MNAC3负调控水稻对稻瘟病抗性的分子机制研究

NAC proteins belong to a plant-specific family of transcription factors, some of which have been demonstrated to play important roles in regulating plant immunity against pathogens. In our previous study, four Magnaporthe oryzae (the causal fungus of blast disease)-induced NAC genes with unknown functions, named MNAC1-4, were identified by combined analyses of publicly available microarray and qRT-PCR data. Preliminary functional analyses using a T-DNA insertion line and overexpression lines showed that, among the four MNAC genes, MNAC3 negatively regulates resistance of rice against blast disease. The goal of the proposed project will investigate the molecular mechanisms by which MANC3 negatively regulates rice resistance to blast disease by combined use of the T-DNA insertion line mnac3-1, CRISPR/Cas9-mediated knockdown lines MNAC3-CRISPR/Cas9 and GFP-tagged MNAC3 overexpression lines MNAC3-GFP-OE. Changes in contents of signaling molecules (e.g. salicylic acid and jasmonic acid), levels of defense responses (e.g. PR gene expression, ROS accumulation and cell wall inforcement) and PAMP (e.g. chiitin or flg22)-induced response will be analyzed and compared the data from the mnac3-1, MNAC3-CRISPR/Cas9 and MNAC3-GFP-OE lines with those from WT to determine the MNAC3-regulated immune responses in rice. RNA-seq-based expression profiling will be carried out and compared the data from the mnac3-1, MNAC3-CRISPR/Cas9 and MNAC3-GFP-OE lines with those from WT to identify the group(s) of MNAC3-regulated genes. ChIP-seq analyses will be performed and compared the data from the MNAC3-GFP-OE and WT plants with or without infection of M. oryzae to characterize genome-wide DNA-binding sites of MNAC3. Putative downstream target genes of MANC3, named as MRTs (MNAC3-regulated targets), will be identified by combined analyses of RNA-seq and ChIP-seq data and some of the MRT genes will be selected to investigate their possible involvement in rice resistance to blast disease. Results from this proposed project will help establish the molecular mechanism by which MNAC3 nagetively regulates rice resistance to blast disease. The expected outputs will be helpful to expand and strengthen our understanding on the molecular basis and regulation network of rice disease resistance as well as will provide a possibility to generate novel rice germplasm resources with improved disease resistance using MNAC3 as a useful functional gene.

NAC蛋白组成一个植物特有的转录因子家族，其中一些NAC蛋白参与植物免疫反应。前期研究中鉴定到4个受稻瘟病菌侵染诱导但功能未知的ONAC基因MNAC1-4。利用T-DNA插入突变体和过表达株系的病害表型分析表明，MNAC3负调控水稻对稻瘟病的抗病性。本项目以T-DNA插入突变体mnac3-1、CRISPR/Cas9敲减突变体MNAC3-CRISPR/Cas9、带有GFP标签MNAC3过表达株系MNAC3-GFP-OE为材料，研究MNAC3负调控抗病性的分子机制，包括：明确MNAC3调控的免疫反应；利用RNA-seq技术鉴定MNAC3调控的基因群组；利用ChIP-seq技术分析MNAC3在基因组上的结合位点；鉴定MNAC3调控的靶标基因MRTs，并分析其抗病功能。研究结果将阐明MNAC3负调控稻瘟病抗性的分子机制，从而加深水稻抗病性分子调控网络的认识，为利用MANC3培育抗病新材料提供支撑。

本课题重点研究了水稻NAC转录因子MNAC3的抗病功能及其作用机制。MNAC3是一个核定位的转录激活因子，但不能形成同源二聚体，能结合NACRS元件，其表达受稻瘟病菌侵染和抗病相关激素诱导。MNAC3-OE和MNAC3-CP株系的表型分析表明，MNAC3负调控水稻对稻瘟病和白叶枯病的抗性以及PTI，而且MNAC3敲除后抑制ABA信号途径基因的表达。DAP-seq分析表明，MNAC3在水稻基因组上结合到CGAG motif序列上，结合RNA-seq与DAP-seq数据，鉴定到12个在MNAC3-OE中表达上调而在MNAC3-CP中表达下调、且MNAC3结合到启动子区域的基因。OsINO80、OsOAT、OsJAZ10和OsJAZ11在MNAC3-OE中表达上调而在MNAC3-CP中表达下调；酵母单杂交、EMSA和双荧光素酶报告系统分析结果表明，MNAC3能结合OsINO80、OsJAZ10和OsJAZ11启动子中的CGAG元件，驱动OsINO80、OsJAZ10和OsJAZ11的转录。MNAC3能与磷酸酶OsPP2C54/OsPP2C41、DnaJ伴侣蛋白OsDjA6互作，而且OsDjA6能增强MNAC3对OsJAZ10和OsJAZ11启动子CGAG元件的结合能力。OsINO80、OsJAZ10和OsJAZ11敲除后增强水稻对稻瘟病的抗性。上述结果表明，MNAC3通过调控OsINO80、OsJAZ10和OsJAZ11免疫相关基因的表达从而负调控水稻对稻瘟病和白叶枯病的抗性。