Functional Analysis Of Ten Small RNAs In Xanthomonas Campestris Pathovar Campestris

Bacterial small RNA (sRNA), also known as non-coding RNA or regulatory RNA, is a kind of RNA in the length of between 50 to 500nt, it does not encode protein, but exerts certain functions independently different from transfer RNA and ribosomal RNA. sRNAs are widely spread in all three kingdoms of life and play an important role in cellular processes. It has been shown that sRNA acts as a post-transcriptional regulator in many important cellular processes such as sugar metabolism, carbon metabolism, quorum sensing, stress response, cell motility, and pathogenesis. In recent years, the identification and functional study of sRNA have become the hot point of molecular biology. As the efficient method of sRNA identification has already been established, thousands of bacterial sRNAs have been identified so far. However, the biological function of these identified sRNA is largely unknown and need further investigation. Furthermore, more studies are required to establish the role of sRNA in the pathogenesis of phytopathogens.Xanthomonas campestris pv.campertris (Xcc) is causal agent of black rot disease of crucifers, and is a model strains for studying the interactions between pathogenic bacteria and their plant host. Previously, although more than 500 sRNA candidates have been identified in XccS004 (at log phase in the minimal medium MMX) by deep RNA sequencing (RNA-seq), the biological functions of these sRNAs are largely unclear. In this work, ten sRNA candidates were choose for functional analysis. Firstly, the existence of these sRNA candidates was determined by using three-primer RT-PCR, and then their biological functions have been determined by deletion and/or over-expression, and phenotype analysis.Three-primer RT-PCR results show that all of the ten sRNA candidates are expressed. Then, the transcriptional direction of these sRNA genes has been determined by chain-specific RT-PCR. Up to date, we have constructed 10 over-expression strains and 9 deletion mutants, and the phenotypes including (1) growth in the minimal medium MMX and the rich medium NYGB, (2) the production of EPS and the activity of extracellular enzymes, (3) cell motility, (4) biofilm formation, (5) hypersensitive response (HR), and (6) virulence, of these strains have been analyzed and compared to that of the wild type strain and the wild type strain carrying an empty over-expression vactor pBBadl8k. The phenotypic analysis results show that:(1) the cell density of the deletion mutant of the sRNA sRXCC-50 is significantly lower than that of the wild-type strain at stationary phase of NYGB medium, (2) the cell density of the deletion mutants of sRXCC-49, sRXCC-51, sRXCC-52 and sRXCC-55 is significantly lower than that of the wild-type strain at log phase and stationary phase of MMX medium, (3) the cell density of OE53, the over-expression strain of sRXCC-53, is significantly lower than that of the control strain WT/pBBad18K at the log and stationary phase of MMX medium, (4) cell density of OE55, the sRXCC-55 over-expression strain, is significantly higher than that of the control strain WT/pBBad18K at the log and stationary phase of MMX medium, (5) DM49, the deletion mutant of sRXCC-49 triggers an enhanced HR compare to the wild type strain, (6) over-expression of sRXCC-52, sRXCC-55 and sRXCC-56 resulted in an enhanced HR compare to wild type strain, (7) except for the phenotype changes described above, no significant phenotype changes were observed in other mutants and over-expression strains.These results indicate that:(1) sRXCC-49, sRXCC-50, sRXCC-52, sRXCC-53 and sRXCC-55 may relate to the bacterial growth, (2) sRXCC-49, sRXCC-52, sRXCC-55 and sRXCC-56 may play an important role in HR of Xcc 8004.In summary, in this work, we confirmed the existence and determined the transcriptional direction of 10 sRNA candidates in Xcc, and more importantly, we investigated the biological functions of the 10 Xcc sRNAs. As a results, we found that sRXCC-49, sRXCC-50, sRXCC-51, sRXCC-52, sRXCC-53, sRXCC-55 may related to bacterial growth and sRXCC-49, sRXCC-52, sRXCC-55, sRXCC-56 may related to HR. Our finding provides the first step towards the understanding of the biological function of sRNA and provides available materials for studying the function and regulatory mechanism of sRNA in Xcc.