A signal transfer system through three compartments transduces the plant cell contact-dependent signal controlling Ralstonia solanacearum hrp genes

Ralstonia solanacearum hrp genes encode a type III secretion system required for disease development in host plants and for hypersensitive response elicitation on non-hosts. hrp genes are expressed in the presence of plant cells through the HrpB regulator. This activation, which requires physical interaction between the bacteria and the plant cell, is sensed by the outer membrane receptor PrhA. PrhA transduces the plant cell contact-dependent signal through a complex regulatory cascade integrated by the PrhJ, HrpG, and HrpB regulators. In this study, we have identified two genes, named prhI and prhR, that belong to the hrp gene cluster and whose predicted products show homology with extracytoplasmic function sigma factors and transmembrane proteins, respectively. Strains carrying a mutation in prhIR show a delayed pathogenic phenotype toward host plants. PrhIR control the plant cell contact-dependent activation of hrp genes. prhIR gene expression is induced by a signal present in the plant cell coculture that is not PrhA-dependent. Genetic evidence shows that PrhIR act upstream of PrhJ in the regulatory cascade, likely transducing the signal sensed by PrhA through the periplasm as described for signal transfer systems through three compartments. This is the first report of such a surface signaling mechanism activating pathogenicity determinants in response to a nondiffusible plant cell wall signal.     

The hrpB and hrpG regulatory genes of Ralstonia solanacearum are required for different stages of the tomato root infection process

hrp genes, encoding type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium Ralstonia solanacearum GMI1000. Here, we show phenotypes of R. solanacearum mutant strains disrupted in the prhJ, hrpG, or hrpB regulatory genes with respect to root infection and vascular colonization in tomato plants. Tests of bacterial colonization and enumeration in tomato plants, together with microscopic observations of tomato root sections, revealed that these strains display different phenotypes in planta. The phenotype of a prhJ mutant resembles that of the wild-type strain. An hrpB mutant shows reduced infection, colonization, and multiplication ability in planta, and induces a defense reaction similar to a vascular hypersensitive response at one protoxylem pole of invaded plants. In contrast, the hrpG mutant exhibited a wild-type level of infection at secondary root axils, but the ability of the infecting bacteria to penetrate into the vascular cylinder was significantly impaired. This indicates that bacterial multiplication at root infection sites and transit through the endodermis constitute critical stages in the infection process, in which hrpB and hrpG genes are involved. Moreover, our results suggest that the hrpG gene might control, in addition to hrp genes, other functions required for vascular colonization.     

Regulation of the type III secretion system in phytopathogenic bacteria

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

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。     

hrp gene-dependent induction of hin1: a plant gene activated rapidly by both harpins and the avrPto gene-mediated signal

Two classes of bacterial genes are involved in the elicitation of the plant hypersensitive response (HR) in resistant plants: hrp genes and avr genes. hrp genes have been shown to be involved in the production and secretion of a new class of bacterial virulence/avirulence proteins, including harpin of Erwinia amylovora and harpin_Pss of Pseudomonas syringae . The ability of avr genes in the elicitation of the HR/resistance is dependent on functional hrp genes. The relationships between harpins and avr gene products are not known. This study investigates the plant genes induced by harpins and the effect of avr genes on the expression of such plant genes. A tobacco gene highly induced by harpins was isolated by a subtractive hybridization method. Induction of hin1 by P.s. pv. syringae 61 (Pss61) was found to be dependent on functional bacterial hrp genes. P. fluorescens (a saprophyte) or hrp mutants defective in the Hrp secretion pathway did not induce hin1 significantly. A hin1 -related gene in tomato cv. Rio Grande-PtoR was found to be rapidly induced by P. s. pv. tomato T1 (a virulent bacterium on Rio Grande-PtoR) containing the avrPto gene, which mediates the elicitation of the HR/resistance in a Pto plant resistance gene-dependent manner. The induction of hin1 by bacteria correlates with production of harpins in planta . The putative open reading frame of hin1 encodes a novel protein of 221 amino acids. The data suggest that harpins and the avrPto -mediated signal induce a common plant gene in the elicitation of the HR.     

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.     

Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome

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.     

Genetic dissection of Ralstonia solanacearum hrp gene cluster reveals that the HrpV and HrpX proteins are required for Hrp pilus assembly

In both plant and mammalian Gram-negative pathogenic bacteria, type III secretion systems (TTSSs) play a crucial role in interactions with the host. All these systems share conserved proteins (called Hrc in plant pathogens), but each bacterium also produces a variable number of additional type III proteins either unique or with counterparts only in a limited number of related systems. In order to investigate the role of the different proteins encoded by the hrp gene cluster of the phytopathogenic bacterium Ralstonia solanacearum, non-polar mutants in all hrp genes (except for hrcQ) were analysed for their interactions with plants, their ability to secrete the PopA protein and their production of the Hrp pilus. In addition to Hrc proteins and the HrpY major component of the Hrp pilus, four additional Hrp proteins are indispensable for type III secretion and for interactions with plants. We also provide evidence that hrpV and hrpX mutants can still target the HrpY pilin outside the bacterial cell but are impaired in the production of Hrp pili, indicating that HrpV and HrpX proteins are involved in the assembly of this appendage.     

The HopPtoF locus of Pseudomonas syringae pv. tomato DC3000 encodes a type III chaperone and a cognate effector

Type III secretion systems are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcF(Pto) and HopF(Pto), that are preceded by a single hrp box promoter. We present evidence here to show that ShcF(Pto) and HopF(Pto) encode a type III chaperone and a cognate effector, respectively. ShcF(Pto) interacts with and stabilizes the HopF(Pto) protein in the bacterial cell. Translation of HopF(Pto) starts at a rare initiation codon ATA that limits the synthesis of the HopF(Pto) protein to a low level in bacterial cells.