稻瘟菌Lon酶切修饰的C3HC-锌指蛋白在线粒体致病途径中的作用机理

The rice blast is a typical fungal disease caused by the rice blast fungus Magnaporthe oryzae. Mitochondrial pathogenicity pathway mediated by the ATP-dependent Lon protease (MAP1) has been confirmed to be essential for blast disease in our previous work. In the pathway, a mitochondrial C3HC type Zinc-finger containing protein (ZFC3), which is controlled by MAP1 and predicted as mitochondrial transcription factor is associated with the expression of mtDNA. Mitochondria are required for pathogenicity in plant fungal pathogens, but the molecular mechanisms for mtDNA transcriptional regulation remain to be further studied. To clarify the function and regulation mechanisms of the zinc-finger protein, this project will focus on the following four points: 1) to systemically analyze the expression patterns of protein-encoding genes of mtDNA, to establish the “RPO41-Mtf1” transcription system for initiation in vitro, and to confirm the transcription activity of ZFC3; 2) to check the mtDNA and mtRNA binding properties of ZFC3, and to identify the promoter or non-specific mtDNA sequences, and mtRNA sequences by ChIP and RIP (RNA-IP) approaches; 3) to check the interactions between ZFC3 and individual of Magnaporthe homologs of RPO41, MTFA or MTFB by using CoIP method, and to identify novel ZFC3-interacting proteins; 4) To check if ZFC3-interacting proteins are involved in pathogenicity. Taken together, the results above will help to verify the actions of the zinc-finger containing protein, and to reveal the mitochondrial pathway mediated by MAP1. Particularly, it will enrich the knowledge of mitochondrial transcription regulation machinery, and provide potential molecular targets for designing novel fungicide.

在稻瘟菌ATP-Lon蛋白酶（MAP1/Lon）介导的线粒体致病途径中， C3HC-锌指蛋白（ZFC3）是受MAP1/Lon酶切修饰的新型致病因子。基于ZFC3敲除突变体毒力下降、且线粒体基因表达下调的结果，推断ZFC3可能以线粒体转录因子或辅因子形式参与稻瘟菌致病过程。为探明ZFC3的作用机理，本项目拟开展如下研究： ①在全面分析线粒体基因结构和表达基础上，建立线粒体转录系统（RPO41-Mtf1），验证ZFC3在线粒体转录中的作用；②用ChIP技术分析ZFC3与线粒体DNA或RNA的互作，鉴定特异序列；③用CoIP和质谱分析技术鉴定ZFC3的互作蛋白；④进一步确认ZFC3-互作蛋白，并通过基因敲除和互补测定部分互作蛋白的致病功能。预期研究结果将揭示ZFC3在调控线粒体致病途径中的作用机理，丰富线粒体基因调控内容，填补丝状真菌线粒体基因转录研究的空白，为新型杀菌剂研制提供参考靶标。

由稻瘟菌侵染水稻造成的稻瘟病是一种重要作物真菌病害。MAP1/Lon作为一种多功能线粒体蛋白酶参与稻瘟菌致病调控。为解析Lon介导的致病途径，本项目针对MAP1/Lon互作蛋白C3HC-锌指蛋白（ZFC3）进行了功能研究。研究证实ZFC3定位于线粒体，通过调控ATP6、ATP8、COX1-3等线粒体基因表达而调控稻瘟菌的生长、发育和致病。此外，本项目还鉴定了ZFC3和MAP1/Lon的互作蛋白PrePL，证实PrePL受MAP1/Lon酶切调控，其特异性酶解产物Pc以分泌蛋白形式进入寄主，参与稻瘟菌的致病性发育。本研究结果丰富了线粒体基因调控内容，为新型杀菌剂研制提供参考靶标。