The Post-transcriptional Regulator rsmA/csrA Activates T3SS by Stabilizing the 5′ UTR of hrpG, the Master Regulator of hrp/hrc Genes, in Xanthomonas

The RsmA/CsrA family of the post-transcriptional regulators of bacteria is involved in the regulation of many cellular processes, including pathogenesis. In this study, we demonstrated that rsmA not only is required for the full virulence of the phytopathogenic bacterium Xanthomonas citri subsp. citri (XCC) but also contributes to triggering the hypersensitive response (HR) in non-host plants. Deletion of rsmA resulted in significantly reduced virulence in the host plant sweet orange and a delayed and weakened HR in the non-host plant Nicotiana benthamiana. Microarray, quantitative reverse-transcription PCR, western-blotting, and GUS assays indicated that RsmA regulates the expression of the type 3 secretion system (T3SS) at both transcriptional and post-transcriptional levels. The regulation of T3SS by RsmA is a universal phenomenon in T3SS-containing bacteria, but the specific mechanism seems to depend on the interaction between a particular bacterium and its hosts. For Xanthomonads, the mechanism by which RsmA activates T3SS remains unknown. Here, we show that RsmA activates the expression of T3SS-encoding hrp/hrc genes by directly binding to the 5′ untranslated region (UTR) of hrpG, the master regulator of the hrp/hrc genes in XCC. RsmA stabilizes hrpG mRNA, leading to increased accumulation of HrpG proteins and subsequently, the activation of hrp/hrc genes. The activation of the hrp/hrc genes by RsmA via HrpG was further supported by the observation that ectopic overexpression of hrpG in an rsmA mutant restored its ability to cause disease in host plants and trigger HR in non-host plants. RsmA also stabilizes the transcripts of another T3SS-associated hrpD operon by directly binding to the 5′ UTR region. Taken together, these data revealed that RsmA primarily activates T3SS by acting as a positive regulator of hrpG and that this regulation is critical to the pathogenicity of XCC.细菌转录后调控因子RsmA/CsrA家族参与调控包括致病过程在内的诸多细胞生命进程。本研究证实，rsmA基因不仅是植物致病细菌柑橘黄单胞菌柑橘亚种（Xanthomonas citri subsp. citri，XCC）实现充分致病力所必需的基因，同时亦参与触发非宿主植物的过敏反应（hypersensitive response，HR）。敲除rsmA会导致宿主植物甜橙的致病力显著降低，同时使非宿主植物本氏烟（Nicotiana benthamiana）中的过敏反应出现延迟且减弱的表型。通过基因芯片（microarray）分析、定量逆转录PCR（quantitative reverse-transcription PCR）、蛋白质印迹（western-blotting）实验以及β-葡萄糖苷酸酶（β-glucuronidase，GUS）报告基因检测，研究发现RsmA可在转录及转录后水平共同调控Ⅲ型分泌系统（type 3 secretion system，T3SS）的表达。RsmA对Ⅲ型分泌系统的调控在携带该系统的细菌中普遍存在，但其具体调控机制似乎因特定细菌与其宿主的相互作用而异。对于黄单胞菌属而言，RsmA激活Ⅲ型分泌系统的具体机制仍未阐明。本研究发现，在XCC中，RsmA可通过直接结合hrpG基因的5'非翻译区（5' untranslated region，UTR），激活编码Ⅲ型分泌系统的hrp/hrc基因的表达——其中hrpG是hrp/hrc基因的主调控因子。RsmA可稳定hrpG的mRNA分子，使其积累量提升，进而促进HrpG蛋白的合成，最终激活hrp/hrc基因的表达。在rsmA缺失突变体中异位过表达hrpG，可恢复其对宿主植物的致病能力以及触发非宿主植物过敏反应的能力，这一结果进一步证实了RsmA通过HrpG激活hrp/hrc基因的调控通路。此外，RsmA还可通过直接结合5'非翻译区，稳定另一个与Ⅲ型分泌系统相关的hrpD操纵子的转录本。综合上述实验数据，本研究揭示：RsmA主要通过作为hrpG的正向调控因子来激活Ⅲ型分泌系统，而该调控通路对于XCC的致病性至关重要。