小麦赤霉菌关键效应蛋白CEP2的功能及其作用机制研究

Fusarium head blight (FHB), caused by Fusarium graminearum, is a great threat to the yields and quality of wheat, barley, and maize that directly related to the survival and health of human beings. F. graminearum can generate mycotoxins, which contaminate the infected kernels and are harmful to humans and livestock. In China, recent epidemics occur frequently and they are extremely destructive to our crops. Due to the lack of resistant cultivars and no-tillage cultivation, the re-emergence of FHB can be worse. Study on the basis of wheat - F. graminearum interaction is therefore urgently required. Effectors play key roles in host - pathogen interaction, however, the functions of F. graminearum effectors are largely unknown. In our previous study, we identified 8 genes that are important for F. graminearum - wheat interaction, via genome-wide comparative analysis of the strains isolated from several countries. Knockout of these genes and subsequent infection assays on wheat revealed that CEP2 is critical for the full virulence of F. graminearum. Further analysis showed that CEP2 is a cytoplasm effector containing DEER-like sequence in the N-terminal. By yeast two hybrid and bimolecular fluorescence complementation experiments, we have confirmed that SGT1 is a candidate host target of CEP2. SGT1 is shown important for both nonhost resistance related cell death and R gene triggered cell death. In this project, we focus on dissecting the functions of N-terminal and C-terminal of CEP2, the mechanism of CEP2 - SGT1 interaction, and the molecular basis of how CEP2 functions as a virulence factor via manipulating host SGT1. This project is expected to reveal the function of a key effector involved in wheat - F. graminearum interaction, and provide theory guideline to the prevention and control of FHB.

赤霉病是小麦、大麦和玉米等禾谷类作物上最严重的病害之一，在导致产量损失的同时还产生多种对人畜有害的真菌毒素，对我国粮食生产和食品安全造成极大威胁。明确赤霉菌与寄主互作的分子机理可为作物抗病遗传改良提供理论基础。效应蛋白是植物与病原互作的关键蛋白，当前对赤霉菌效应蛋白的认知非常有限。前期研究中，申请人通过重测序和比较基因组学鉴定到8个赤霉菌候选效应蛋白基因，并通过基因敲除分析证明其中CEP2是赤霉菌侵染小麦的必需基因。进一步工作表明CEP2是一个N端含有DEER-like结构域的胞内效应蛋白。酵母双杂交和双分子荧光互补实验显示CEP2可与小麦细胞坏死相关蛋白SGT1互作。本项目拟进一步解析CEP2 N端和C端的功能，明确CEP2与候选靶标SGT1的互作机理，并探究CEP2如何通过SGT1调控植物的防卫反应。研究结果有助于揭示赤霉菌效应蛋白在病原与寄主互作中的作用，为赤霉菌的防控提供理论基础。

赤霉病是小麦、大麦和玉米等禾谷类作物最严重的真菌病害之一，其直接危害作物穗部，不仅引起严重的产量损失，还可导致籽粒中真菌毒素超标，对我国粮食生产和食品安全造成极大的威胁。明确赤霉菌与寄主互作的分子机理可为作物抗病性的遗传改良提供理论基础。效应蛋白是病原菌与植物互作的关键蛋白，然而我们当前对赤霉菌效应蛋白的认知非常有限。本项目鉴定到赤霉菌一个关键效应蛋白基因CEP2（FGRRES_11564，OSP24），其缺失导致病原菌在小麦穗部的侵染与扩展严重受阻。携有核定位信号的融合效应蛋白Cep2-GFP-NLS可在侵染时进入寄主植物细胞核内，表明Cep2是一个胞内效应蛋白。烟草瞬时表达实验表明，Cep2可以抑制Bax和Inf1引发的植物细胞坏死，表明Cep2可以干预植物基础防卫反应。为了探究Cep2在植物细胞内的功能，我们通过酵母双杂交筛选，双分子荧光互补和免疫共沉积验证，发现赤霉菌Cep2可与小麦TaSnRK1和TaSgt1互作。RNA-seq分析发现许多与植物能量代谢、环境互作和细胞防卫反应相关的基因在CEP2敲除突变体侵染小麦时上调，这与SnRK1的功能相一致，我们因此认为TaSnRK1是赤霉菌效应蛋白Cep2的主要靶标蛋白。为了研究TaSnRK1是否参与小麦对赤霉病的抗性，我们构建了该基因的沉默植株和过表达植株。接种分析发现TaSnRK1沉默植株对赤霉病抗性减弱，而TaSnRK1过表达植株对赤霉病抗性增强，表明TaSnRK1是小麦对赤霉病抗性的一个正调控子。为了研究赤霉菌效应蛋白Cep2是如何操纵小麦TaSnRK1蛋白的，我们比较了cep2敲除突变体和PH1侵染组织总蛋白对TaSnRK1的影响，发现Cep2可在侵染过程中加速TaSnRK1的降解。进一步研究发现Cep2加速TaSnRK1的降解需要小麦26S蛋白酶体的参与。综上所述，该项目的研究结果表明小麦赤霉菌关键效应蛋白Cep2可通过靶向并加速TaSnRK1的降解来促进小麦感病。该研究发现了小麦赤霉菌致病的新机制，并为赤霉病新型防控策略的开发提供了新的理论依据。.

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