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.     

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.     

Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors

Harpins are a subset of type III secretion system (T3SS) substrates found in all phytopathogenic bacteria that utilize a T3SS. Pseudomonas syringae pv. tomato DC3000 was previously reported to produce two harpins, HrpZ1 and HrpW1. DC3000 was shown here to deploy two additional proteins, HopAK1 and HopP1, which have the harpin-like properties of lacking cysteine, eliciting the hypersensitive response (HR) when partially purified and infiltrated into tobacco leaves, and possessing a two-domain structure similar to that of the HrpW1 class of harpins. Unlike the single-domain harpin HrpZ1, the two-domain harpins have C-terminal enzyme-like domains: pectate lyase for HopAK1 and lytic transglycosylase for HopP1. Genetic techniques to recycle antibiotic markers were applied to DC3000 to generate a quadruple harpin gene polymutant. The polymutant was moderately reduced in the elicitation of the HR and translocation of the T3SS effector AvrPto1 fused to a Cya translocation reporter, but the mutant was unaffected in the secretion of AvrPto1-Cya. The DC3000 hrpK1 gene encodes a putative translocator in the HrpF/NopX family and was deleted in combination with the four harpin genes. The hrpK1 quadruple harpin gene polymutant was strongly reduced in HR elicitation, virulence, and translocation of AvrPto1-Cya into plant cells but not in the secretion of representative T3SS substrates in culture. HrpK1, HrpZ1, HrpW1, and HopAK1, but not HopP1, were independently capable of restoring some HR elicitation to the hrpK1 quadruple harpin gene polymutant, which suggests that a consortium of semiredundant translocators from three protein classes cooperate to form the P. syringae T3SS translocon.     

Pseudomonas syringae HrpJ is a type III secreted protein that is required for plant pathogenesis, injection of effectors, and secretion of the HrpZ1 Harpin

The bacterial plant pathogen Pseudomonas syringae requires a type III protein secretion system (TTSS) to cause disease. The P. syringae TTSS is encoded by the hrp-hrc gene cluster. One of the genes within this cluster, hrpJ, encodes a protein with weak similarity to YopN, a type III secreted protein from the animal pathogenic Yersinia species. Here, we show that HrpJ is secreted in culture and translocated into plant cells by the P. syringae pv. tomato DC3000 TTSS. A DC3000 hrpJ mutant, UNL140, was greatly reduced in its ability to cause disease symptoms and multiply in Arabidopsis thaliana. UNL140 exhibited a reduced ability to elicit a hypersensitive response (HR) in nonhost tobacco plants. UNL140 was unable to elicit an AvrRpt2- or AvrB1-dependent HR in A. thaliana but maintained its ability to secrete AvrB1 in culture via the TTSS. Additionally, UNL140 was defective in its ability to translocate the effectors AvrPto1, HopB1, and AvrPtoB. Type III secretion assays showed that UNL140 secreted HrpA1 and AvrPto1 but was unable to secrete HrpZ1, a protein that is normally secreted in culture in relatively large amounts, into culture supernatants. Taken together, our data indicate that HrpJ is a type III secreted protein that is important for pathogenicity and the translocation of effectors into plant cells. Based on the failure of UNL140 to secrete HrpZ1, HrpJ may play a role in controlling type III secretion, and in its absence, specific accessory proteins, like HrpZ1, may not be extracellularly localized, resulting in disabled translocation of effectors into plant cells.     

HopPtoN is a Pseudomonas syringae Hrp (type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associated with both compatible and incompatible plant interactions

Pseudomonas syringae pv. tomato DC3000 causes bacterial speck disease in tomato, and it elicits the hypersensitive response (HR) in non-host plants such as Nicotiana tabacum and Nicotiana benthamiana. The compatible and incompatible interactions of DC3000 with tomato and Nicotiana spp., respectively, result in plant cell death, but the HR cell death occurs more rapidly and is associated with effective plant defense. Both interactions require the Hrp (HR and pathogenicity) type III secretion system (TTSS), which injects Hop (Hrp outer protein) effectors into plant cells. Here, we demonstrate that HopPtoN is translocated into tomato cells via the Hrp TTSS. A hopPtoN mutant produced eightfold more necrotic 'speck' lesions on tomato leaves than did DC3000, but the mutant and the wild-type strain grew to the same level in infected leaves. In non-host N. tabacum leaves, the hopPtoN mutant produced more cell death, whereas a DC3000 strain overexpressing HopPtoN produced less cell death and associated electrolyte leakage in comparison with wild-type DC3000. Transient expression of HopPtoN via infection with a PVX viral vector enabled tomato and N. benthamiana plants to tolerate, with reduced disease lesions, challenge infections with DC3000 and P. syringae pv. tabaci 11528, respectively. HopPtoN showed cysteine protease activity in vitro, and hopPtoN mutants altered in the predicted cysteine protease catalytic triad (C172S, H283A and D299A) lost HR suppression activity. These observations reveal that HopPtoN is a TTSS effector that can suppress plant cell death events in both compatible and incompatible interactions.     

Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast

The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.     

The Pseudomonas syringae HopPtoV protein is secreted in culture and translocated into plant cells via the type III protein secretion system in a manner dependent on the ShcV type III chaperone

The bacterial plant pathogen Pseudomonas syringae depends on a type III protein secretion system and the effector proteins that it translocates into plant cells to cause disease and to elicit the defense-associated hypersensitive response on resistant plants. The availability of the P. syringae pv. tomato DC3000 genome sequence has resulted in the identification of many novel effectors. We identified the hopPtoV effector gene on the basis of its location next to a candidate type III chaperone (TTC) gene, shcV, and within a pathogenicity island in the DC3000 chromosome. A DC3000 mutant lacking ShcV was unable to secrete detectable amounts of HopPtoV into culture supernatants or translocate HopPtoV into plant cells, based on an assay that tested whether HopPtoV-AvrRpt2 fusions were delivered into plant cells. Coimmunoprecipitation and Saccharomyces cerevisiae two-hybrid experiments showed that ShcV and HopPtoV interact directly with each other. The ShcV binding site was delimited to an N-terminal region of HopPtoV between amino acids 76 and 125 of the 391-residue full-length protein. Our results demonstrate that ShcV is a TTC for the HopPtoV effector. DC3000 overexpressing ShcV and HopPtoV and DC3000 mutants lacking either HopPtoV or both ShcV and HopPtoV were not significantly impaired in disease symptoms or bacterial multiplication in planta, suggesting that HopPtoV plays a subtle role in pathogenesis or that other effectors effectively mask the contribution of HopPtoV in plant pathogenesis.     

A Pseudomonas syringae pv. tomato avrE1/hopM1 mutant is severely reduced in growth and lesion formation in tomato

The model plant pathogen Pseudomonas syringae pv. tomato DC3000 grows and produces necrotic lesions in the leaves of its host, tomato. Both abilities are dependent upon the hypersensitive response and pathogenicity (Hrp) type III secretion system (TTSS), which translocates multiple effector proteins into plant cells. A previously constructed DC3000 mutant with a 9.3-kb deletion in the Hrp pathogenicity island conserved effector locus (CEL) was strongly reduced in growth and lesion formation in tomato leaves. The ACEL mutation affects three putative or known effector genes: avrE1, hopM1, and hopAA1-1. Comparison of genomic sequences of DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a revealed that these are the only effector genes present in the CEL of all three strains. AvrEl was shown to carry functional TTSS translocation signals based on the performance of a fusion of the first 315 amino acids of AvrE1 to the Cya translocation reporter. A DC3000 delta avrE1 mutant was reduced in its ability to produce lesions but not in its ability to grow in host tomato leaves. AvrE1 expressed from the 35S promoter elicited cell death in nonhost Nicotiana tabacum leaves and host tomato leaves in Agrobacterium-mediated transient expression experiments. Mutations involving combinations of avrE1, hopM1, and hopAA1-1 revealed that deletion of both avrE1 and hopM1 reproduced the strongly reduced growth and lesion phenotype of the delta CEL mutant. Furthermore, quantitative assays involving different levels of inoculum and electrolyte leakage revealed that the avrE1/hopM1 and deltaCEL mutants both were partially impaired in their ability to elicit the hypersensitive response in nonhost N. benthamiana leaves. However, the avrE1/hopM1 mutant was not impaired in its ability to deliver AvrPto1(1-100)-Cya to nonhost N. benthamiana or host tomato leaves during the first 9 h after inoculation. These data suggest that AvrE1 acts within plant cells and promotes lesion formation and that the combined action of AvrE1 and HopM1 is particularly important in promoting bacterial growth in planta.     

