Institute for Biology, Department of Genetics, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
PLoS Pathog. 2013 Sep;9(9):e1003626. doi: 10.1371/journal.ppat.1003626. Epub 2013 Sep 12.
Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of sX13 impinged on Xcv virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of in vitro transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected Xcv virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the hfq mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding Xcv genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen Xanthomonas.
小非编码 RNA（sRNA）是普遍存在的基因表达转录后调控因子。我们使用模式植物病原细菌丁香假单胞菌 pv.vesicatoria（Xcv），详细研究了高度表达和保守的 sRNA sX13。sX13 的缺失影响了 Xcv 的毒力和编码 Hrp 型 III 型分泌（T3S）系统组件和底物的基因的表达。qRT-PCR 分析显示，sX13 促进了 T3S 系统关键调节因子 HrpX 的 mRNA 积累，而主调节因子 HrpG 的 mRNA 水平不受影响。互补研究表明，sX13 作用于 HrpG 的上游。微阵列分析鉴定了 63 个 sX13 调控的基因，它们涉及信号转导、运动、转录和转录后调节以及毒力。体外转录的 sX13 结构分析显示，它具有三个稳定的茎和三个顶端 C 富集环。对推定调节基序的计算搜索表明，sX13 抑制的 mRNA 主要在翻译起始位点附近含有富含 G 的基序。sX13 环的突变对 Xcv 的毒力和推定靶标 mRNA 的丰度产生了不同的影响。使用 GFP 报告系统，我们证明 sX13 介导的蛋白质合成抑制需要 sX13 中的 C 富集基序和潜在靶标 mRNA 中的富含 G 的基序。虽然 RNA 结合蛋白 Hfq 对 sX13 的活性不是必需的，但 hfq mRNA 和 Hfq::GFP 的丰度受到 sX13 的负调控。此外，我们发现 sX13 抑制的 mRNA 中的富含 G 的基序可以作为翻译增强子，并且位于 5%的所有编码 Xcv 基因的核糖体结合位点。我们的研究表明，sX13 代表了一类新的毒力调节因子，并提供了对植物病原菌丁香假单胞菌中 sRNA 介导的适应性过程调节的深入了解。
