小麦TaAGO4a参与白粉病抗性的分子路径解析

ARGONAUTE (AGO) proteins are the central components of various small RNA (sRNA)-induced gene silencing complexes (RISCs). They are encoded by a small gene family,among which AGO4 is known to be involved in sRNA-directed target site DNA methylation, but the moleclar pathway mediated by AGO4 for disease resistance is not clear. Our previous results showed that down regulation of TaAGO4a causes loss of powery mildew (PM) resistance in wheat, suggesting that TaAGO4a is required for PM resistance. In this proposal, we plan to obtain TaAGO4a-bound small RNAs that are differentially expressed under PM infection using available TaAGO4a-D antibodies. These sRNAs will be aligned to newly finished Aegilops taushii (DD) draft genome sequence so that the potential target sites can be determined. We will then assay the methylation status of these sites and the expression patterns of the neighboring disease resistance related genes, some of which will be further tested for their functions in disease resistance using virus induced gene silencing technique. We also propose to study the mode of coordination between three TaAGO4a homoeologues in the hexaploid wheat and over express TaAGO4a to study its in planta function in PM resistance. This work will be the first in wheat to dissect the PM resistance mechanims from the epigenetic point of view and should provide valuable knowledge for wheat disease resistance breeding.

ARGONAUTE (AGO) 蛋白是小分子RNA (sRNA) 介导的基因沉默复合物的最核心组分，由一个小基因家族编码。AGO4主要参与sRNA靶位点的DNA甲基化，但其参与抗病的分子机制尚不清楚，在小麦中更是空白。前期工作证明下调TaAGO4a导致小麦丧失白粉病抗性，表明TaAGO4a为小麦白粉病抗性所必需。本项目拟利用已获得的小麦D基因组特异TaAGO4a抗体，获取与其特异结合并显著应答白粉菌侵染的sRNA，结合D基因组草图寻找差异sRNA靶位点，研究靶位点DNA甲基化水平与其邻近抗病相关基因表达的关系，验证部分候选基因的抗病功能；本项目还将针对小麦六倍体特点，利用前期获得的TaAGO4a三个部分同源基因研究它们在白粉病抗性中的协同工作模式；并通过TaAGO4a小麦过表达探索其应用的可能性。本研究首次从表观遗传学层面剖析小麦白粉病抗性机制，为小麦抗病遗传育种提供重要的理论依据。

ARGONAUTE (AGO) 蛋白是小分子RNA (small RNA, sRNA) 介导的基因沉默复合物的最核心组分，由一个小基因家族编码。其中AGO4主要参与sRNA靶位点的DNA甲基化(RdDM)。尽管有文献报道DNA去甲基化参与拟南芥细菌抗性，但其是否参与活体寄生的真菌抗性尚不清楚。首先，利用小麦二倍体供体种粗山羊草抗病品系(Y2280)与感病品系 (AL8/78)确认白粉菌侵染后12 h是侵染的关键时期，并用qPCR检测发现RdDM路径基因 (Pol IV、RDR2、DCL3a、AGO4a、AGO4b、Pol V、DRM2) 接菌后均下调表达，表明RdDM路径参与了白粉菌应答。接着，高通量测序侵染0 h和12 h粗山羊草叶片的Total sRNA和AGO4a 捕获的sRNA，发现接菌后AGO4a捕获的24 nt小分子RNA显著下调，且抗病材料中的下调强于感病材料，而Total sRNA在接菌后无明显变化，表明白粉菌减少了AGO4a捕获的24 nt小分子RNA。进一步高通量测序侵染12 h 对照和接菌的粗山羊草叶片的全基因组甲基化和转录组，发现CHH甲基化下调位点区域显著多于CG和CHG甲基化下调位点，对CHH甲基化下调位点区域覆盖且侵染后上调表达的基因，进行GO富集分析，发现这些基因参与刺激应答和脂质代谢路径。此外，CHH甲基化水平下调的节律核心基因Circadian Clock Associated1 (CCA1)，在持续光照条件下接种白粉菌后，其节律不变，振幅增强；发现活性氧路径基因Peroxidase (POX5, POX22) 和茉莉酸Methyl Jasmonate (JA) 路径基因TPS3也表现出CHH甲基化下降，基因上调表达。最后利用VIGS方法，在感病材料中，下调DRM2表达，发现随着DRM2基因下调程度的增强，感病表型变弱。进而在DRM2的VIGS植株中，发现 POX5，POX22，TPS3等基因上调表达，从而证明这些基因受DRM2介导的CHH甲基化调节。在感病材料中瞬时过表达POX5，增强了感病材料对白粉病菌的抵御能力。上述工作首次在小麦二倍体供体种中揭示：白粉菌侵染通过下调AGO4a捕获的24 nt小分子及其相关的CHH甲基化，使节律基因振幅增强，活性氧路径基因和JA路径基因上调表达，进而参与植物抗病性。