Characterization of small regulatory RNAs with a putative function in the virulence of Xanthomonas campestris pv. vesicatoria

野油菜黄单胞菌致病性研究水疱病（Xcv）依赖于III型分泌系统（T3 SS），其将效应蛋白注入宿主细胞胞质溶胶中。T3 SS在植物中被两种调节蛋白HrpG和HrpX特异性激活。HrpG控制全基因组调节子的表达，该调节子也受到调节细菌毒力的小RNA（sRNA）的影响。在第一个资助期内，对Xcv菌株85-10的转录组进行了深度测序、生物信息学和北方分析，确定了27种sRNA。一个例子是sR72，一种高度结构化的sRNA，它是完全毒力所必需的，并且可能作用于HrpG的上游。此外，实验鉴定了具有毒力功能的另外的sRNA和顺式编码的反义RNA。在拟议的项目中，我们的目标是从Xcv中选择的sRNA的功能表征和识别其目标。此外，选定的顺式编码的反义RNA和RNA结合蛋白Hfq和CsrA将在遗传和分子水平上进行表征。这些方法将有助于阐明Xcv在转录后水平上控制毒力的机制。

Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) depends on the type III secretion system (T3SS), which injects effector proteins into the host cell cytosol. The T3SS is specifically activated in planta by two regulatory proteins, HrpG and HrpX. HrpG controls expression of a genome-wide regulon which is also affected by small RNAs (sRNAs) that modulate bacterial virulence. In the first funding period, deep sequencing, bioinformatics and Northern analyses of the transcriptome of Xcv strain 85-10 identified 27 sRNAs. One example is sR72, a highly structured sRNA, which is required for full virulence and presumably acts upstream of HrpG. Furthermore, additional sRNAs and cis-encoded antisense RNAs with a virulence function were experimentally identified. In the proposed project we aim at the functional characterization of selected sRNAs from Xcv and the identification of their targets. In addition, selected cis-encoded antisense RNAs and the RNA-binding proteins Hfq and CsrA will be characterized genetically and on the molecular level. These approaches should help to elucidate the mechanisms that control virulence of Xcv on the posttranscriptional level.