Function Analysis Of Type VI Secretion System In Ralstonia Solanacearum Pathogenicity And Secretion Protein

The newly discovered type VI secretion system (T6SS) is related to many plants, animals and human diseases. Therefore, studing the type VI secretion system will help uncover the mechanism of bacterial pathogenesis.Firstly, transposon Tn5 mutagenesis Ralstonia solanacearum race 3, Po41, obtained with the reduction of extracellular protein secretion mutants. On this basis, the Tn5 insertion sites were analyzed, in order to find type VI secretion system-related mutants. At the same time, the type VI secretion system will be studied through the 8-core gene cloning and sequence analysis, constructing type VI secretion system mutants, doing functional analysis about the type VI secretion system genes. Therefore, analyze Type VI secretion system virulence gene and secretion protein.1. Tn5 insertion site analysis of R. solanacearum mutants Through screening the Tn5-insertion mutant library of Ralstonia solanacearum srain Po41 constructed by one of the former studends in this lab, find the mutants whose virulence is reduced.2. The pathogenicity analysis of R. solanacearum mutants The inoculation experiment showed reduced virulence of the mutant strain PM2594. In the root colonization ability of the study, compared with wild-type strain Po41, at the third day after inoculation, PM2594 in bacteria per gram of root number were equal to 1/50 of Po41. Proliferation capacity in the stem study, compared with wild-type strain Po41, 3 days after inoculation, PM2594 stems in 1 cm equal to 1/10 of Po41 in the number of bacteria. That mutant PM2594 gene function was related with host plants of R. solanacearum in the proliferation and movement.3. Virulence analysis of R. solanacearum imp gene Further analysis of the mutant PM2594 has determined that the positions of the Tn5 insertion are located in open reading frame Rsp1610. Rsp1610 was inactive because of Tn5 insertion. The gene was named as imp (impaired motility and proliferation) gene. Construct the GMI1000 imp gene mutant. Similar function of imp gene in GMI1000 strain was verified.4. The gene cloning and functional analysis of R. solanacearum type VI secretion system gene cluster Construct the T6SS mutant in order to identify the T6SS function in R. solanacearum. The result of tomato inoculation test showed that the GMI1000-m strain was much less virulent on tomato than the wild type GMI1000, which indicated that the vasK gene is a very important factor during the pathogenesis of R. solanacearum. Statistical analysis showed that, the GMI1000 incidence significantly higher than GMI1000-m, which after onset 2 to 7 days of differential highly significant.5. Two-dimensional electrophoresis analysis of secretion protein R. solanacearum vasK gene mutant Thirty-eight protein spots were obtained and identified in the supernatants of the wild-type GMI1000 and vasK mutant strain by two-dimensional (2D) gel electrophoresis and peptide mass fingerprint (PMF). Including fifteen proteins in GMI1000, ten proteins in vasK mutant, three protein up-regulated in GMI1000 and ten protein down-regulated in vasK mutant.Protein spots 592, which is one of components of the type VI secretion system, had a expression level three times less than the wild-type GMI1000. This suggested that the deletion of vasK gene had certain impact on the expression of type VI secretion system in R. solanacearum. The remaining proteins are still function unknown. The structure and function of these proteins have to be further studied.