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.     

Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato

Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell. Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive. Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions. Pseudomonas syringae pv. tomato T1 causes an HR-like cell death on Nicotiana benthamiana. This rapid cell death was delayed significantly in plants inoculated with P. syringae pv. tomato expressing avrPto. In addition, P. syringae pv. tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines. Transient expression of avrPto in both N. benthamiana and tomato prf3 plants also was able to suppress nonhost HR. Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens. AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions. Experiments with avrPto mutants revealed several residues important for the suppression effects. AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N. benthamiana. These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion.     

A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000.     

Contributions of the effector gene hopQ1-1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

To study the role of type III-secreted effectors in the host adaptation of the tobacco ( Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci , a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean ( Phaseolus vulgaris ) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1-1 _Pph1448A were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1-1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1-1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine-tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non-host species.     

Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana

The Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf. Prf is absolutely required for specific recognition of both effectors. Here, using stable transgenic lines, we show that expression of Pto from its genomic promoter in susceptible tomatoes was sufficient to complement recognition of Pseudomonas syringae pv. tomato (Pst) bacteria expressing either avrPto or avrPtoB. Pto kinase activity was absolutely required for specific immunity. Expression of the Pto N-myristoylation mutant, pto(G2A), conferred recognition of Pst (avrPtoB), but not Pst (avrPto), although bacterial growth in these lines was intermediate between resistant and susceptible lines. Overexpression of pto(G2A) complemented recognition of avrPto. Transgenic tomato plants overexpressing wild-type Pto exhibited constitutive growth phenotypes, but these were absent in lines overexpressing pto(G2A). Therefore, Pto myristoylation is a quantitative factor for effector recognition in tomato, but is absolutely required for overexpression phenotypes. Native expression of Pto in the heterologous species Nicotiana benthamiana did not confer resistance to P. syringae pv. tabaci (Pta) expressing avrPto or avrPtoB, but recognition of both effectors was complemented by Prf co-expression. Thus, specific resistance conferred solely by Pto in N. benthamiana is an artefact of overexpression. Finally, pto(G2A) did not confer recognition of either avrPto or avrPtoB in N. benthamiana, regardless of the presence of Prf. Thus, co-expression of Prf in N. benthamiana complements many but not all aspects of normal Pto function.     

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.     

Role of a xyloglucan-specific endo-beta-1,4-glucanase inhibitor in the interactions of Nicotiana benthamiana with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

A xyloglucan-specific endo-β-1,4-glucanase inhibitor cDNA, NbXEGIP1 , was amplified from diseased leaves of Nicotiana benthamiana . The sequence was similar to the tomato xyloglucan-specific endo-β-1,4-glucanase inhibitor (XEGIP) and tobacco nectarin IV genes that have been described as binding and inactivating fungal Family 12 xyloglucan-specific endo-β-1,4-glucanases. Expression of NbXEGIP1 was not detected in healthy leaves, but the gene was induced during the later stages of infection by the fungi Colletotrichum destructivum and C. orbiculare . Induction of NbXEGIP1 also occurred during disease development by the bacterium Pseudomonas syringae pv. tabaci and during the hypersensitive response produced by P. syringae pv. tabaci expressing avrPto . A portion of NbXEGIP1 was cloned into a tobacco rattle virus vector for virus-induced gene silencing in N. benthamiana . Silencing NbXEGIP1 did not affect the interactions with either Colletotrichum species but did significantly reduce population levels of P. syringae pv. tabaci in the compatible interaction and P. syringae pv. tabaci expressing avrPto in the incompatible interaction. In the susceptible response to P. syringae pv. tabaci , silencing of NbXEGIP1 also resulted in visibly wilted leaves several hours prior to necrosis, which was not observed in control plants. This was related to a significantly higher level of electrolyte leakage and higher expression of a defensin gene from infected NbXEGIP1 -silenced leaves compared with control leaves. Silencing appeared to be specific as it did not affect expression of a related gene, NbXEGIP2 . NbXEGIP1 may act as an inhibitor of a bacterial enzyme that degrades the xyloglucan–cellulose plant cell-wall network, and degradation of the cell wall results in host membrane disruption and signalling of defence responses.     

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.     

The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in pseudomonas syringae

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene, avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical 'hrp box,' which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabidopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.     

Leaf stage-associated resistance is correlated with phytohormones in a pathosystem-dependen manner

It has been reported in several pathosystems that disease resistance can vary in leaves at different stages. However, how general this leaf stage-associated resistance is, and the molecular mechanism(s) underlying it, remain largely unknown. Here, we investigated the effect of leaf stage on basal resistance, effectortriggered immunity(ETI) and nonhost resistance, using eight pathosystems involving the hosts Arabidopsis thaliana, Nicotiana tabacum, and N. benthamiana and the pathogens Sclerotinia sclerotiorum, Pseudomonas syringae pv. tabaci, P. syringae pv. tomato DC3000, and Xanthomonas oryzae pv. oryzae(Xoo). We show evidence that leaf stage-associated resistance exists ubiquitously in plants, but with varying intensity at different stages in diverse pathosystems. Microarray expression profiling assays demonstrated that hundreds of genes involved in defense responses, phytohormone biosynthesis and signaling, and calcium signaling, were differentially expressed between leaves at different stages. The Arabidopsis mutants sid1, sid2-3, ein2, jar1-1, aba1 and aao3 lost leaf stage-associated resistance to S. sclerotiorum, and the mutants aba1 and sid2-3 were affected in leaf stage-associated RPS2/Avr Rpt2t-conferred ETI, whereas only the mutant sid2-3 influenced leaf stage-associated nonhost resistance to Xoo. Our results reveal that the phytohormones salicylic acid,ethylene, jasmonic acid and abscisic acid likely play an essential, but pathosystem-dependent, role in leaf stageassociated resistance.     

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.     

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.     

Expression of the Tomato Pto Gene in Tobacco Enhances Resistance to Pseudomonas syringae pv tabaci Expressing avrPto

The Pto gene encodes a serine-threonine kinase that confers resistance in tomato to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. We examined the ability of Pto to function in tobacco, a species that is sexually incompatible with tomato. Evidence that a heterologous Pto-like signal transduction pathway is present in tobacco was suggested by the fact that tobacco line Wisconsin-38 exhibits a hypersensitive response after infection with P. syringae pv tabaci expressing avrPto. We introduced a Pto transgene into cultivar Wisconsin-38 and assessed the ability of transformed plants to further inhibit growth of the P. s. tabaci strain expressing avrPto. The Pto-transformed tobacco plants exhibited a significant increase in resistance to the avirulent P. s. tabaci strain compared with wild-type tobacco as indicated by (1) more rapid development of a hypersensitive resistance response at high inoculum concentrations (108 colony-forming units per mL); (2) lessened severity of disease symptoms at moderate inoculum concentrations (106 and 107 colony-forming units per mL); and (3) reduced growth of avirulent P. s. tabaci in inoculated leaves. The results indicate that essential components of a Pto-mediated signal transduction pathway are conserved in tobacco and should prompt examination of resistance gene function across even broader taxonomic distances.     

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.     

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.     

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.     

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.     

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.