大豆孢囊线虫效应蛋白G20E03抑制植物免疫的分子机制

Soybean cyst nematode (SCN) is an important agricultural pathogen. Effectors secreted by SCN play an important role in the interaction between soybean and SCN. Secretory protein G20E03 was first identified in SCN, but its function and mechanism remain unknown. In our previous work, based on transcriptome analysis before and after SCN infecting soybean, the expression level of the G20E03 gene with significant difference was screened out. G20E03 has a signal peptide and no transmembrane domain. Moreover, it can inhibit the HR response in tobacco caused by Burkholderia glumae. Interfering with the expression level of the G20E03 gene significantly reduced the pathogenicity of SCN to soybean. In order to elucidate the pathogenesis of G20E03, this project intends to first study localization, secretion and transmembrane characteristics of G20E03 through subcellular localization, yeast secretion system and barley fusarium wilt system. Secondly, the overexpression test of this gene in soybean will be carried out to clarify the pathogenicity changes of SCN. Then transcriptome sequencing will be performed with the interference and overexpression lines to determine the signal pathway which the gene regulates in host. Finally, the interaction protein of G20E03 will be identified by yeast double hybridization and bimolecule fluorescence complementary system to illuminate the virulent mechanism of G20E03. The result will have the vital significance for the prevention and management of SCN.

大豆孢囊线虫(SCN)是重要农业病原物。由SCN分泌的效应蛋白在其侵染大豆过程中发挥重要作用。分泌蛋白G20E03最初在SCN中鉴定，但其功能和作用机制尚未明确。在前期工作中，基于SCN侵染大豆前后的转录组分析，筛选出差异显著表达的G20E03基因；G20E03有信号肽，不含跨膜结构域，能够抑制谷枯菌在烟草上形成的过敏性坏死反应；干扰该基因的表达显著降低了SCN对大豆的致病性。为阐明G20E03致病机制，本项目拟首先通过亚细胞定位、酵母分泌系统和大麦枯萎病菌系统研究G20E03的作用位点、分泌和跨膜特性；其次开展该基因在大豆内的超表达试验，明确超表达该基因对SCN致病性的影响；然后对该基因的干涉和超表达大豆株系进行转录组测序，确定该基因调控寄主的信号途径；最后利用酵母双杂交和双分子荧光互补系统鉴定G20E03的互作蛋白，阐明G20E03毒性作用机理，研究结果对于防治SCN具有重要意义。

大豆孢囊线虫是大豆生产上最重要的植物病原线虫之一，其食道腺分泌效应蛋白能抑制寄主的防卫反应。申请人前期研究已鉴定到一个大豆孢囊线虫效应蛋白G20E03, 与线虫的寄生相关，调控寄主植物的免疫反应。然而，G20E03与寄主互作的机理仍然未知。本项目采用亚细胞定位研究G20E03在寄主内的作用位点，明确其位于寄主的细胞膜上；由酵母分泌系统验证G20E03的信号肽具有分泌功能；在大豆中过量表达G20E03增加大豆了对线虫的敏感性，二龄幼虫侵染率和孢囊数均显著增加；采用酵母双杂交技术鉴定了效应蛋白G20E03的互作蛋白GmCYP71D9，通过BIFC体内实验证明G20E03可与大豆GmCYP71D9发生特异性互作；分别采用过表达和CRISPR/Cas9技术研究GmCYP71D9对线虫寄生能力的影响，转录组数据表明G20E03和CYP71D9共调控大豆活性氧物质相关基因的差异表达从而影响SCN对大豆的致病性。研究结果将为探究大豆孢囊线虫致病分子机理提供新的证据，为大豆孢囊线虫的绿色防控提供新靶标，对大豆孢囊线虫的抗性育种具有十分重要的理论和实践意义。