水稻白叶枯病菌第二信使c-di-GMP信号对无毒基因avrXa21的调控研究

Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterial pathogen, causes leaf blight, one of the most devastating diseases of rice worldwide. The interaction between Xoo and rice is a model system to study pathogen-host interaction. The avirulence protein AvrXa21 interacts with rice resistance protein XA21 and induces rice resistance to Xoo. AvrXa21 is sulfurated by RaxST and secerected by type I secretion system (T1SS). Recently, we have found that T1SS genes (raxA and raxB) and raxST were tightly regulated by the two-component signaling system (TCSS) RavA/RavR. TCSS is the major system for response to a wide range of environmental and cellular stimuli (including host signals) that trigger adaptive responses by reprogramming gene expression, altering cellular behavior, regulating proteolysis or triggering differentiation. The prototype of TCSS consists of a histidine kinase (HK) sensor and a response regulator (RR). HK usually autophosphorylates by ATP hydrolysis and then transfers a phosphoryl group to a conserved aspartic acid residue of the cognate RR. The RR protein RavR is an enzyme involved in c-di-GMP turnover and its activity switch from hydrolase to synthase is regulated by phosphorylation, which was controled by the cognate HK, RavA. C-di-GMP is an intracellular signaling molecule that acts as a cellular second messenger in bacteria. Mutation of either ravA or ravR decreased bacterial virulence in non-XA21 rice but increased in XA21-expressing rice. As RavA/RavR is involved in regulation of Xoo and rice recognization, RavA might sense signals from the host. In this study, we will focus on the roles of c-di-GMP signaling mediated by RavA/RavR in the regulation of AvrXa21 modification, secretion and function. We will also explore the roles of RavA/RavR in sensing of signals from rice host and in the XA21-mediated immunity. Our study will highlight the importance of c-di-GMP signaling in pathogen and host interaction.

由水稻黄单胞菌水稻致病变种(Xoo)引起的白叶枯病是重要的水稻病害之一。水稻与Xoo互作是研究宿主与病原菌相互识别的模式系统。水稻抗性蛋白XA21特异性识别Xoo无毒蛋白AvrXa21。AvrXa21由I型分泌系统分泌，并在分泌前或分泌过程中被硫化。近期我们发现双组分系统RavA/RavR调控了I型分泌系统和AvrXa21硫化的基因表达。RavR参与第二信使c-di-GMP的合成与分解，调控细菌的致病力。因此，RavA/RavR介导的c-di-GMP信号可能对AvrXa21的加工、分泌和功能起重要的调控作用。本项目将研究：(1) c-di-GMP信号调控I型分泌系统和硫化基因表达的途径；(2) RavA/RavR是否作为感应宿主信号的感应系统调控Xoo与水稻的互作。此研究将阐明 c-di-GMP调控AvrXa21的机制，拓展 c-di-GMP调控的细胞网络，为Xoo防治提供一定的理论基础。

水稻白叶枯病是由黄单胞菌水稻致病变种（Xanthomonas oryzae pv. oryzae，Xoo）引起的一种细菌性病害，对水稻产量构成较大的威胁。水稻抗性蛋白XA21通过感应Xoo效应蛋白AvrXa21，激活自身磷酸化反应，引起植物抗性表型。研究表明AvrXa21活动受3个操纵子（raxSTAB，raxPQ和raxRH）。其中RaxST负责AvrXa21的翻译后修饰；RaxA和RaxB负责分泌成熟的AvrXa21。RaxP/RaxQ和RaxR/RaxH为两对双组分系统，调控raxSTAB的表达。本研究主要分析了RavA/RavR双组分系统调控I型分泌系统进而调控AvrXa21活性实现的机制以及RaxR/RaxH与RavA/RavR的关系。我们首先构建了RaxR/RaxH与RavA/RavR的突变体及其互补菌株，然后分析了这些菌株的致病性特征及I型分泌系统基因raxSTAB和鞭毛结构基因fliP的表达情况。结果表明RaxR/RaxH可调控RavA/RavR的表达，进而影响raxSTAB的表达，但不调控AvrXa21的表达。我们还通过体外实验证实RaxR可以通过结合ravA和ravR启动子直接调控其表达，影响RavA/RavR介导的c-di-GMP信号传导。此外，我们还筛选了可能参与RavR介导的c-di-GMP信号传导的细胞组分，为进一步分析RavA/RavR如何调控rax基因表达提供了材料。重要的是，我们通过遗传和分子生物学方法还鉴定了一个调控AvrXa21表达的调控因子RaxM。RaxM可能直接或间接调控raxM自身及avrXa21启动子活性，进而影响Xoo与水稻的互作。以上研究结果拓展了AvrXa21的调控网络，加深我们对Xoo与水稻互作的理解，对建立新型病害防控策略具有重要的理论意义。