组蛋白去乙酰化酶OsHDT701介导水稻先天免疫抗稻瘟病的分子机理及其调控网络的解析

Post-translational modifications, such as methylation,ubiquitination, and acetylation play an important role for various biological function. The histone acetylation and deacetylation regulate gene transcription. In general, acetylation of histone is associated with transcriptional activation whereas deacetylation of histione is contributed to gene repression. Epigenetic regulations such as histone modifications and chromatin remodeling are critical for host defense against pathogens. Further understanding of the epigenetic mediated plant resistance, signals and networks and identification of the extent and nature of epigenomic changes induced by different pathogens remain to be elucidated. Our preliminary data showed that OsHDT701 negatively regulates rice blast fungus PAMPs triggered innate immunity by modulating the levels of histone H4 acetylation and the expression of pattern recognition receptor (PRR) and defense-related genes in rice. Therefore, the overall aim of this proposal is understanding the molecular mechanism of OsHDT701 regulating innate immunity in rice. To this aim, firstly,we will perform yeast-two-hybrid assay to screen the OsHDT701 interaction protein. Secondly, identify the OsHDT701 binding DNA sites by a whole-genome ChIP-seq assay. Thirdly, to analysis the OsHDT701 modulated transcriptome by RNA-seq approach. Finally, we will comparing the yeast-two-hybrid, ChIP-seq, and RNA-seq data and find out the signals,networks, and defense-related genes regulated by OsHDT701. Understanding the mechanism of plant epigenetic regulating resistance to pathogens will provide a new strategy for crop disease control.

甲基化，泛素化和乙酰化等翻译后修饰在生命体中具有十分重要的作用。其中，组蛋白的乙酰化和去乙酰化与转录调节密切相关。由表观遗传介导的寄主对病原菌的抗性是目前国际上植物与病原菌互作研究领域的一个热点、难点问题。本课题组前期研究结果表明，水稻组蛋白去乙酰化酶OsHDT701通过抑制组蛋白乙酰化水平及PTI相关基因的表达，负调控对稻瘟病菌PAMP诱导PTI的抗性。基于此，本项目将采用分子植物病理学、遗传学、生物化学及生物信息学方法，着重研究OsHDT701在参与抗稻瘟病的调控机制。首先，通过酵母双杂交手段鉴定OsHDT701互作水稻蛋白HDIPs；然后，利用全基因组ChIP-seq分析鉴定OsHDT701蛋白结合的基因启动子区DNA位点；再次，使用RNA-seq分析鉴定OsHDT701的调控谱；最后，系统解析OsHDT701调控水稻抗稻瘟病的信号网络。研究结果将为农作物病害防控策略提供新思路。

由稻瘟菌引起的稻瘟病是水稻生产中最为严重的真菌病害，研究稻瘟菌与水稻的互作，揭示其致病及抗性机理，对保障粮食安全具有不容忽视的意义。由表观遗传介导的寄主对病原菌的抗性是目前国际上植物与病原菌互作研究领域的一个热点、难点问题。甲基化，泛素化和乙酰化等翻译后修饰在生命体中具有十分重要的作用，其中，组蛋白的乙酰化和去乙酰化与基因转录调节密切相关。本课题组前期研究结果表明，水稻组蛋白去乙酰化酶OsHDT701通过抑制组蛋白乙酰化水平及PTI相关基因的表达，负调控对稻瘟病菌PAMP诱导PTI的抗性。基于本课题组已有的研究基础，本项目以水稻组蛋白去乙酰化酶蛋白OsHDT701为诱饵，筛选水稻cDNA文库，获得了其在水稻中的8个候选互作蛋白；通过酵母双杂、GST Pull-down等实验手段在体内与体外验证了OsHDT701与水稻RNase P的p30亚基（Rpp30）的互作。RNase P是一类保守的内源剪切酶，它可以去除tRNA前体的5´序列，对tRNA成熟必不可少。tRNA是一类长度大约为75nt的非编码RNAs，其在蛋白翻译合成过程中起着至关重要的作用。进一步通过Rpp30的过量表达（OX）与基因沉默（RNAi）转基因水稻株系的创建及抗病表型鉴定等实验证明了Rpp30与水稻抗病反应的正相关性；同时在Rpp30蛋白N端保守功能结构中鉴定到了3个乙酰化修饰位点，为OsHDT701与Rpp30互作并调控其修饰水平提供了有力证据；综上所述，该部分实验结果揭示了Rpp30与OsHDT701互作后共同调控水稻先天免疫反应的作用机理，丰富了tRNA生物学功能在水稻抗病及植物免疫方面的认识。此外，我们还利用全基因组ChIP-seq分析鉴定OsHDT701蛋白结合的基因启动子区DNA位点；使用RNA-seq分析鉴定OsHDT701的基因表达调控谱；综合所有研究结果，我们较为系统地解析了OsHDT701调控水稻抗稻瘟病的信号网络。