhibit GUS activity in culture and yet triggered an HR when inoculated onto tobacco (Table 1). Thus, as the majority of the ipx fusions characterized in this study are induced in hrp-inducing or minimal media, our data suggest that these genes are expressed in response to environmental or nutritional cues present in plant tissue. In the case of genes regulated by HrpL, in planta expression is governed by hrpR and hrpS, whose products are related to the NtrC family of regulatory proteins (Grimm and Panopoulos, 1989; Xiao et al., 1994). However, as HrpR and HrpS lack the regulatory domain found in other members of the NtrC family of two-component response regulators, the mechanism by which HrpR and HrpS sense and respond to environmental cues is unclear (Xiao et al., 1994). In this regard, it will be interesting to analyse the role of the two putative two-component sensors (ipx48, 49) isolated in this screen (Table 1). Future studies on the regulation of ipx genes may help to elucidate the mechanisms that govern how P. syringae senses and responds to the host environment.
Our results indicate that there is at least one additional class of in planta-expressed genes, those whose expression is independent of (or is not solely regulated by) HrpL (Table 1). These genes are especially exciting, as they may define novel virulence factors that constitute one or more new regulatory groups. Further analysis of these novel genes, their regulation and their potential role in pathogenesis should contribute to our understanding of the complex interactions between plant pathogens and their hosts.