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Many plant pathogenic bacteria utilize a conserved type III secretion system (TTSS) to deliver effector proteins into the host tissue. Indirect evidence has suggested that at least some effector proteins are translocated from the bacterial cytoplasm into the plant cell. Using an immunocytochemical approach, we demonstrate that the type III effector AvrBs3 from Xanthomonas campestris pv. vesicatoria localizes to nuclei of infected pepper leaves. Importantly, AvrBs3 translocation was observed in situ in native tissues of susceptible and resistant plants. AvrBs3 was detected in the nucleus as soon as 4 h post infection, which was dependent on a functional TTSS and the putative translocator HrpF. N-terminal AvrBs3 deletion derivatives are no longer secreted by the TTSS in vitro and could not be detected inside the host cells, suggesting that the N-terminus of AvrBs3 is important for secretion. Deletion of the nuclear localization signals in the AvrBs3 C-terminus, which are required for the AvrBs3-mediated induction of the hypersensitive reaction in resistant pepper plants, abolished AvrBs3 localization to the nucleus. This is the first report on direct evidence for translocation of a native type III effector protein from a plant pathogenic bacterium into the host cell. 相似文献
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Effectors of the bacterial type III secretion system provide invaluable molecular probes to elucidate the molecular mechanisms of plant immunity and pathogen virulence. In this report, we focus on the AvrBs2 effector protein from the bacterial pathogen Xanthomonas euvesicatoria (Xe), the causal agent of bacterial spot disease of tomato and pepper. Employing homology-based structural analysis, we generate a three-dimensional structural model for the AvrBs2 protein and identify catalytic sites in its putative glycerolphosphodiesterase domain (GDE). We demonstrate that the identified catalytic region of AvrBs2 was able to functionally replace the GDE catalytic site of the bacterial glycerophosphodiesterase BhGlpQ cloned from Borrelia hermsii and is required for AvrBs2 virulence. Mutations in the GDE catalytic domain did not disrupt the recognition of AvrBs2 by the cognate plant resistance gene Bs2. In addition, AvrBs2 activation of Bs2 suppressed subsequent delivery of other Xanthomonas type III effectors into the host plant cells. Investigation of the mechanism underlying this modulation of the type III secretion system may offer new strategies to generate broad-spectrum resistance to bacterial pathogens. 相似文献
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Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria and most other Gram-negative bacterial plant pathogens largely depends on a type III secretion (TTS) system which is encoded by hypersensitive response and pathogenicity (hrp) genes. These genes are induced in the plant and are essential for the bacterium to be virulent in susceptible hosts and for the induction of the hypersensitive response (HR) in resistant host and non-host plants. The TTS machinery secretes proteins into the extracellular milieu and effector proteins into the plant cell cytosol. In the plant, the effectors presumably interfere with cellular processes to the benefit of the pathogen or have an avirulence activity that betrays the bacterium to the plant surveillance system. Type III effectors were identified by their avirulence activity, co-regulation with the TTS system and homology to known effectors. A number of effector proteins are members of families, e.g., the AvrBs3 family in Xanthomonas. AvrBs3 localizes to the nucleus of the plant cell where it modulates plant gene expression. Another family that is also present in Xanthomonas is the YopJ/AvrRxv family. The latter proteins appear to act as SUMO cysteine proteases in the host. Here, we will present an overview about the regulation of the TTS system and its substrates and discuss the function of the AvrRxv and AvrBs3 family members in more detail. 相似文献
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Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana 总被引:8,自引:0,他引:8
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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. 相似文献
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DOREEN GÜRLEBECK SIMONE JAHN NORMAN GÜRLEBECK ROBERT SZCZESNY BORIS SZUREK SIMONE HAHN GERD HAUSE ULLA BONAS 《Molecular Plant Pathology》2009,10(2):175-188
Xanthomonas campestris pv. vesicatoria secretes at least 20 effector proteins through the type III secretion system directly into plant cells. In this study, we uncovered virulence activities of the effector proteins AvrBs1, AvrBs3 and AvrBs4 using Agrobacterium‐mediated transient expression of the corresponding genes in Nicotiana benthamiana, followed by microscopic analyses. We showed that, in addition to the nuclear‐localized AvrBs3, the effector AvrBs1, which localizes to the plant cell cytoplasm, also induces a morphological change in mesophyll cells. Comparative analyses revealed that avrBs3‐expressing plant cells contain highly active nuclei. Furthermore, plant cells expressing avrBs3 or avrBs1 show a decrease in the starch content in chloroplasts and an increased number of vesicles, indicating an enlargement of the central vacuole and the cell wall. Both AvrBs1 and AvrBs3 cause an increased ion efflux when expressed in N. benthamiana. By contrast, expression of the avrBs3 homologue avrBs4 leads to large catalase crystals in peroxisomes, suggesting a possible virulence function of AvrBs4 in the suppression of the plant defence responses. Taken together, our data show that microscopic inspection can uncover subtle and novel virulence activities of type III effector proteins. 相似文献
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Functional analysis of HrpF, a putative type III translocon protein from Xanthomonas campestris pv. vesicatoria
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Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen Xanthomonas campestris pv. vesicatoria, encoded by the hrp (hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an hrpF null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane. 相似文献
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Zwiesler-Vollick J Plovanich-Jones AE Nomura K Bandyopadhyay S Joardar V Kunkel BN He SY 《Molecular microbiology》2002,45(5):1207-1218
Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins. 相似文献
