Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island

We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 micro M in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.     

Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.     

Identification of Erwinia amylovora genes induced during infection of immature pear tissue

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In this study, we used a modified in vivo expression technology system to identify E. amylovora genes that are activated during infection of immature pear tissue, a process that requires the major pathogenicity factors of this organism. We identified 394 unique pear fruit-induced (pfi) genes on the basis of sequence similarity to known genes and separated them into nine putative function groups including host-microbe interactions (3.8%), stress response (5.3%), regulation (11.9%), cell surface (8.9%), transport (13.5%), mobile elements (1.0%), metabolism (20.3%), nutrient acquisition and synthesis (15.5%), and unknown or hypothetical proteins (19.8%). Known virulence genes, including hrp/hrc components of the type III secretion system, the major effector gene dspE, type II secretion, levansucrase (lsc), and regulators of levansucrase and amylovoran biosynthesis, were upregulated during pear tissue infection. Known virulence factors previously identified in E. (Pectobacterium) carotovora and Pseudomonas syringae were identified for the first time in E. amylovora and included HecA hemagglutinin family adhesion, Peh polygalacturonase, new effector HopPtoC(EA), and membrane-bound lytic murein transglycosylase MltE(EA). An insertional mutation within hopPtoC(EA) did not result in reduced virulence; however, an mltE(EA) knockout mutant was reduced in virulence and growth in immature pears. This study suggests that E. amylovora utilizes a variety of strategies during plant infection and to overcome the stressful and poor nutritional environment of its plant hosts.     

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv. tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions. Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors. Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes. Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue. Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P. syringae. Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.     

HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family.

HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora. The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family. Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv. syringae, HrpO from Pseudomonas solanacearum, and HrpC2 from Xanthomonas campestris pv. vesicatoria. Cells of E. amylovora containing mutated hrpI genes or cells of Escherichia coli containing the cloned hrp gene cluster with mutated hrpI produce but do not export harpin. When similar cells with functional hrpI genes were grown at 25 degrees C, but not at 37 degrees C, harpin was exported to the culture supernatant. Direct evidence that HrpI is involved in the secretion of a virulence protein has been offered. Two other loci of the hrp gene cluster are involved in the regulation of harpin, and four other loci also are involved in the secretion of harpin. Since harpin and other proteins likely to be secreted by the LcrD family of proteins lack typical signal peptides, their secretion mechanism is distinct from the general protein export pathway.     

Novel exchangeable effector loci associated with the Pseudomonas syringae hrp pathogenicity island: evidence for integron-like assembly from transposed gene cassettes

Pseudomonas syringae strains use a type III secretion system (TTSS) to translocate effector proteins that assist in the parasitism of host plant cells. Some genes for effector proteins are clustered in the exchangeable effector locus (EEL) associated with the hrp pathogenicity island. A polymerase chain reaction-based screen was developed to amplify the EEL from P. syringae strains. Of the 86 strains screened, the EEL was successfully amplified from 30 predominately North American P. syringae pv. syringae strains using hrpK and queA-derived primers and from an additional three strains using hrpL and queA-derived primers. Among the amplified EEL, ten distinct types of EEL were identified that could be classified into six families distinguishable by genetic composition, but other types of EEL may be present in strains isolated in other geographical regions. No linkage with the host range of the source strain was apparent. Gene cassettes carrying conserved flanking, coding, and intergenic sequences, present in different combinations, were identified in the characterized EEL. Six new alleles of known effectors were identified that differed from the homolog in sequence, size, or both of the gene. One of these apparently novel effector proteins, HopPsyB, retained a strongly conserved amino terminus similar to that of HopPsyA, but other regions of the two polypeptides were only weakly similar. hopPsyB was expressed from an apparent operon that included hrpK and a shcA homolog, shcB. Escherichia coli MC4100 expressing the hrp TTSS, ShcB, and HopPsyB elicited the hypersensitive response (HR) in tobacco, consistent with effector production. Indicative of translocation as an effector, P. syringae pv. tomato DC3000 expressing a HopPsyB':'AvrRpt2 fusion elicited the HR in RPS2+ Arabidopsis thaliana. P. syringae pv. tomato DC3000 carrying HopPsyB exhibited slightly enhanced virulence in several Brassica spp. These results are consistent with the hypotheses that the EEL is a source of disparate effectors functioning in pathogenicity of P. syringae strains and that it evolved independently of the hrp pathogenicity island central conserved region, most likely through integron-like assembly of transposed gene cassettes.     

