禾谷镰刀菌SR蛋白调控有性生殖和致病性的分子机制

In human, SR protein-specific kinase SRPK1 regulates RNA splicing by phosphorylating SR proteins and plays an essential role in cellular differentiation and organs development. In filamentous fungi, RNA splicing was found in diverse development stages, but little is known about its regulatory mechanism. Previous studies indicated that the only SR protein-specific kinase (FgSrpk1) from Fusarium graminearum may regulate sexual reproduction, DON production and pathogenicity by modulating RNA splicing and gene transcription. Recently, we found that two SR protein FgHrb1 and FgSrp1 are also involved in sexual reproduction, DON production and pathogenicity, and can interact with FgSrpk1. In this study, we will identify the genes whose RNA splicing or transcriptional level is regulated by SR protein. Subsequently, we will try to identify SR protein-associated RNA sequence and proteins including splicing factors and components of spliceosome, and detect whether SR proteins are phosphorylated by FgSrpk1 or other kinases. Our goal is to determine the molecular mechanism of SR proteins in regulating sexual reproduction, DON production and pathogenicity in F. graminearum. Over all, results for proposed project will be helpful not only to better understand the pathogenesis of Fusarium head blight but also to provide a novel drug target for control of the Fusarium head blight.

人体SR蛋白特异激酶SRPK1通过磷酸化SR蛋白调控了RNA剪接，进而在细胞分化、器官发育等过程中扮演了重要的角色。在丝状真菌的不同发育时期也存在RNA剪接现象，但是对其调控机制还不太清楚。前期研究工作显示：禾谷镰刀菌中唯一的SR蛋白特异激酶FgSrpk1能通过调控RNA剪接和基因转录影响有性生殖、产毒和致病力。近期又发现SR蛋白FgHrb1和FgSrp1也都参与了有性生殖、产毒和致病性且与FgSrpk1互作。本项目拟以FgHrb1和FgSrp1为研究对象进一步寻找RNA剪接和转录水平受SR蛋白调控的靶基因，鉴定出SR蛋白特异结合的RNA序列、剪接因子以及剪接体组分等蛋白，检测SR蛋白是否被FgSrpk1或其它蛋白激酶激活，从而最终揭示SR蛋白调控禾谷镰刀菌有性生殖、产毒和致病力的分子机制。本研究将加深我们对禾谷镰刀菌致病机制的认识，并为小麦赤霉病的防治提供新的药物靶标。

由禾谷镰刀菌引起的小麦赤霉病是一种毁灭性的小麦真菌病害，在世界范围内对小麦生产造成了巨大的破坏。其不仅造成小麦产量的严重损失，还会产生大量真菌毒素对食品安全构成极大的威胁。明确禾谷镰刀菌的致病机理，可以为该病害的防控提供重要的理论基础。本项目鉴定到一个对RNA剪接和基因转录具有重要调控作用的SR的蛋白FgHrb1，该基因缺失不但会影响病原菌的营养生长、无性和有性生殖，还会导致病原菌在小麦穗上侵染结构的形成以及侵染菌丝的扩展。共聚焦显微镜观察结果显示，FgHrb1主要在细胞核中发挥作用，而其中的RS结构域而非RRM结构域在其核质转运过程中扮演了重要的角色。另外我们还发现FgHrb1的三个RRM结构域中，RRM3对于FgHrb1的功能具有更重要的作用。RNA-seq数据分析显示，FgHRB1基因的缺失不但改变了许多下游基因RNA的可变剪接效率，还影响了许多基因的转录水平，这其中包含许多对禾谷镰刀菌生长发育、DON毒素合成和致病十分重要的基因。为了进一步探究FgHrb1的功能，通过酵母双杂交和双分子荧光互补证明了禾谷镰刀菌的FgHrb1可以与蛋白激酶Srk1和核转运蛋白FgMtr10互作，暗示Srk1可能介导了FgHrb1的磷酸化修饰。然而Srk1并未参与FgHrb1蛋白的亚细胞定位，推测Srk1可能影响了FgHrb1与其它靶蛋白或者靶标RNA的互作。本研究结果进一步加深我们对禾谷镰刀菌致病机制的认识，有望为小麦赤霉病的防治提供新的作用靶标。