Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

Gene expression profile of Ralstonia Solanacearum for the rhizosphere ecological niche of Solanum tuberosum

Plant root secretion can be regarded as signal molecules, which exerts impact on microorganisms in the rhizosphere ecological niche. We obtained gene expression profile of Ralstonia solanacearumPO41 under the root secretions environment of Solanum tuberosum at the time points of 8 hrs, 16 hrs and 24 hrs, respectively, after infection with RNA microarray technology. Bioinformatics tools of differential genes expression analysis, GO functional analysis, cluster analysis and pathway analysis were conducted to find out the pathogenic genes and other related genes. We found that the virulence factors of R. solanacearum mainly focused on the output pathways of toxic protein (Sec pathway, Tat pathway and type III secretion system (T3SS)), the aggregation and transfer of exopolysaccharides and the chemotactic movement and adhesion of flagellum in the potato root secretion ecological niche, while the virulence factors in the atypical output pathway mainly distributed in Sec (secB, secDF, yidc) and Tat (tatA, tatC) pathways to promote the output of folded and unfolded toxic proteins. The fliIATPase was obviously upregulated 8 hrs postinoculation, suggesting that type III secretion system was only active at the early stage of PO41 infection. The upregulated expression of phosphoglucomutase and epimerase showed that the virulence factor of exopolysaccharides (EPS) was synthesized at the early stage of R. solanacearum infection. Chemotactic receptor and motor protein were obviously upregulated within 24 hrs postinoculation. Our study revealed that R. solanacearumPO41 had already colonized to the roots within 24 hrs with the stimulating of root secretion. Some pathogenic genes were upregulated during this period.     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

Phylogenetic Characterization lcrD Gene Family: Molecular Evolutionary Aspects of Pathogen-Induced Hypersensitivity in plants

Abstract — Hrp(hypersensitivity response and pathogenicity) genes encode signal-peptide independent transporter molecules that function in the Type III secretion pathway and are present in a number of plant pathogenic bacterial species. These Hrp transporter molecules largely export harpin and other virulence factors across the bacterial membrane and onto theHrploci are part of a largerlcrD family which encode the low calcium response proteins. Members of this family serve to transport a number of diverse virulence factors in a variety of enteric and other purple bacteria species both pathogenichrp-induced pathogenicity by different plant pathogenic bacterial species is the result of a single evolutionary event or evolved independently, cladistic analyses were performedlcrD gene family. The results of these studieslcrD orhrpgeneslcrD homologues which comprised the other twohrptransporter genes do not capture the phylogenetic history of their host bacteriallcrD gene was horizontally introduced into each of four different plant pathogenic species which may have resulted from four independent transfer events. This monophyletic partitioning ofhrpgenes precludes their use as reliable taxonomic markers while further supporting the current notion thathrptransport     

Flagellar biosynthesis exerts temporal regulation of secretion of specific Campylobacter jejuni colonization and virulence determinants

The Campylobacter jejuni flagellum exports both proteins that form the flagellar organelle for swimming motility and colonization and virulence factors that promote commensal colonization of the avian intestinal tract or invasion of human intestinal cells respectively. We explored how the C. jejuni flagellum is a versatile secretory organelle by examining molecular determinants that allow colonization and virulence factors to exploit the flagellum for their own secretion. Flagellar biogenesis was observed to exert temporal control of secretion of these proteins, indicating that a bolus of secretion of colonization and virulence factors occurs during hook biogenesis with filament polymerization itself reducing secretion of these factors. Furthermore, we found that intramolecular and intermolecular requirements for flagellar‐dependent secretion of these proteins were most reminiscent to those for flagellin secretion. Importantly, we discovered that secretion of one colonization and virulence factor, CiaI, was not required for invasion of human colonic cells, which counters previous hypotheses for how this protein functions during invasion. Instead, secretion of CiaI was essential for C. jejuni to facilitate commensal colonization of the natural avian host. Our work provides insight into the versatility of the bacterial flagellum as a secretory machine that can export proteins promoting diverse biological processes.     

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 type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as-yet-unidentified avirulence factor possessed by the Japanese tobacco-avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector-triggered immunity (ETI). The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence-related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid-resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco-virulent strain BK1002 contains mutations in ripB and expresses a C-terminal-truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine-tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB-induced resistance against R. solanacearum was abolished in Roq1-silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.     

Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum

Ralstonia solanacearum is a Gram‐negative soil‐borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid‐resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp‐dependent manner. An Agrobacterium‐mediated transient expression system revealed that Rip36, a putative Zn‐dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn‐binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002‐derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn‐dependent protease motif is essential for this activity.     

Pseudomonas syringae lytic transglycosylases coregulated with the type III secretion system contribute to the translocation of effector proteins into plant cells

Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH_1-241. DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH_1-241 variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection.     

Characterisation and diversity of Indian isolates of Ralstonia solanacearum causing bacterial wilt of Capsicum annuum L.

Genetic diversity of 13 isolates of Ralstonia solanacearum causing bacterial wilt in hot pepper and bell pepper (Capsicum annuum L.) from 6 states of India was assessed. All isolates of R. solanacearum belonged to biovar 3, race 1 and phylotype I. These isolates consisted of 4 distinct DNA types at 75% similarity coefficient using ERIC, BOX and REP-PCRs techniques. Multilocus sequence analysis of hrpB, fliC and egl genes of 6 isolates of R. solanacearum along with 2 out group bacteria was done and they showed high level of variability within these three regions of the genome involving in pathogenicity.     

Erwinia amylovora Secretes DspE, a Pathogenicity Factor and Functional AvrE Homolog, through the Hrp (Type III Secretion) Pathway

Erwinia amylovora was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of Pseudomonas syringae pv. tomato. DspE was identified among the supernatant proteins isolated from cultures grown in an hrp gene-inducing minimal medium by immunodetection with a DspE-specific antiserum. Secretion required an intact Hrp pathway.     

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.     

Secretion and regulation Hrp mutants of Erwinia amylovora trigger different responses in apple

The interaction between two Hrp mutants of Erwinia amylovora, the parental virulent strain, and a host plant (Malus sp.) was studied with apple seedlings and apple flowers, using inoculation procedures which reduce the possibility of injury. We showed that one hrp regulatory mutant protected apple tissue against the virulent strain to a greater degree than a secretion mutant. The dynamics of bacterial growth in this interaction indicated that the protection was associated with the inhibition of the multiplication of the virulent strain. The activity of two enzymes involved in plant defence responses, phenylalanine-ammonia lyase and guaiacol peroxidase, increased in leaves treated with the regulatory mutant, but not in the leaves treated with the secretion mutant. The role of regulatory genes of the hrp cluster in this interaction is discussed.