调控丁香假单胞菌致病性的关键双组分系统的信号传导机理研究

Pseudomonas syringae, the leading cause of crop diseases world-wide, uses type III secretion system (T3SS) to invade host plants. T3SS of plant pathogenic bacteria is induced in the plant and minimal media (MM). Although a number of components regulating the T3SS gene expression have been identified, how bacteria sense the environmental conditions and further transduce the signal(s) to the known regulatory components remain largely unknown. We have found a two-component system (TCS) RhpRS as a master regulator and a switch of T3SS in P. syringae. In our previous studies, we have identified a plant cutin monomer cis-9,10-epoxystearic acid (ESA) and a histidine kinase EnvZ in P. syringae that inhibit T3SS via activating RhpRS. We also have done ChIP-seq analyses revealing key RhpR-binding loci, including the promoter regions of hrpR and a putative small RNA PSPPH1805^6. Based on these preliminary results, we hypothesize that RhpRS directly senses ESA to inhibit T3SS, which is via the crosstalk with EnvZ and the direct regulation of hrpR and PSPPH1805^6. In order to test this central hypothesis, we have three key objectives: 1. to determine that RhpS senses ESA to inhibit T3SS; 2. to examine the crosstalk between RhpRS and EnvZ; and 3. to study how RhpRS directly regulates hrpR and PSPPH1805^6? This proposal is aimed at understanding the signaling process governing the regulation of T3SS genes via RhpRS. Genetic and biochemical characterizations of RhpRS-pathway is the first step toward identifying signals to which the T3SS responds, and toward the development of new strategies for disease control based on T3SS manipulation. Our long-term goal is to leverage the outcomes of the herein proposed studies to develop better therapies against P. syringae infection.

丁香假单胞菌属（Pseudomonas syringae）是全世界农作物的主要病原菌之一，其致病性主要由III型分泌系统（T3SS）介导。虽然目前已经鉴定了一些调节T3SS的组份，但是细菌如何感应环境信号并作出相应的反应还知之甚少。我们在前期实验中发现了第一套直接调控T3SS的双组分系统RhpRS，并且初步分析了它的信号通路和调控机理。根据初步实验结果推测：1.环氧硬脂酸ESA是RhpS识别的信号分子；2.EnvZ与RhpRS交联作用共同调控T3SS；3.RhpR通过直接抑制hrpR启动子和激活新基因PSPPH1805^6从而抑制T3SS。本课题旨在围绕“探索RhpRS抑制T3SS的信号通路”这一关键问题，鉴定ESA为RhpS的信号分子，解析EnvZ在RhpRS-T3SS信号通路中的关键作用，同时研究RhpR抑制T3SS的分子机理，为丁香假单胞菌的防控提供新策略。

丁香假单胞菌是一种典型的植物病原细菌，在世界范围内造成了农业疾病和巨大的经济损失。III型分泌系统(T3SS)是其毒力的关键决定因素，其中双组分系统RhpRS是T3SS的关键调控因子。RhpS对RhpR同时具有激酶和磷酸酶活性，并具有自身激酶的功能。然而，RhpRS如何使用外部信号和磷酸化状态来行使其调节功能，以及靶向RhpRS的植物源化合物仍然未知。在本项目中，我们发现磷酸化的RhpR通过直接结合hrpR启动子来抑制T3SS基因的表达。并首先证明了植物源性多酚是RhpS直接感知的信号。在1680个不同的小分子中，发现有三种多酚(鞣酸、1,2,3,4,6-五没食子酰葡萄糖和表没食子儿茶素没食子酸酯)对丁香假单胞菌T3SS和毒力有抑制作用。磷酸化的RhpR (P-RhpR)通过抑制hrpR的表达负向调控T3SS基因，但直接激活小基因grlp的表达。我们发现grlp编码位于外膜(OM)的富含甘氨酸脂蛋白(grlp)，并在过表达时对细菌的行为具有多效作用，如降低致病性、游动动力、生物膜形成和对各种应激的耐受性，但当grlp被删除时可起诱导作用。丰富培养基中grlp的转录水平高于最小培养基，在营养条件有限的情况下，grlp的表达减少有利于细菌种群的长期存活。过表达grlp使胞周间隙增大，敲除grlp使胞周间隙减小。由于质周空间和OM机制在细菌生理调节中起着核心作用，我们推测GrlP可能是通过调节质周空间和OM机制进行细菌生理的调节。这些结果表明，T3SS调控的细菌毒力与RhpRS介导的细菌胞膜特性密切相关。总之，我们揭示了丁香假单胞菌RhpRS的分子调控机制，发现了RhpS感知植物源信号的关键位点，为未来后续的药物筛选和研发提供重要的理论基础。

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