Pseudomonas syringae type III chaperones ShcO1, ShcS1, and ShcS2 facilitate translocation of their cognate effectors and can substitute for each other in the secretion of HopO1-1

The Pseudomonas syringae type III secretion system (TTSS) translocates effector proteins into plant cells. Several P. syringae effectors require accessory proteins called type III chaperones (TTCs) to be secreted via the TTSS. We characterized the hopO1-1, hopS1, and hopS2 operons in P. syringae pv. tomato DC3000; these operons encode three homologous TTCs, ShcO1, ShcS1, and ShcS2. ShcO1, ShcS1, and ShcS2 facilitated the type III secretion and/or translocation of their cognate effectors HopO1-1, HopS1, and HopS2, respectively. ShcO1 and HopO1-1 interacted with each other in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, ShcS1 and ShcS2 were capable of substituting for ShcO1 in facilitating HopO1-1 secretion and translocation and each TTC was able to bind the other's cognate effectors in yeast two-hybrid assays. Moreover, ShcO1, ShcS1, and ShcS2 all bound to the middle-third region of HopO1-1. The HopS2 effector possessed atypical P. syringae TTSS N-terminal characteristics and was translocated in low amounts. A site-directed HopS2 mutation that introduced a common N-terminal characteristic from other P. syringae type III secreted substrates increased HopS2 translocation, supporting the idea that this characteristic functions as a secretion signal. Additionally, hopO1-2 and hopT1-2 were shown to encode effectors secreted via the DC3000 TTSS. Finally, a DC3000 hopO1-1 operon deletion mutant produced disease symptoms similar to those seen with wild-type DC3000 but was reduced in its ability to multiply in Arabidopsis thaliana. The existence of TTCs that can bind to dissimilar effectors and that can substitute for each other in effector secretion provides insights into the nature of how TTCs function.     

Pseudomonas syringae pv. tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that injects virulence effector proteins into host cells via a type III secretion system (TTSS). TTSS-deficient mutants have a Hrp- phenotype, that is, they cannot elicit the hypersensitive response (HR) in non-host plants or pathogenesis in host plants. Mutations in effector genes typically have weak virulence phenotypes (apparently due to redundancy), but deletion of six open reading frames (ORF) in the DC3000 conserved effector locus (CEL) reduces parasitic growth and abolishes disease symptoms without affecting function of the TTSS. The inability of the DeltaCEL mutant to cause disease symptoms in tomato was restored by a clone expressing two of the six ORF that had been deleted: CEL ORF3 (HopPtoM) and ORF4 (ShcM). A DeltahopPtoM::nptII mutant was constructed and found to grow like the wild type in tomato but to be strongly reduced in its production of necrotic lesion symptoms. HopPtoM expression in DC3000 was activated by the HrpL alternative sigma factor, and the protein was secreted by the Hrp TTSS in culture and translocated into Arabidopsis cells by the Hrp TTSS during infection. Secretion and translocation were dependent on ShcM, which was neither secreted nor translocated but, like typical TTSS chaperones, could be shown to interact with HopPtoM, its cognate effector, in yeast two-hybrid experiments. Thus, HopPtoM is a type III effector that, among known plant pathogen effectors, is unusual in making a major contribution to the elicitation of lesion symptoms but not growth in host tomato leaves.     

Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis: The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE(Pto) are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.     

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.     

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.     

Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins

Basal resistance in plants is induced by flagellin and several other common bacterial molecules and is implicated in the immunity of plants to most bacteria and other microbes. However, basal resistance can be suppressed by effector proteins that are injected by the type III secretion system (TTSS) of pathogens such as Pseudomonas syringae. This study demonstrates that basal resistance in the leaves of Nicotiana benthamiana is accompanied by reduced vascular flow into minor veins. Reduced vascular flow was assayed by feeding leaves, via freshly excised petioles, with 1% (weight in volume, w/v) neutral red (NR) and then observing differential staining of minor veins or altered levels of extractable dye in excised leaf samples. The reduced vascular staining was localized to tissues expressing basal resistance and was observable when resistance was induced by either the non-pathogen Pseudomonas fluorescens, a TTSS-deficient mutant of P. syringae pv. tabaci, or flg22 (a flagellin-derived peptide elicitor of basal resistance). Nicotiana benthamiana leaf areas expressing basal resistance no longer elicited the hypersensitive response when challenge inoculated with P. syringae pv. tomato DC3000. The reduced vascular staining effect was suppressed by wild-type P. syringae pv. tabaci and P. fluorescens heterologously expressing a P. syringae TTSS and AvrPto1(PtoJL1065). TTSS-proficient P. fluorescens was used to test the ability of several P. syringae pv. tomato DC3000 effectors for their ability to suppress the basal resistance-associated reduced vascular staining effect. AvrE(PtoDC3000), HopM1(PtoDC3000) (formerly known as HopPtoM), HopF2(PtoDC3000) (HopPtoF) and HopG1(PtoDC3000) (HopPtoG) suppressed basal resistance by this test, whereas HopC1(PtoDC3000) (HopPtoC) did not. In summary, basal resistance locally alters vascular function and the vascular dye uptake assay should be a useful tool for characterizing effectors that suppress basal resistance.     

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.     

Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector proteins of Pseudomonas syringae pv. phaseolicola 1448A

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.     

Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.