禾谷镰刀菌DON毒素合成相关G蛋白偶联受体的作用机理

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most destructive diseases of wheat. In addition to severe yield losses and grain quality reduction, F. graminearum produces trichothecene mycotoxin deoxynivalenol (DON) in infested grains which is harmful to human health. Trichothecene (TRI) synthesis gene expression was detected as early as 12 hpi in infection cushions or penetration pegs. However, it remains unclear how F. graminearum recognizes signals from the host to regulate DON biosynthesis. G protein-coupled receptors (GPCRs) are considered to be important for signal recognition. In the preliminary study, we functionally characterized all the 85 GPCR genes and found three of them were important for DON production. In this study, we aim to further characterize the role of these three GPCR genes in DON synthesis and identify their downstream signal transduction pathways. Regarding functional redundancy between GPCRs, we will characterize their functional relationship by generating double or triple mutants and mutants deleted of multiple GPCR genes with similar expression profiles or structures with these three GPCRs, then assayed for their defects in DON biosynthesis. To determine the roles of these three GPCRs in the recognition to host-derived signals, we will assay for their functions in the recognition of known DON inducing plant compounds and other novel DON-stimulating factors in wheat floral tissues. The specific ligand-receptor relationship between a fungal GPCR and a DON inducing host factor will be further characterized with the yeast expression system. Overall, results from the proposed project will not only improve our understanding of host recognition and DON regulation, but also lead to the development of novel strategies for reducing mycotoxin contamination in wheat.

禾谷镰刀菌引起的赤霉病不仅是小麦上的重大致灾病害，而且产生的DON毒素严重威胁人畜健康。已知在禾谷镰刀菌侵染早期即可检测到DON合成基因的表达，然而禾谷镰刀菌如何识别宿主信号并启动DON合成的机制仍不清楚。G蛋白偶联受体（GPCR）是负责信号识别的重要膜受体和药物靶标，项目前期通过敲除禾谷镰刀菌85个GPCR基因，鉴定到其中3个与DON合成相关（G6、G22和G59）。我们拟对这3个DON合成相关GPCR以及与它们在序列和表达模式上类似的GPCR进行多基因敲除，分析这些GPCR在DON合成中的功能及相互关系，并明确其介导的下游信号通路。为了研究DON合成相关GPCR在寄主信号识别中的作用，我们将从寄主组织中分离并筛选出DON合成诱导物，进一步确定病原真菌GPCR和寄主信号物质的对应关系。研究结果对明确禾谷镰刀菌的寄主识别和产毒调控机制具有重要价值，为防治小麦赤霉病毒素污染提供候选药物靶标。

小麦赤霉病由禾谷镰刀菌引起，是威胁我国粮食产量和食品安全的重大真菌病害。禾谷镰刀菌侵染反应的起始以及DON毒素合成的发生都依赖于病菌对外界环境和寄主物质的识别。鉴定侵染和产毒过程中的关键膜受体并揭示其介导的信号传导途径，对认识禾谷镰刀菌的侵染和产毒机制至关重要。通过对禾谷镰刀菌所有G蛋白偶联受体的系统敲除，其中3个突变体致病力严重降低，其对应的G蛋白偶联受体被命名为Giv1，Giv2和Giv3。对这三个G蛋白偶联受体进行进一步的功能分析，发现Giv1主要负责侵染结构形成时期的分子识别，Giv2则在穿透植物细胞时发挥重要作用。而Giv2和Giv3还介导了侵染菌丝的分化和扩展过程。通过磷酸化实验鉴定到cAMP-PKA和Gpmk1信号通路是G蛋白偶联受体的下游传导途径。通过对G蛋白偶联受体进行进化关系和表达模式的分析，发现禾谷镰刀菌的G蛋白偶联受体中存在阶段特异性表达的亚家族，且该亚家族成员在其他病原真菌中鲜有同源蛋白，基于此，提出病原真菌G蛋白偶联受体的适应性进化理论。除此之外，项目还针对G蛋白偶联受体的下游信号通路进行研究，通过构建MAPK激酶的三敲突变体，获得了一个完全不含任何MAPK激酶的真菌菌株，发现该菌株不仅生长发育受阻，致病力严重降低，且无法合成DON毒素。进一步对三个MAPK激酶在DON毒素合成过程中的功能进行探究，其中gpmk1突变体产毒能力下降，而mgv1和Fghog1突变体中，TRI基因表达严重降低，产毒能力完全丧失。进一步说明MAPK信号通路对DON毒素合成的关键调控作用。除此之外，项目还发现组蛋白乙酰转移酶FgHat1通过调控TRI基因表达及产毒时期的细胞分化以调控DON毒素合成，在表观修饰和DON毒素合成之间建立了联系。通过本项目的研究，鉴定了禾谷镰刀菌侵染致病及产毒过程中的关键G蛋白偶联受体，揭示了其下游信号传导途径，并探明了信号传导及表观修饰在DON毒素合成过程中的重要作用及调控方式。研究不仅拓展了对禾谷镰刀菌侵染和产毒分子基础的认识，同时也为防病抑毒药物的研发提供了候选基因资源及靶点。