Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.     

New protein-protein interactions identified for the regulatory and structural components and substrates of the type III Secretion system of the phytopathogen Xanthomonas axonopodis Pathovar citri

We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.     

The type III-dependent Hrp pilus is required for productive interaction of Xanthomonas campestris pv. vesicatoria with pepper host plants

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

An hrcU-homologous gene mutant of Xanthomonas campestris pv. glycines 8ra that lost pathogenicity on the host plant but was able to elicit the hypersensitive response on nonhosts.

Transposon mutagenesis was used to isolate nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra, which causes bacterial pustule disease in soybean. A 6.1-kb DNA region in which a mutation gave loss of pathogenicity was isolated and found to carry six open reading frames (ORFs). Four ORFs had homology with hrcU, hrcV, hrcR, and hrcS genes of Ralstonia solanacearum and X. campestris pv. vesicatoria. One nonpathogenic mutant, X. campestris pv. glycines H80, lost pathogenicity on soybean but was able to elicit the hypersensitive response (HR) on nonhost pepper and tomato plants. This mutant still multiplied as well as the wild type in the leaves or cotyledons of soybean. Although the DNA and amino acid sequences showed high homology with known hrp genes, the hrcU-homolog ORF is not required for HR induction on nonhost plants, pepper and tomato, or for the multiplication of bacteria in the host plant. This gene was only required for the pathogenic symptoms of X. campestris pv. glycines 8ra on soybean.     

Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv. oryzae

We have cloned a hrp gene cluster from Xanthomonas oryzae pv. oryzae. Bacteria with mutations in the hrp region have reduced growth in rice leaves and lose the ability to elicit a hypersensitive response (HR) on the appropriate resistant cultivars of rice and the nonhost plant tomato. A 12,165-bp portion of nucleotide sequence from the presumed left end and extending through the hrpB operon was determined. The region was most similar to hrp genes from Xanthomonas campestris pv. vesicatoria and Ralstonia solanacearum. Two new hrp-associated loci, named hpa1 and hpa2, were located beyond the hrpA operon. The hpa1 gene encoded a 13-kDa glycine-rich protein with a composition similar to those of harpins and PopA. The product of hpa2 was similar to lysozyme-like proteins. Perfect PIP boxes were present in the hrpB and hpa1 operons, while a variant PIP box was located upstream of hpa2. A strain with a deletion encompassing hpa1 and hpa2 had reduced pathogenicity and elicited a weak HR on nonhost and resistant host plants. Experiments using single mutations in hpa1 and hpa2 indicated that the loss of hpa1 was the principal cause of the reduced pathogenicity of the deletion strain. A 1,519-bp insertion element was located immediately downstream of hpa2. Hybridization with hpa2 indicated that the gene was present in all of the strains of Xanthomonas examined. Hybridization experiments with hpa1 and IS1114 indicated that these sequences were detectable in all strains of X. oryzae pv. oryzae and some other Xanthomonas species.     

Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants

HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp. The secondary structure of the peptide is predicted to be an alpha-helix, as is the HrpW region that is homologous to HpaG. The predicted alpha-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a xopA gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.     

Immunogold labeling of Hrp pili of Pseudomonas syringae pv. tomato assembled in minimal medium and in planta

Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.     

Domain structure of HrpE, the Hrp pilus subunit of Xanthomonas campestris pv. vesicatoria

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3Delta2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.     

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.     

水稻条斑病细菌hrp基因诱导表达系统的建立

水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。     

Determinants of pathogenicity in Xanthomonas campestris pv. vesicatoria are related to proteins involved in secretion in bacterial pathogens of animals.

One of the model systems investigated for studying plant bacterial pathogenesis is Xanthomonas campestris pv vesicatoria, the causal agent of bacterial spot disease of pepper and tomato. Genes necessary for both basic pathogenicity and the induction of the hypersensitive response in resistant plants (hrp genes) were previously isolated from X. c. pv. vesicatoria and characterized genetically. As a first step toward functional analysis, part of the hrp gene cluster, making up several loci, was sequenced. Here, we report the first indications of the function of hrp genes. Striking similarities to proteins from the mammalian pathogens Shigella flexneri, Yersinia enterocolitica, Y. pestis, and other bacteria were discovered. Proteins encoded by genes within the X. c. pv. vesicatoria loci hrpA, hrpB, and hrpC are similar to ATPases and to Yersinia Ysc and LcrD proteins, which are involved in secretion of Yop proteins, a particular class of essential pathogenicity factors produced by Yersinia species. This finding indicates, for the first time, that the fundamental determinants of pathogenicity may be conserved among bacterial pathogens of plants and animals. We hypothesize that hrp genes are involved in the secretion of molecules essential for the interaction of X. c. pv. vesicatoria with the plant.     

水稻条斑病菌hrcC、hpa3和hrpE基因表达不依赖hrpG和hrpX基因调控

摘要：【目的】决定水稻条斑病菌（Xanthomonas oryzae pv. oryzicola）在非寄主植物上激发过敏反应（hypersensitive response, HR）和在寄主水稻上致病性（pathogenicity）的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌III型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析III型分泌途经的形成提供了线索。