杨树溃疡病菌Lonsdalea quercina调节蛋白RRT1参与致病过程和逆境胁迫应答的调控机制研究

A new canker disease of Populus × euramericana caused by Lonsdalea quercina subsp. Populi was first discovered in China in 2006. The disease causes much more devastating damage and severely economic loss to poplar plants than other types of poplar canker diseases. We have known nothing about the pathogenesis of this phytobacteria. In the latest study, we identified and characterized a regulation protein of two-component systems, RRT1, which is involved in responses to oxidative and osmotic stresses and virulence in L. quercina. RRT1 probably function as a transcription factor, since it has a C-terminal DNA-binding domain. The regulatory mechanism and the targets of RRT1 involved in these processes need to be explored. In this study, comparative transcriptomic and molecular genetic methods will be employed to identify the differential expression target genes of transcription factor RRT1 treated with or without oxidative and osmotic stresses; and proteomic techniques will be furthered adopted to identify the binding proteins of RRT1 under normal or stress conditions mentioned above. The interactions between candidate binding proteins and RRT1 will be confirmed by bacterial two-hybrid system, pull-down or MST. The confirmed candidate targets and binding proteins of RRT1 will be furthered investigated, as well as their effects on stress responses and pathogenicity of L. quercina by heterogenous expression, knock-out or knock-in mutation analysis. The results will propose a regulatory network mediated by RRT1 to address the mechanisms on growth and development, stress responses and virulence of L. quercina. This study will not only help us better understand the regulatory mechanisms of RRT1-mediated signaling network and screen for antibiotic drug targets, but also help to design efficient disease management strategies for poplar trees.

由Lonsdalea quercina引起的欧美杨溃疡病于2006年在国内首次发现，不同于水泡型与冰核细菌型溃疡病，该病害对欧美杨速生林造成毁灭性破坏和严重经济损失，但其致病机制尚不明确。前期鉴定到一个参与氧化和渗透压胁迫应答与致病过程的双组分系统调节蛋白RRT1。RRT1的C端具有DNA结合域，推测其以转录调控方式参与信号网络调节。为了深入解析RRT1作用机制，本项目拟利用转录组学和分子遗传学等方法，在胁迫与非胁迫条件下，鉴定和确认RRT1直接转录调控的靶标基因及其互作蛋白。充分利用异源表达和缺失突变的分析方法，研究这些生物分子在病原菌的胁迫适应和致病过程中的功能，明确它们在信号网络中的地位。研究结果将揭示RRT1介导的信号调控网络在L. quercina生长、逆境胁迫应答及致病过程中的分子机制，同时对筛选以该网络关键信号分子为靶标的农用抗生素、设计高效低毒林木细菌病害控制策略提供指导。

欧美杨细菌性溃疡病是革兰氏阴性细菌Lonsdalea populi引起的杨树枝干病害，其危害严重，已造成欧美杨人工林的重要经济损失。双组分系统是细菌最重要的信号转导通路之一，在细菌的生长繁殖、环境适应、胁迫耐受性以及致病过程中发挥重要作用。目前，欧美杨细菌性溃疡病菌的双组分系统如何调控致病过程仍缺乏系统研究。因此，本项目集中开展解析欧美杨细菌性溃疡病菌双组分系统基因参与致病过程和逆境胁迫应答的调控机制。通过全基因组测序和毒力相关基因、抗药性基因等生物学功能的注释分析，揭示了其在致病性和环境适应性方面的分子机制。通过构建双组分系统基因的缺失突变体库，初步鉴定了欧美杨溃疡病菌不同双组分系统基因的生物学功能。通过深入研究DcuS-DcuR系统，明确了该双组分系统是调控病原菌致病性的重要基因，并且其也调控细菌鞭毛和游动性、生物膜及过氧化氢和氯霉素胁迫耐受性等生理过程，同时发现DcuS的不同结构域和His326位点及DcuR的REC结构域和Asp56位点在突变体表型缺陷及基因表达模式改变中发挥了重要作用；通过对TCS基因lqp0812-lqp0813的深入探索，发现lqp0812参与病原菌的游动性，lqp0812与lqp0813共同负调控菌株生物膜的形成和对氧化胁迫、抗生素胁迫的耐受性，揭示了lqp0812-lqp0813在病原菌抵御外源抗生素和过氧化氢上的作用机制；对Cpx系统、Rst系统、Rcs系统、rssB等基因的转录组分析也进一步阐述了欧美杨溃疡病菌双组分系统基因在病原菌致病能力、生物膜形成、游动性以及对外源抗生素和过氧化氢耐受性的调控机制。本项目证明了双组分系统在L. populi N-5-1 生命活动中占据重要地位，为后续病原菌的分子机制研究提供了遗传材料，为阐述欧美杨细菌性溃疡病致病机制提供了数据支撑。