转录激活因子PilR在σ54调控水稻白叶枯病菌毒性中的功能研究

Sigma 54 is an important transcriptional regulator dependent on the transcriptional activators, and regulates the virulence of Xanthomonas oryzae pv. oryzae (Xoo). However, the regulation mechanism of the σ54 still remains unclear. In various pathogenic bacteria, PilR regulates the biosynthesis of pili, which plays an important role in the bacterial virulence. In Xoo, PilR encoded by pilR, which is located at type IV pili gene cluster, has three conserved domains that are similar with σ54 transcriptional activators. Downstream of the pilR, the gene pilA which the promoter contains conserved binding sites with σ54 was regulated by σ54. These results implied that PilR as σ54 transcriptional activator regulates the virulence of Xoo by controlling the type IV pili biosynthesis. Therefore, the characterization of PilR will be further proposed in this project, including identification of σ54 transcriptional activator PilR, investigation of the mechanism that the σ54 and PilR regulate the transcription levels of type IV pili genes, and determination of the effects of PilR on Xoo vireluence. The objectives of this project are to reveal the pathway that σ54 and PilR regulate the transcription of type IV pili gene in Xoo, which may mediate the mechanism for σ54 regulate the virulence of Xoo.

σ54是一类依赖于转录激活因子的细菌重要转录调控因子，调控着水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae, Xoo）毒性，但其作用机制尚不清楚。PilR控制细菌菌毛形成，而菌毛的缺失严重削弱病菌的毒性。Xoo IV型菌毛基因簇中基因pilR编码蛋白具有σ54转录激活因子保守结构域，同时pilR下游基因pilA启动子含有σ54保守结合位点，且其活性受σ54的调控。这些结果暗示PilR可能作为σ54转录激活因子通过调控IV型菌毛基因的转录进而影响Xoo毒性。因此，本项目将深入的对PilR进行功能分析，包括：（1）确证PilR为σ54的转录激活因子；（2）明确σ54与PilR对IV菌毛基因转录的调控作用；（3）分析PilR对Xoo毒性的调控作用。本研究旨在揭示σ54与PilR对Xoo IV型菌毛基因转录的调控途径，并解析此途径可能介导了σ54对Xoo毒性的调控机制。

IV型菌毛是一类特殊的细菌表面细丝，在病原细菌的表面粘附，运动性，生物膜形成和毒性中起着至关重要的作用。然而，在水稻白叶枯病的病原体稻黄单胞菌稻致病变种中，对IV型菌毛的调节机制及其与细菌毒力的关系仍然知之甚少。我们的前期研究明确了转录调控因子RpoN2对白叶枯病菌鞭毛合成的调控机制，但对其毒性的调控机制还不清楚。在本研究中，我们采用酵母双杂交和GST pull-down 技术证明了RpoN2能直接和特异性的与PilRX相互作用，而PilRX是菌毛基因簇中双组分系统PilS-PilR反应调节因子PilR的同源物。生物信息学分析和RT-PCR显示白叶枯病菌菌毛基因簇由13个转录调控单元组成，包含25个编码基因，在大多数基因的启动子序列中鉴定出一个共有的RpoN2结合序列GGN10GC。凝胶阻滞实验证实RpoN2与主要的菌毛蛋白基因pilAX，内膜平台蛋白基因pilCX和pilRX的启动子直接结合。启动子活性和荧光定量PCR分析证明了RpoN2对菌毛基因的直接和间接调控。此外，单独突变pilAX，pilCX和pilRX会导致病菌运动性、生物膜形成和毒性显着降低。综上所述，RpoN2-PilRX通过调控菌毛基因转录来控制白叶枯病菌的运动性和毒性，这一研究结果加深了对病原细菌和作物互作的分子机制的理解，为植物细菌病害的防控提供了新的思路和理论指导。