水稻对稻瘟病菌免疫反应的miRNA调控网络

The rice-rice blast fungus phytopathosystem has become a model in the study of plant-fungus interactions because rice is one of the most important staple foods for most of the world’s population and rice blast is the most devastating disease for rice production. Current strategies for study on rice immunity against the blast fungus Magnaporthe oryzae are focused on the screening, identification and functional characterization of disease resistance genes. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression through silencing of target mRNAs in a sequence-specific manner. One miRNA is usually able to target multiple mRNAs. One miRNA is also regulated by multiple endogenous target mimics (eTMs) in which a decoy RNA bind to a miRNA via complementary sequences and therefore block the interaction between the miRNA and its authentic targets. Thus, there are regulatory networks composed of eTMs, miRNAs and miRNA targets that are involved in a variety of biological processes including plant innate immunity. However, up to date, it is not clear how many and which miRNAs are involved in rice immunity against the blast fungus. Previously, through deep sequencing small RNA libraries from susceptible and resistant lines, we identified a set of miRNAs that were differentially responsive to the infection of the blast fungus. Intriguingly, ectopic expression of either miR160a or miR398b leads to elevated resistance to the blast disease. To reveal the miRNA regulatory network that is involved in fine tuning rice immunity against the blast fungus, we propose the following experiments. First, through ectopic expression of miRNAs combining with over-expressing endogenous or artificial target mimics in transgenic plants, the regulatory roles of 27 miRNAs in rice immunity against the blast fungus will be characterized. Second, the function of more than 100 authentic miRNA targets will be analyzed through over- or down-regulation in transgenic plants. Third, the roles of miRNA pathway components, including some OsDCLs and OsAGOs, in rice immunity against the blast fungus will be analyzed. The relationship between eTMs and miRNAs, miRNAs and their targets, and their roles in rice immunity will be clarified. Finally, comparative transcriptome analyses will be conducted among several monogenic resistant lines, combining with quantitative real-time PCR analysis, the dynamic expression of eTMs and the miRNA targets will be revealed. Data from this proposed project will significantly contribute to our knowledge on rice immunity against the blast fungus and be helpful with engineering resistance to rice blast disease in breeding programs.

稻瘟病免疫机理一直是分子植物病理学的一个研究热点，传统思路是筛选鉴定克隆抗病基因，研究抗病基因的作用机理。miRNA通过调控靶基因行驶多种生物学功能，一个miRNA调控多个靶基因并受多个内源诱捕靶标的调控，由内源诱捕靶标、miRNA和靶基因构成miRNA调控网络。目前尚不清楚哪些miRNA调控稻瘟病免疫反应。前期研究中，我们发现一批miRNA可能与稻瘟病免疫反应有关，上调表达其中的两个均可明显提高对稻瘟病的抗性。本项目拟通过超表达miRNA或超表达内源及人工诱捕靶标来全面研究这批miRNA在稻瘟病免疫反应中的功能；同时，通过超表达或下调表达靶基因和miRNA路径元件基因来研究它们对稻瘟病免疫反应的影响，建立稻瘟病免疫调控的内源诱捕靶标—miRNA—靶基因网络。我们还将对多个稻瘟病抗病单基因系进行比较分析，验证miRNA调控网络，研究结果可为构筑稻瘟病抗病性和创制抗病育种新种质提供理论指导。

miRNA是一类长度为20至24个核苷酸的非编码RNA，通过碱基配对原则调控基因的表达。在前一项目中，通过分析抗、感材料对稻瘟菌侵染响应的差异，获得一批差异表达的miRNA。在此基础上，本项目分别对30个miRNA进行了研究，初步明确miR160、miR162、miR166、miR172、miR393、miR398、miR1320和miR1425等8个miRNA正向调控水稻对稻瘟菌的免疫应答，而miR156、miR159、miR164、miR167、miR168、miR169、miR171、miR396、miR435、miR439、miR444b.2、miR528、miR530、miR535、miR812、miR1432、miR1863、miR1871、miR1873、miR2869和miR24584等21个miRNA负向调控水稻对稻瘟菌的免疫应答；miR827的作用不明显。明确了miR156、miR162、miR164、miR166、miR167、miR168、miR396、miR398、miR812和miR1873等miRNA对稻瘟菌的免疫调控起作用的靶基因。其中miR396-GRFs调控模块在协调稻瘟菌免疫和产量性状上起关键作用。而且，miR168-AGO1调控模块同时协调对稻瘟菌的免疫、产量性状和生育期，该模块可能通过调控上述多个miRNA来发挥作用。此外，还鉴定到一个环RNA，即circR5g05160，该环RNA可能通过miR168-AGO1模块正向调控对稻瘟菌的免疫应答。因此，本项目鉴定到从circR5g05160到miR168-AGO1模块再到多个miRNA及其靶基因的分子调控网络，为改良水稻的抗瘟性、产量相关性状和生育期提供了具有精密调控作用的基因资源，对创制抗病、高产、优质水稻新种质有重要的指导意义和应用价值。