水稻中XA21同源蛋白对条斑菌分子模式Ax21的识别研究

The pattern recognition receptor XA21 originated from the wild rice species Oryza longistaminata recognizes the highly conserved peptide Ax21 secreted by Xanthomonas oryzae pv. oryzae (Xoo) and then induces hypersensitive responses in rice. Like Xoo, the Xanthomonas oryzae pv. oryzicola (Xoc) genome carries a whole set of genes involved in the biosynthesis, modification, secretion and perception of Ax21. However, rice plants with Xa21 show no significant resistance to Xoc after infection. Unexpectedly, our experimental results demonstrated that the ax21 gene deletion mutant of Xoc had increased virulence on rice plants regardless of the existence of XA21, indicating that Xoc secretes Ax21 which can be perceived by other pattern recognition receptors besides XA21 in rice. Therefore, we set up a series of experiments to confirm the hypothesis and to explore molecular mechanism of the perception of Xoc Ax21 in rice. First, the secretion of Ax21 from Xoc will be detected through immunoblotting analyses. Second, we will construct a set of Xoc deletion mutants for genes that are involved in the secretion, modification and perception of Ax21. These mutant strains will be then evaluated for virulence on rice and for the phenotypes including the secretion of extracellular polysaccharide and proteases, biofilm formation and motility. Third, homologous genes of Xa21 in rice will be investigated to determine if these genes are also involved in the perception of Ax21 using rice T-DNA insertion mutants and/or the transgenic rice and Arabidopsis plants with altered expression of these genes. Fourth, whole-genome expression profiling in rice before and after Ax21 treatment will be compared to analyze the regulatory gene network in rice PTI. Last but not least, protein-protein interaction will be also studied in Ax21-triggered immunity in rice via yeast two-hybridization screening and co-immunoprecipitation. The resultant data in the project are expected to be useful for rice breeding with broad-spectrum and persistent resistance to Xoc.

源自野生稻的模式识别受体XA21对白叶枯菌分泌蛋白Ax21的识别会激发水稻的超敏抗病反应。条斑菌与白叶枯菌均具有一套与Ax21合成、修饰与分泌相关的基因，但携有Xa21的水稻对条斑菌不表现明显的抗性。我们研究发现条斑菌ax21突变体对含或不含Xa21水稻的致病力都增强，表明条斑菌Ax21能够被除XA21外的其他受体识别。基于此，项目将探讨水稻识别条斑菌Ax21的分子机理。首先，通过生化手段和对ax21及相关基因敲除体的表型与致病力分析，确定条斑菌分泌Ax21的能力与功能；其次，通过转基因技术在水稻与拟南芥中调控水稻Xa21同源基因的表达或利用水稻插入突变体，明确这些蛋白能否识别Ax21；并检测Ax21诱导水稻全基因组表达谱的变化，分析参与水稻基础抗性的关键基因；最后，通过酵母双杂或免疫沉淀等方法筛选与XA21同源蛋白互作并参与水稻基础抗性的新组分。研究结果可望应用于水稻广谱抗条斑病育种。

黄单胞菌是一类广泛存在、严重威胁作物生产的重要病原细菌，揭示其致病机理对这类病害的防控有着重要的指导意义。已有报道，稻黄单胞菌稻属致病变种Xoo中，能被水稻抗病蛋白XA21识别并诱导免疫的病原相关分子为Ax21，后被订正为RaxX。RaxX分泌、修饰和感知受rax基因簇的调控。RaxX和rax基因簇在细菌性条斑病菌Xoc基因组中都存在，通过对Xoc的rax基因突变体表型检测，发现这些基因的缺失不影响Xoc的生长、胞外酶与胞外多糖的产生以及在本生烟上的超敏反应；但除raxR突变体外，其它突变体产生生物膜的量均显著下降；raxH和raxR突变体的游动性也明显下降，其它突变体则没有变化。这些结果表明Xoc中rax基因簇对该细菌的致病因子起到重要的调控作用。.此外，病原细菌延伸因子EF-Tu的氮端高度保守的elf18多肽能被拟南芥特有的模式识别受体AtEFR识别。本研究构建了AtEFR转基因水稻，并发现该转基因水稻能识别elf18，激活免疫反应，且表现出对水稻褐条病增强的抗性。还发现，用elf18预处理水稻叶片能显著抑制Xoo在水稻叶片中的生长；但并不诱导水稻对真菌性病害稻瘟病的抗性。另外，Xoo的EF-Tu的第246至280个氨基酸区域能激发水稻悬浮细胞强烈的免疫反应，表明单子叶植物水稻也能识别EF-Tu，该发现为研究水稻识别EF-Tu的机理奠定了基础。.最后，该项目还研究了黄单胞菌野油菜致病变种Xcc中AvrXccB蛋白的功能。证实了AvrXccB是由III型分泌系统分泌到植物细胞中，并通过豆蔻酰化修饰定位在细胞质膜上。在拟南芥中异源表达AvrXccB能抑制植物的免疫反应，促进Xcc和PstDC3000的侵染能力等。其细胞质膜定位和酶催化位点对其发挥功能起到重要作用。此外，揭示了AvrXccB与拟南芥蛋白SAM-MT1及其同源蛋白SAM-MT2都互作，表明AvrXccB 与SAM-MT1/2形成互作复合体。SAM-MT1部分定位于植物的细胞膜上，也存在于细胞质中与膜相关、间断的点状结构中。SAM-MT1受PAMP和病原菌侵染早期诱导表达；而SAM-MT2则在侵染后晚期上调表达。这些研究表明Xcc 效应因子AvrXccB可能通过作用于SAM-MT1/2的复合体来抑制植物的免疫反应。