Identification of a novel Pseudomonas syringae Psy61 effector with virulence and avirulence functions by a HrpL-dependent promoter-trap assay

The hrp pathogenicity island of Pseudomonas syringae encodes a type III secretion system (TTSS) that translocates effectors into plant cells. Most genes encoding effectors are dispersed in the P. syringae genome. Regardless of location, all are regulated coordinately by the alternative sigma factor HrpL. An HrpL-dependent promoter-trap assay was developed to screen genomic libraries of P. syringae strains for promoters whose activity in Escherichia coli is dependent on an inducible hrpL construct. Twenty-two HrpL-dependent promoter fragments were isolated from P. syringae Psy61 that included promoters for known HrpL-dependent genes. One fragment also was isolated that shared no similarity with known genes but retained a near consensus HrpL-dependent promoter. The sequence of the region revealed a 375-amino acid open reading frame encoding a 40.5-kDa product that was designated HopPsyL. HopPsyL was structurally similar to other secreted effectors and carried a putative chloroplast-targeting signal and two predicted transmembrane domains. HopPsyL':'AvrRpt2 fusions were translocated into host cells via the P. syringae pv. tomato DC3000 hrp TTSS. A hopPsyL::kan mutant of Psy61 exhibited strongly reduced virulence in Phaseolus vulgaris cv. Kentucky Wonder, but did not appear to act as a defense response suppressor. The ectopically expressed gene reduced the virulence of Pseudomonas syringae DC3000 transformants in Arabidopsis thaliana Col-0. The gene was shown to be conserved in 6 of 10 P. syringae pv. syringae strains but was not detected in 35 strains of other pathovars. HopPsyL appears to be a novel TTSS-dependent effector that functions as a host-species-specific virulence factor in Psy61.     

Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence

Phage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non-pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp-) while six still could (Dsp-). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp- mutants were all clustered on the left part of this region, while the MudIIPR13 insertions of Dsp- mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E. amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N.J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.     

A pseudomonas syringae pv. tomato DC3000 Hrp (Type III secretion) deletion mutant expressing the Hrp system of bean pathogen P. syringae pv. syringae 61 retains normal host specificity for tomato

The plant pathogenic species Pseudomonas syringae is divided into numerous pathovars based on host specificity. For example, P. syringae pv. tomato DC3000 is pathogenic on tomato and Arabidopsis, whereas P. syringae pv. syringae 61 is pathogenic on bean. The ability of P. syringae strains to elicit the hypersensitive response (HR) in non-hosts or be pathogenic (or parasitic) in hosts is dependent on the Hrp (type III secretion) system and effector proteins this system is thought to inject into plant cells. To test the role of the Hrp system in determining host range, the hrp/hrc gene cluster (hrpK through hrpR) was deleted from DC3000 and complemented in trans with the orthologous cluster from strain 61. Mutant CUCPB5114 expressing the bean pathogen Hrp system on plasmid pCPP2071 retained the ability of wild-type DC3000 to elicit the HR in bean, to grow and cause bacterial speck in tomato, and to elicit a cultivar-specific (gene-for-gene) HR in tomato plants carrying the Pto resistance gene. However, the symptoms produced in compatible tomato plants involved markedly reduced chlorosis, and CUCPB5114(pCPP2071) did not grow or produce symptoms in Arabidopsis Col-0 although it was weakly virulent in NahG Arabidopsis. A hypersensitive-like collapse was produced by CUCPB5114(pCPP2071) in Arabidopsis Col-0 at 1 x 10(7) CFU/ml, but only if the bacteria also expressed AvrB, which is recognized by the RPM1 resistance gene in Col-0 and confers incompatibility. These observations support the concept that the P. syringae effector proteins, rather than secretion system components, are the primary determinants of host range at both the species and cultivar levels of host specificity.     

WtsE, an AvrE-family effector protein from Pantoea stewartii subsp. stewartii, causes disease-associated cell death in corn and requires a chaperone protein for stability

The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxol. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.     

Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora

Erwinia amylovora is the causal agent of fire blight, a disease affecting members of subfamily Maloideae. In order to analyze mechanisms leading to compatible or incompatible interactions, early plant molecular events were investigated in two genotypes of Malus with contrasting susceptibility to fire blight, after confrontation with either E. amylovora or the incompatible tobacco pathogen Pseudomonas syringae pv. tabaci. Many defense mechanisms, including generation of an oxidative burst and accumulation of pathogenesis-related proteins, were elicited in both resistant and susceptible genotypes by the two pathogens at similar rates and according to an equivalent time course. This elicitation was linked with the functional hypersensitive reaction and pathogenicity (hrp) cluster of E. amylovora, because an hrp secretion mutant did not induce such responses. However, a delayed induction of several genes of various branch pathways of the phenylpropanoid metabolism was recorded in tissues of the susceptible genotype challenged with the wild-type strain of E. amylovora, whereas these genes were quickly induced in every other plant-bacteria interaction, including interactions with the hrp secretion mutant. This suggests the existence of hrp-independent elicitors of defense in the fire blight pathogen as well as hrp-dependant mechanisms of suppression of these nonspecific inductions.