水稻内生菠萝泛菌YJ76中吲哚的上游调控通路与信号感受器

Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa cv. Yuefu) showing multiple growth-promoting effects on host plant, is a typical model bacterium for endophyte-host association studies. Recently, indole has been identified as an important signal molecule to induce a variety of physiological behaviors of bacteria, provoking great attention among bacteriologists. Nevertheless, most efforts focused on its downstream regulations. In our previous studies, it is found that YJ76 can spontaneously synthesize indole and actively regulate the yield of indole to respond to adverse stresses, and a new gene that mediates the upstream regulation of indole was screened out. In this project, the gene will be taken as a breakthrough point, and the adversity sensing mechanism of the bacterium and activating mechanism of indole signaling pathway will be revealed via exploration on its regulatory role on the key genes' transcription of indole synthesis in response to adversity. Now, scholars around the world cannot answer the question of "how bacteria sense indole to regulate gene expression" since no receptor protein of indole has been found so far. Here, basing on the discovery of the core region of pcnB promoter that senses indole, we propose a new idea to try to solve this difficulty by exploring the natural aptamer of indole and its NMR structure from the perspective of riboswitches, which will essentially throw light on the molecular mechanism of indole regulation. Also, the particular significance of indole for the endophyte-plant associating micro-ecosystem will be explained via the research on the relationship between symbiotic adaptability of the bacterium and indole activation mechanism and its receptor.

菠萝泛菌YJ76是具有多种促生作用的水稻优势内生固氮菌，是研究内生菌与植物互作的良好模式材料。近年来吲哚被确认是一种重要的信号分子，可诱发菌体多种生理行为而受到高度关注，但主要集中在其下游调控。课题组前期研究，发现YJ76能自发合成吲哚且能主动应答逆境调节吲哚产量，筛选到一个介导吲哚上游调控的新基因。本项目拟以该基因为切入点，研究其响应逆境对吲哚合成关键酶基因转录的调控作用与通路，揭示细菌对逆境的感受机制及吲哚信号通路的激活机制。由于迄今未发现吲哚信号的受体蛋白，造成所有学者都不能回答“细菌究竟如何感知吲哚来调控基因表达”这一问题。我们拟换一条思路，基于课题组发现的pcnB启动子感知吲哚的核心区，尝试从核糖开关的角度探索吲哚天然核酸适配体及其NMR结构，揭示吲哚调控的分子机制，攻克这一难题。通过研究吲哚激活机制和受体及与菌体共生适应性之关系，阐释其在内生菌-宿主互作微生态系统中的特殊意义。

本项目选取水稻内生菠萝泛菌YJ76为研究材料，以L-色氨酸代谢途径为基础，探究了细菌对逆境的感受机制及吲哚信号通路的激活机制。研究发现逆境因子胁迫能促进吲哚合成通路中关键酶基因的表达，证明了YJ76能主动感受逆境并自主激活吲哚合成途径。利用mTn5gusA-pgfp21转座子插入突变技术，筛选出三个基因即编码葡萄糖-6-磷酸脱氢酶的zwf、一种新发现的编码新型L-Ser孤儿底物结合蛋白的sbp和编码甲基趋化受体蛋白的mcp-5685是吲哚合成调控通路的关键基因，这些基因突变后显著降低了YJ76吲哚产量、抗逆能力、对宿主水稻的定殖和促生能力等。本项目明确了全局性碳存储调控因子A（CsrA）介导了吲哚信号对菌体抗饥饿生存的调控通路，其机制是吲哚抑制csrA的表达，借由CsrA在翻译水平上调控鞭毛合成调控蛋白FlhDC、糖原合成蛋白GlgCAP、抗饥饿调控蛋白CstA及其他靶标蛋白的合成，进而抑制菌体运动性、促进糖原积累和提高细菌抗饥饿性状等，实现对细菌各种生理生化过程的广泛调控。鉴定了YJ76中转录因子NtrC对zwf基因的调控位点，发现该转录因子有多种逆境响应能力且可以介导吲哚的调控。发现pcnB尽管在5'-UTR存在感受吲哚的关键核心区域，但它与吲哚并不能直接结合，吲哚可能不以核酸开关的形式来调控基因表达，即所期望揭示的核酸开关模式的吲哚信号感受器并不存在。此外，本研究还发现菠萝泛菌YJ76参与并促进水稻内生成团泛菌YS19定殖特异性结构共质体的形成，两菌共培养时增强了内生菌在宿主中的定殖与促生能力；发现成团泛菌YS19可产生eDNA，而YJ76产生的吲哚能够增加eDNA的释放，增强共质体形成，从而揭示了两菌共栖息协同作用的一种机制。上述研究成果构筑了吲哚信号的上游调控网络，阐释了吲哚调控通路及与菌体共生适应性之关系，初步揭示了吲哚信号调节在内生菌-宿主互作的微生态系统中的特殊意义。