| The bacterial wilt of plant, caused by Ralstonia solanacearum, is a widely-existed disease around the world. The reason for this is because that the R. solanacearum has a wide geographic distribution and a wide host range, which can cause huge economic losses. With the development and progress of genetic engineering technology, it became easier to analyze pathogenesis of R. solanacearum at the genetic level. This provided new ways and methods to control the diseases caused by R. solanacearum more effectively. What is more, it provided theoretical foundation of controlling R. solanacearum in the field.The type Ⅵ secretion system(T6SS) has been identified in various bacteria. Recently, the research on T6 SS was mainly focused on human and animal pathogenic bacteria. It is reported that T6 SS was closely tied with the pathogenicity, competition, interactions and other life activities of bacteria. In the earlier study, we found that T6 SS core genes have certain effect on the pathogenicity of R. solanacearum. In this study, gene deletion mutants of T6 SS gene cluster of R. solanacearum strain Po82 were constructed in order to evaluate the function of the T6 SS gene cluster.There are two type Ⅰ signal peptidase in the genome of R. solanacearum, which plays a vital role in protein secretion. In order to better understand the role of type Ⅰ signal peptidase in the plant pathogen, and explore the molecular mechanism of pathogenic bacteria of R. solanacearum, we carried out experiments to study the functions of two type Ⅰ signal peptidase genes in R. solanacearum Po82 strains.1. Generation of the gene deletion mutant and the complementary strainsBioinformatic analysis showed that, type Ⅰ signal peptide enzyme of R. solanacearum have two conserved domains, wherein the protein encoded by the 1716 gene, having a signal peptide and two transmembrane domains; while the Lep B protein may have a transmembrane domain, but not a signal peptide.By the method of homologous recombination, we have obtained gene deletion mutant strains and the complementary strains.2. Functional characterization of T6 SS gene clusterDuring the early stage of inoculation, mutant Po82ΔⅥ was significantly reduced in virulence. While in the late stage, its virulence was gradually recovered when compared to the wild-type strain. The growth rate of Po82ΔⅥ was dramatically reduced at log phase. In addition, the motility of the mutants was increased, and no significant difference in biofilm formation was detected, q RT-PCR analysis showed that, the expression of flagellar genes flh C, flh D and fnr, but not fli A were down-regulated in the mutant Po82ΔⅥ; There were no significant differences between the mutant and the wild-type strains in type Ⅲ effector genes expression. These results showed that the T6 SS gene cluster is involved in pathogensis of R. solanacearum, but the deletion of T6 SS gene cluster had no or little impact on the expression of type Ⅲ effector genes in the Po82 strains.3. Functional characterization of type Ⅰ signal peptide enzyme geneThe mutant strain Po82△1716 had significantly attenuated its virulence, growth rate and the motility. The expression of flagellar genes flh C、flh D and fnr were down-regulated, the expression of type Ⅲ effector genes were down-regulated revealed by q RT-PCR. These results indicated that the 1716 gene positively regulated the expression of T3 E genes and flagellar genes; the mutant strain Po82△lep B was significantly attenuated its virulence on tomato plants, there are no significant differences in the motility. However, the expression of flagellar genes were up-regulated significantly, especially the flh C and fli A genes. the expression of type Ⅲ effector genes were up-regulated revealed by real-time PCR. These results suggested that the lep B gene negatively regulated the expression of T3 E genes and flagellar genes. |
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