Identifiction Of SRNA Direct Targets In Xanthomonas Campestris Pathovar Campestris

Bacteria small RNA （sRNA） is a kind of RNA in the length between 50 to 500 nt, usually does not encode protein but has independent functions. It has been shown that bacterial sRNA play an important role in heavy metal ion metabolism, environmental response, quorum sensing, biofilm formation and virulence. In most case, bacterial sRNA acts a regulator to regulates expression of its target genes at the post-transcriptional level by base pairing with its target mRNA and thus affect the translation or stability of the mRNA.Xanthomonas campestris pv. Campestris （Xcc） is the causal agent of black rot disease of cruciferous crops such as Chinese cabbage, cabbage, radish, rape and other important crops and is a model bacteria strains for studying the molecular basis of pathogenesis of phytopathogen. To investigate the role of sRNA in Xcc, we started sRNA identification and functional analysis work in Xcc several years ago, and many sRNAs have been identified, and by using deletion and over-expression method, we investigated the biological function of these identified sRNA. However, up to date, only a few sRNAs’ biology function has been determined. For most sRNAs, no phenotype changes were observed in their deletion and/or over-expression strain, indicating that they play a minor role in the cell. In this study, we try to study the cellular function of sRNA by identification of their direct targets. Our strategy is, firstly, we predicted the direct targets of sRNA of interested by bioinformatics analysis, and then validate the direct target candidates by electrophoretic mobility shift assay （EMSA）.Of the dozens of Xcc sRNAs, we chose four high abundant sRNAs （sRNA-Xcc1, sRNA-Xcc2, sRNA-Xcc3 and sRNA-Xcc4） for our experiments. Firstly, we use CopraRNA （Comparative prediction algorithm for small RNA target） software to predict the direct targets of the four sRNAs. Prediction results show that, using the cutoff value of p values< 0.01 and q values< 0.5, there are 27 target genes for sRNA-Xcc1,2 for sRNA-Xcc2,3 for sRNA-Xcc3 and 35 for sRNA-Xcc4. Then we chose five sRNA-Xccl targets （XC₂374, XC₁589, XC₀611, XC₁788, XC₀721）, two sRNA-Xcc2 targets （XC₄321, XC）1078), three sRNA-Xcc3 targets （XC₁548, XC₀725, XC₀690） and five sRNA-Xcc4 targets （XC 0760, XC₃379, XC₃499, XC₃561, XC₂308） for EMSA. Before doing EMSA, the transcription start site of candidate target genes was determined by using 5’RACE （5’Rapid Amplification of cDNA Ends）. EMSA results show that the transcription start site of XC₀725 was located upstream of the predicted sRNA-Xcc3 banding region. The transcription start site of XC₄321, which has been determine in a previous work, was located upstream of the predicted sRNA-Xcc2 predicted banding region. Base on these results, we carried out EMSA to test whether sRNA-Xcc2 and sRNA-Xcc3 binds to their predicted target mRNA. EMSA results show that both sRNA-Xcc2 and sRNA-Xcc3 can not bind to their prediction target in vitro, indicating that XC₄321 is not the direct target of sRNA-Xcc2, and XC₀725 is not the direct target of sRNA-Xcc2. But it is also possible that binding of to sRNA-Xcc2 and sRNA-Xcc3 to the predicted targets required other factor（s）.To determine whether the sRNA-Xcc2 and sRNA-Xcc3 regulate the expression of their predicted target genes, we detected the expression level of the target gene XC₀725 in sRNA-Xcc3 over-expression strain, and the expression level of the target gene XC₄321 in sRNA-Xcc2 over-expression strain and its deletion strain （constructed by Ruiping Jang）, by semi-quantitative RT-PCR. RT-PCR results show that the expression of XC₄321 decreased in the over-expression strain and increased in the deletion strain, indicating that sRNA-Xcc2 negatively regulates XC₄321. But over-expression of sRNA-Xcc3 did not affect the expression of XC₀725.In this study, we try to combine bioinformatics prediction and EMSA to identify the direct target of four Xcc sRNAs. Although we failed to get their direct target, we found that sRNA-Xcc2 negatively regulates XC₄321. Since sRNA-Xcc2 can not bind to XC₄321 mRNA in vitro, it is possible that sRNA-Xcc2 regulates XC₄321 indirectly. More work is need to determine how sRNA-Xcc2 regulates XC₄321.