Identification And Functional Analysis Of Ten Small Rnas Whose Expressions Are Affected By Acid And Alkali In Xanthomonas Campestris Pathovar Campestris

Bacterial small RNA (sRNAs), also known as non-coding RNAs (ncRNAs) and regulatory RNA, are the RNA molecules usually in the size of between50-500nt, which do not encod protein but have biological functions. It has been sown that bacterial sRNA plays important roles in a number of cellular processes such as glucose metabolism, metal ion transport, quorum sensing, environmental stress response and pathogenesis. Due to the effective sRNA indetification methods were established, more than one thousand of bacterial sRNAs have been identified during the last ten years. However, little is known about the biological function of these sRNAs.Xanthomonas campestris pv.campertris (Xcc) is the causal agent of the black rot of cruciferous plants (including important vegetables such as radish, the Chinese cabbage, kale and important oil crops rape). To identify the sRNAs involved in acid and alkali stress response of Xcc, our laboratory carried out genome-wide identification of the sRNAs whose expression are affected by acid and alkali by high-throughput RNA deep sequencing (RNA-seq), and more than500sRNA candidates has been identified. However, the existence, expression manner and the biological function of these sRNA candidates need further ivestegation. In this work, ten sRNA candidates (renamed as sRXcc-25to sRXcc-34) were selected for further study. Firstly, the existence of these sRNA candidates were confirm by a method which is based on RT-PCR, and then the affect of their expression by acid and alkali was confirmed by semi quantitative RT-PCR, and finally, their biological functions were determined by construction and phenotype analysis of their overexpression strains and deletion mutants.As a result, the RT-PCR results demonstrated that all ten target sRNAs are really existing. Their transcription orientations were determined by using chain specific RT-PCR. The semi quantitative RT-PCR results show that the expressions of all ten target sRNAs are affected by acid conditions and three of them (sRXCC-26to sRXCC-30) are affected by alkali. Up to date, the over-expression strains (named OE25to OE34) of all ten target sRNAs and the deletion mutants (named DM25, DM29, DM30, DM31, DM33and DM34) of six target sRNAs (sRsXCC-25,sRACC-29,sRXCC-30,sRXCC-31,sRXCC-33and sRXCC-34) were constructed.The phenotypes including (1) acid and alkali tolerance,(2) extracellular polysaccharides production and extracellular enzyme activity,(3) SDS and NaCl tolerance,(4) metal ion tolerance,(5) swimming motility,(6) biofilm formation,(7) growth in rich and minimal medium,(8) virulence in host plants and hypersensitive response (HR) in non-host plants were tested, and compared to that of the wild type strain Xcc%004and the wild type strain carrying the over-expressin vactor plasmid WT/pBBad18K, The results show that, no phenotype changes were observed in all of the over-expressin strains and deletion mutants tested except that the over-expression strains of all ten target sRNAs displayed a reduced Zn2+and Ni2+tolerance compare to that of the control strain WT/pBBad18K in the plate assay, indicating that the ten sRNAs is involved in Zn2+and Ni2+tolerance of Xcc.In summary, in this work, we have identified ten Xcc sRNAs whose expression was affected by acid and alkali, and found that they play a role in Zn2+and Ni2+tolerance of Xcc. Our results provide useful materials for the studying the role and regulatory mechanisms of sRNA in Xcc.