Functional Analysis Of12Small Rnas In Xanthomonas Campestris Pathovar Campestris

Bacterial small RNA (sRNA), also known as non-coding RNA (non-coding RNA, ncRNAs), is a kind of RNA molecules ranging in length from approximately50to500nucleotides. They do not encode proteins and have independent functions in the cell, but are different from mRNA, rRNA and tRNA. It has been shown that sRNAs are widely distributed in all living organisms, acting as post-transcriptional regulators and playing important role in bacterial cellular process including iron metabolism, carbon metabolism, quorum sensing, stress respone and pathogenesis. It has been known that sRNAs play important role in the pathogenesis of animal and human bacterial pathogens. However, little is known about that role of sRNA in the pathogenesis of plant bacterial pathogensRecently, several hundreds of sRNAs candidates have been identified in Xcc8004by deep RNA sequencing in our lab, and over80sRNAs have been validated by Northorn Bioting and reverse transcription-PCR (RT-FCR). To investigate the biological function of these newely identified sRNAs, in this study, we try to construct the deletion mutants and over-expression strains of12selected sRNAs (sRNA-Xcc7. sRNA-Xcc8.sRNA-Xcc9.sRNA-xcc10.sRN Xcc11,sRNA-Xcc12,sRNA-Xcc13,sRNA-Xcc14,sRNA-Xcc15, sRNA-Xcc16, sRNA-Xcc17and sRNA-Xcc18).As a results,the deletion mutants of four sRNAs(sRNA-Xcc7,sRNA-Xcc8,sRNA-Xcc9and sRNA-Xcc10),and the over-expression strains of night sRNA(sRNA-Xcc9, sRNA-Xcc11,sRNA-Xcc12,sRNA-Xcc13,sRNA-Xce14, sRNA-Xcc15,sRNA-Xcc16,sRNA-Xcc17and sRNA-Xcc18)were successfully constructed.The phenotypes including growth in minimal medium,extracelluar enzymes activiy；extracelluar polysaccharide production,virlence,hypersensitive reaction,stress tolerance, swimming mobility and aggregate growth were tested and compared to that of the wild type strain.The results show that,(i)the virulence of the deletion mutant and the over-expression strain of sRNA-Xcc9is reduced compared to that of the wild type strain；(ii)the extracellular protease and amylase activiy of the over-expression strain of sR:NA-Xcc9is reduced compared to that of the wild t),pe straill:(iii)the extracellular amylase activiy of the over-expression strain of sRNA-Xcc17is reduced compared to that of the wild type strain:(iv)the tested phenotypes of both the deletion mutants and the over-expression strains of other sRNAs(sRNA-Xcc7,sRNA-Xcc8.and sRNA-Xcc10,sRN-Xcc11. sRNA-Xcc12,sRNA-Xcc13,sRNA-Xcc14,sRNA-Xcc15.sRNA-Xcc16, sRNA-Xcc18are similar to that of the wild type strain.These results indicated that①the sRNA sRNA Xcc9may be invelved in the mathovenvis and oxtracellular protease and amviase aetivity of Ncc1②the sRNA sRNA-Ccc17may be involved in the extracellular amvlase actiyity of Xcc. In summary, this study provide a firest stept towards the understanding the biological function and their regulatory machanisms of sRNA-Xcc9and sRNA-Xcc17.