RaxM调控AvrXa21实现功能的机制

Sensing of the extracellular cues by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. The XA21 protein recognizes Xoo strains carrying AvrXa21 activity. The AvrXa21 activity (a) depends on a bacterial type one secretion system (TOSS) not on a TTSS, (b) is regulated by two-component regulatory systems RaXH/RaxR, PhoP/PhoR and RavA/RavR, (c) is regulated by cell density, and (d) is dependent on posttranslational modification of sulfuration by tyrosine sulfotransferase RaxST. Sulfated RaxX or the derivative 21–amino acid synthetic peptide (RaxX21-sY) can trigger hallmarks of the plant immune response in an XA21-dependent manner. Therefore, RaxX might act as AvrXa21 in Xoo strains. But there are not direct evidence to support this hypothesis because (a) no direct interaction between sulfated RaxX and XA21 was found, and (b) the molecule weight of sulfated RaxX or the derivative RaxX21-sY is higher than that of real AvrXa21 (<1500 Dalton). One possibility is that AvrXa21 is a RaxX derivative that is less than 14 amino acid produced by a peptidase in vivo. We have found a gene (raxM) upstream of raxX is essential to AvrXa21 activity. Deletion of raxM results in similar phenotypes to that of raxX, but does not change the gene expression of TOSS and raxST, and slightly increases its own expression. Therefore, RaxM regulates AvrXa21 activity is independent of these neighbors gene expression. The function of RaxM involved in AvrXa21 activity remains obscure. Therefore, the objective of this study is to ascertain the mechanism of that RaxM regulates AvrXa21 activity. We will answer three questions to uncover the mechanism: 1) Whether and what AvrXa21-related genes are regulated by RaxM, 2) Whether and what cellular components interact with and are required for the activity of RaxM, 3) whether RaxM can function as a modification enzyme for AvrXa21 maturation from RaxX? Our study will not only identify RaxM function but also draw a global regulatory network of AvrXa21 activity. It will give us comprehensive understanding of AvrXa21-mediated immune response.

水稻受体激酶XA21识别带有AvrXa21的水稻白叶枯病菌，进而对其产生抗性。AvrXa21由I型分泌系统分泌，受双组分系统和细胞密度的调控。AvrXa21须经硫化修饰才具有活性。硫化的RaxX及其21氨基酸衍生肽可激发XA21依赖的免疫反应，可能是AvrXa21。我们发现raxX上游基因raxM突变的表型与raxX突变一致，但不影响相邻基因raxSTAB的表达。RaxM负调控自身操纵子raxMXY的表达。这表明突变体表型不是由于邻近基因表达变化造成的。本研究的目标是明确RaxM调控AvrXa21活性的机制。我们将通过实验回答下面3个问题来揭示RaxM的调控作用：（1）RaxM是否调控其它基因表达？（2）RaxM是否通过其它蛋白影响AvrXa21的活性？（3）RaxM是否参与AvrXa21的成熟？此研究将揭示AvrXa21活性调控的新机制，有助于深入解析AvrXa21介导的免疫反应。

以前研究发现，RaxX需经过翻译后加工才能形成成熟的无毒蛋白AvrXA21，诱发水稻（表达XA21）的免疫反应。这些加工修饰过程包括酶切和硫化，最后再分泌到胞外与植物XA21结合，诱导XA21介导的免疫反应。但RaxX是否在转录水平也受调控并不清楚。本项目通过遗传和生物信息学分析，发现了RaxM蛋白可以调控自身及RaxX的mRNA含量，进一步分析发现RaxM影响自身及RaxX的启动子活性。但是RaxM并不作为转录因子直接结合到自身及RaxX的启动子上，暗示其可能通过其它方式调控自身和RaxX启动子的活性。在此过程中RaxM也不与RaxX直接互作，在体内也未发现RaxM影响RaxX的剪切。因此，RaxM可能通过与其它蛋白互作来调控RaxX启动子活性。通过酵母双杂交筛选和pulldown等实验发现RaxM能与MutL互作，且MutL的活性位点突变会降低它们之间的结合能力，说明MutL可能介导RaxM调控raxX的表达。启动子活性分析结果显示mutL突变导致raxX表达量降低和raxM表达量升高，这种调控作用受RaxM的影响。致病力分析结果表明mutL突变与raxM突变都导致病原菌在表达XA21的水稻上的致病能力增强。这些结果说明RaxM可能通过MutL调控自身和raxX的表达，进而影响AvrXA21介导的免疫反应。我们的研究发现了除翻译后修饰调控外，RaxX也能在转录水平被调控，拓展了我们对病原菌调控自身毒力因子表达及其与宿主互作的认识，为白叶枯病的防控奠定了一定的理论基础。