稻瘟病菌转录因子MoAp1介导的基因调控网络生物学功能解析

Reactive oxygen species (ROS) are active in the critical zone where pathogens and plants interaction happens and play important roles in defense mechanisms. Recent studies demonstrated that it is necessary for full virulence of the rice blast fungus Magnaporthe oryzae by suppressing plant-generated ROS during early stages of infection to impair ROS-mediated rice defense. Our study revealed that MoAp1, a transcription factor as a positive ROS-stress sensor and regulated gene expression to detoxify rice-generated ROS and eventually blocked ROS-triggered plant defense. Thus, research on gene regulatory network mediated by MoAp1 could help to elucidate molecular aspects of pathogenicity of M. oryzae. .One of our specific objectives is to characterize biological function of MoAp1-binding proteins and evaluate the effect of these proteins on MoAp1-GFP localization after identified by co-immunoprecipitation in M. oryzae. The other specific object is to use Chromatin Immunoprecipitation followed by Illumina sequencing (ChIP-Seq) to identify the MoAp1 binding motifs and evaluate the putative MoAp1 binding motifs by electro mobility shift assays (EMSA). MoAp1 binding motifs as well as our previous RNA-sequencing data of the Moap1 mutant will used to identify potential target genes regulated by MoAp1. Finally our specific objective is to analyze the potential targets regulated by MoAp1 by bioinformatics and fully characterize the biological funtion of some target genes by gene knock-out strategy in M. oryzae. These studies will help us to predict potential MoAp1-mediated gene regulatory network and elucidate the roles of this network in response to ROS stress, growth, development and pathogenicity of M. oryzae. .Taken together, our aim is to enhance understanding of molecular basis of rice blast fungus pathogenesis, and allow us to design new disease control strategies for this phytophathogen and other fungi.

植物与病菌互作时，植物细胞产生的活性氧在植物防卫中具有非常重要的作用。已证明稻瘟病菌要成功侵染水稻，必须清除互作早期水稻细胞产生的活性氧。我们前期研究发现转录因子MoAp1作为活性氧胁迫的感受器，调控稻瘟病菌一系列基因的表达，清除活性氧，从而抑制其介导的防卫反应。因此解析MoAp1调控的基因网络有助于揭示该病菌的致病分子机制。本项目拟采用co-IP技术鉴定MoAp1的结合蛋白，并鉴定其生物学功能及其对MoAp1定位的影响。采用染色体免疫共沉淀技术鉴定MoAp1在该病菌中的DNA结合序列，结合项目组已有的转录组数据，鉴定MoAp1的靶基因，并揭示其生物学功能，从而提出MoAp1调控的基因网络，解析该网络在稻瘟病菌应答活性氧胁迫、及生长发育和致病中的分子机制。上述工作不仅对揭示MoAp1在该病菌生长发育及致病中的分子机理具有重要的理论价值，而且对设计以网络为靶标的病害控制策略具有重要指导意义。

稻瘟病菌引起的稻瘟病是水稻上最重要的病害。病菌必须清除其与水稻互作早期水稻中积累的活性氧(ROS)与克服ROS诱发的免疫反应，但病菌如何清除ROS仍不清楚。项目组前期研究发现MoAp1是调控稻瘟病菌生长发育、致病及对 ROS 应答的一个重要的转录因子。本项目深入研究了该转录因子MoAp1介导的基因网络如何调控病菌清除寄主活性氧，如何调控自身的发育。系统解析了bZIP家族蛋白在稻瘟病菌中的生物学功能，发现bZIP家族蛋白在病菌生长发育及致病中的调控作用，其中MoHac1和MoMetR是重要的致病相关因子。采用co-IP与蛋白质谱测序技术鉴定到42个MoAp1的结合蛋白，发现小G蛋白MoRas2和MoAp1是相互作用，其他互作蛋白正在进一步深入分析。采用转录组学方法发现1200个受MoAp1调控的基因，其中上调表达基因497个（460个有AP1结合位点），下调表达682个（658个有AP1结合位点）。系统解析了MoAp1调控蛋白MoGti1和MoPac2的生物学功能。发现MoGti1调控稻瘟病菌的生长、产孢和致病性；而MoPac2缺失突变体在侵染过程中能够引起水稻强烈的防卫反应，致病性显著降低。表明MoAp1通过调控Gti1/Pac2家族蛋白的表达，参与稻瘟病菌的生长，产孢及致病过程。进一步鉴定到一系列受MoAp1调控的靶蛋白。采用凝胶迁移实验（EMSA）证明MoAp1可分别与MoYCP4和MoTRX2启动子区域结合，表明MoYcp4和MoTrx2是受MoAp1调控的靶蛋白。.对MoYCP4和MoTRX2分别进行敲除突变发现，MoYcp4参与调控病菌的生长、产孢，附着胞膨压及致病过程。MoTrx2通过参与清除寄主体内的活性氧，抑制活性氧诱发的免疫反应，帮助病菌侵染水稻，引起水稻发病，MoTrx2也参与产孢和硫源同化等生物学过程。发现蛋白磷酸酶MoYvh1在遗传上作用于转录因子MoAp1上游，正调控MoAp1和磷酸二酯酶MoPdeH的表达，参与病菌的生长、产孢、活性氧清除及致病力等生物学过程。上述研究结果揭示转录因子 MoAp1介导的对稻瘟病菌生长发育及致病过程调控的基因网络，对认识水稻及其他重要作物的病原真菌的致病机理具有重要的借鉴意义；同时对开发以转录因子 MoAp1相关的蛋白为靶标的新型杀菌剂，设计高效、低毒的真菌病害控制策略具有重要的实践指导意义。