Interactions Analysis between the Gsp Proteins of Type Ⅱ Secretory System in Ralstonia Solanacearum

Abstract: The bacteria, by specific transmissible system of the proteins, secrete toxins and effectors, and communicate with the parasite by molecules. Among the total, the type II secretory system is widely distributed among Gram-negative bacteria. The probable general secretory pathway transmembrane protein (GSP) gene cluster is the center of the type II secretory system. Bacterial wilt caused by Ralstonia solanacearum, is one of the most important and widespread bacterial diseases of plants in the world. Type II secretory system is associated with secretory of pathogenic proteins of R. solanacearum. In this paper, the interactions between Gsp proteins of type II secretory system in R. solanacearum GMI 1000 strain were analysed by yeast two-hybrid system.According to upstream and downstream sequences of gsp genes, we designed 12 the best primers of gsp genes with the primer design softwares. The high content of GC of gsp genes (67.1-74.4%) resulted in difficulty of PCR amplifications, we cloned successfully all of 12 gsp genes by optimizing PCR reaction system.For constructing bait pool and prey pool, the 12 gsp genes were ligated into bait vector-pGBKT7 and prey vector-pGADT7, respectively. The open reading-frames of 12 gsp genes were verified by sequening, which ensure that gsp genes can be expressed rightly in yeast system.The bait pool and prey pool were cotransformed into yeast AH 109 cell. In yeast two- hybrid system, interactions between Gsp L and Gsp E, Gsp L and Gsp M, Gsp G and Gsp E, Gsp C and Gsp L, Gsp I and Gsp J were detected. After exchanging the vectors, the interactions between Gsp E and Gsp L, Gsp M and Gsp L, Gsp E and Gsp G had been reverified.We first applied yeast two-hybrid system to research interactions between Gsp proteins of type II secretory system of R. solanacearum. Three pair Gsp protein interactions were obtained. These results suggested that yeast two-hybrid is an effective technology on research interactions between Gsp proteins of R, solanacearum. In the type II secretory system of human pathogen bacteria and Erwinia spp, Gsp L, Gsp E and Gsp M proteins constitute a complex in the inner membrane that is used as a platform for assembling other parts of the secretion machinery. Our results of are in good agreement with above-mentioned.These researches on the type II secretory system of R. solanacearum will offer great potential for understanding the molecular basis of how R. solanacearum excretes and regulates pathogenic proteins, searching acting targets of hindering excretion of pathogenic proteins, exploiting new way to control bacterial wilt diseases…
Key words: Ralstonia solanacearum; Type II secretory system; Gsp Protein; Yeast two-hybrid system; Interactions between proteins

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