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Gibly A Bonshtien A Balaji V Debbie P Martin GB Sessa G 《Molecular plant-microbe interactions : MPMI》2004,17(11):1212-1222
The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. Plants of the tomato line Hawaii 7981 are resistant to race T3 of X. campestris pv. vesicatoria expressing the type III effector protein AvrXv3 and develop a typical hypersensitive response upon bacterial challenge. A combination of suppression subtractive hybridization and microarray analysis identified a large set of cDNAs that are induced or repressed during the resistance response of Hawaii 7981 plants to X. campestris pv. vesicatoria T3 bacteria. Sequence analysis of the isolated cDNAs revealed that they correspond to 426 nonredundant genes, which were designated as XRE (Xanthomonas-regulated) genes and were classified into more than 20 functional classes. The largest functional groups contain genes involved in defense, stress responses, protein synthesis, signaling, and photosynthesis. Analysis of XRE expression kinetics during the tomato resistance response to X. campestris pv. vesicatoria T3 revealed six clusters of genes with coordinate expression. In addition, by using isogenic X. campestris pv. vesicatoria T2 strains differing only by the avrXv3 avirulence gene, we found that 77% of the identified XRE genes were directly modulated by expression of the AvrXv3 effector protein. Interestingly, 64% of the XRE genes were also induced in tomato during an incompatible interaction with an avirulent strain of Pseudomonas syringae pv. tomato. The identification and expression analysis of X. campestris pv. vesicatoria T3-modulated genes, which may be involved in the control or in the execution of plant defense responses, set the stage for the dissection of signaling and cellular responses activated in tomato plants during the onset of spot disease resistance. 相似文献
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Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island 总被引:6,自引:0,他引:6
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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. 相似文献
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AvrXa7 is a member of the avBs3/pthA gene family and the only known type III secretion system effector gene from Xanthomonas oryzae pv. oryzae with a major contribution to bacterial growth and lesion formation in bacterial blight disease of rice. We examined the general requirement for effectors of the AvrBs3/PthA family in bacterial blight of rice by identifying effectors from diverse strains of the pathogen. Inactivation of single effector genes in representative strains from Japan, Korea, and the Philippines resulted in severely limited growth in plants. Five strains harbored one gene of the avrBs3/pthA family, while one strain had two genes with the equivalent virulence activity of avrXa7. Sequence analysis revealed three genes with unique repeat arrangements in comparison to avrXa7. Comparison of the repetitive regions revealed a potential motif for the group that was also present in the repetitive region of avrBs3. However, the repetitive region of avrBs3 could not support virulence activity but, in combination with the C-terminal coding region of avrXa7, triggered a Xa7-dependent avirulence reaction. The results revealed diverse members of the avrBs3/pthA gene family with virulence activity in X. oryzae pv. oryzae and supported the hypothesis that bacterial blight disease of rice is highly dependent on a single class of type III effectors. The results also indicated that avrXa7 avirulence specificity is separable from virulence activity. 相似文献
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Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades. 相似文献
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The hrp (hypersensitive response and pathogenicity) gene cluster of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria encodes a type III secretion (TTS) system, which injects bacterial effector proteins into the plant cell. Here, we characterized hpaB (hpa, hrp-associated), which encodes a pathogenicity factor with typical features of a TTS chaperone. We show that HpaB is important for the efficient secretion of at least five effector proteins but is dispensable for the secretion of non-effectors such as XopA and the TTS translocon protein HrpF. GST pull-down assays revealed that HpaB interacts with two unrelated effector proteins, AvrBs1 and AvrBs3, but not with XopA. The HpaB-binding site is located within the first 50 amino acids of AvrBs3. This region also contains the targeting signal for HpaB-dependent secretion, which is missing in HrpF and XopA. Intriguingly, the N-termini of HrpF and XopA target the AvrBs3Delta2 reporter for translocation in a DeltahpaB mutant but not in the wild-type strain. This indicates that HpaB plays an essential role in the exit control of the TTS system. Our data suggest that HpaB promotes the secretion of a large set of effector proteins and prevents the delivery of non-effectors into the plant cell. 相似文献
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Xanthomonas campestris pv. vesicatoria is an economically important pathogen of pepper and tomato and has been established as a model organism to study bacterial infection strategies. In the last two decades, intensive genetic and molecular analyses led to the isolation of many genes that play a role in the intimate molecular relationship with the host plant. Essential for pathogenicity is a type III protein secretion system, which delivers bacterial effector proteins into the host cell. Currently, the genome of X. campestris pv. vesicatoria is being sequenced. The availability of genomic sequence information will pave the way for the identification of new bacterial virulence factors by bioinformatic approaches. In this article, we will present preliminary data from the genomic sequence analysis and describe recent and novel studies to identify bacterial type III effector genes. 相似文献
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Zhao Y Thilmony R Bender CL Schaller A He SY Howe GA 《The Plant journal : for cell and molecular biology》2003,36(4):485-499
Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato. The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA). We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants. Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes. These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants. 相似文献