Research On The Function Of Genes In Type Ⅵ Secretion System Of Ralstonia Solanacearum GM1000Strain

Ralstonia solanacearum is probably the most destructive plant pathogenic bacterium worldwide.One of the reasons for this is that the R. solanacearum species are composed of a very large group ofstrains varying in their geographical origin, host range and pathogenic behavior. Elucidation of itspathogenesis and the regulatory mechanism will contribute to effective control of the disease. Thesequences of11R. solanacearum strains including GM1000, UM551have been announced, whichprovided the basis for further investigation on the molecular mechanism of R. solanacearum.The type Ⅵ secretion system (T6SS) has been discovered recently in gram-negative bacteria. Thebacteria which harbor the T6SS show a serious threat for animal and human health. The research ofT6SS was mainly focused on animal and human pathogens. Bioinformatics analysis shows that R.solanacearum also harbor the T6SS gene cluster. However, the molecular mechanism of T6SS in R.solanacearum remains unclear. On the basis of prophase studies in the laboratory, this paper furtherinvestigates the molecular mechanism of T6SS in R. solanacearum.1. Study on the pathogenicity and phenotypic characteristic of the tssM gene.Pathogenicity tests showed that the disease symptoms caused by tssM mutant were delayed whencompared to the wild-type strains, while the complementary strains rescue the pathogenisis of thebacteria. The phenotypic characteristics of tssM gene mutant were tested. Significant impairments ofroot colonization, proliferation, biofilm formation and motility were observed in tssM-mutant strain,compared with wild-type strain. qRT-PCR analysis showed flagellar genes crp, fliA, flgM and flhC weredown-regulated in GMI1000ΔtssMRS. Furthermore, qRT-PCR analysis also showed type III secretionsystem (T3SS) effector genes popP1, popA, popB and popC were up-regulated in GMI1000ΔtssMRS.2. Construction of the tssB gene deletion mutant and their correspongding complementarystrain.Bioinformatic analysis shows that, the full-length sequence of tssB gene, which belongs to R.solanacearum GM1000strain, is1491bp. The molecular weight of TssB protein is55kDa. TssB,which contains a domain of unknown function, DUF877, belongs to a family of uncharacterizedbacterial proteins that are associated with the T6S locus. One transmembrane domain could be predictedin TssB protein. However, TssB lacks a signal peptide sequence. The mutant of tssB gene wasconstructed by homologous recombination. Based on the mutant strain, the complement strain was alsoconstructed.3. Study on the pathogenicity and phenotypic characteristic of the tssB gene.Pathogenicity tests showed that the disease symptoms caused by tssB mutant were delayed whencompared to the wild-type strains, while the complementary strains rescue the pathogenisis of thebacteria. The phenotypic characteristics of tssB gene mutants were tested. Significant impairments ofroot colonization, proliferation, biofilm formation and motility were observed in tssB-mutant strain,compared with wild-type strain. qRT-PCR analysis showed flagellar genes crp, fliA, flgM, flhD and flhCwere down-regulated in GMI1000ΔtssB. Furthermore, qRT-PCR analysis also showed type III secretion system (T3SS) effector genes popP1、popA、popB and popC were down-regulated in GMI1000ΔtssB.4. The interaction between TssB and TssC proteinThe bait and prey recombinant vectors of TssB/TssC were constructed respectively. The Y2Hexperiments showed that TssB interacted with TssC. A series of plasmids expressing truncated mutantsof TssB were generated to interact with TssC by Yeast Two-Hybrid system. The result showed that TheN-teiminal amino acid positions63~112of TssB protein were associated with TssC protein.5. Analysis of Hcp secretion and localization of TssBThe result of Hcp secretion showed that wild type GMI1000strain and complementary strain couldproduce and secrete Hcp. However, the mutant strain GMI1000ΔtssB could produce but could notsecrete Hcp. The sub-cellular localization of TssB showed that TssB was found in the cell andmembrane of GMI1000, but was not found in soluble fraction. This suggested that TssB was localizedin the cell membrane.