小麦蓝矮植原体RNA沉默抑制子的功能及其作用机理研究

Wheat blue dwarf (WBD) disease is a new destructive disease, which frequently appears in the winter wheat of arid areas of Northwest China and results in significant yield losses. However, it is still unclear about the virulence factors and pathogenesis, which hinder the effective prevention of WBD disease. In previous studies, we have sequenced the complete genome of WBD phytoplasma, and identified SWP16 as a RNA suppressor through transient expression assays in Nicotiana benthamiana plants from 37 phytoplasma secreted proteins, which provide the groundwork for identification of virulence factors to better understand the pathogenic mechanisms. So that, we will focus on the following studies: First, functional analysis of secreted protein SWP16 interaction between wheat and phytoplasma by gene overexpression and silencing techniques; Second, identification of wheat target protein interacting with SWP16 using Y2H, Co-IP and BiFC as well as function of wheat target protein via transgene and VIGS; Third, the molecular mechanism of SWP16 as RNA silencing suppressors. Through the above study, the work will provide new approaches for WBD disease control.

小麦蓝矮病（Wheat blue dwarf, WBD）是我国小麦上一种新的危险性病害，由植原体侵染引起。目前，该病原的致病因子与致病机理尚不明确。我们前期首次完成了我国小麦蓝矮植原体的全基因组，从37个分泌蛋白中筛选获得了可以系统抑制RNA沉默的分泌蛋白SWP16。为揭示植原体SWP16蛋白抑制RNA沉默的作用机理，本课题拟采用基因过表达和基因沉默技术研究SWP16在植原体与小麦互作过程中的功能；采用Y2H、Co-IP和BiFC方法鉴定SWP16与小麦的互作靶标蛋白；通过转基因和VIGS方法研究靶标蛋白的功能；通过深度测序分析小麦中SWP16抑制RNA沉默的miRNA表达谱，鉴定关键差异miRNA的靶基因，并研究靶基因的功能，以期明确SWP16抑制RNA沉默的机理,为植原体病害的防控探索新的途径，具有重要的科学意义与实践应用价值。

小麦蓝矮病（Wheat blue dwarf, WBD）是我国小麦上一种重要病害，由植原体（Phytoplasma）侵染引起。目前，国际上对植原体如何攻克寄主的防御系统，入侵寄主的机理尚不清楚，是植原体学（Phytoplasmology）研究的热点问题。前期，实验室首次完成了我国小麦蓝矮植原体（‘Candidatus Phytoplasma tritici’）的全基因组草图绘制，在此基础上注释了37个分泌蛋白，并从植原体编码的37个分泌蛋白筛选获得了一个具有RNA沉默抑制子（RNA suppressor）功能的效应蛋白SWP16，可以抑制寄主对其基因的沉默，进行反防御，以达到侵染寄主植物的目的。本课题研究揭示了植原体分泌的效应蛋白SWP16与寄主有丝分裂S-期激酶相关蛋白1（Skp1）的Cullin-1结合结构域互作，影响植物内源 miRNA 积累和 RNA 沉默信号通过维管束韧皮部胞间连丝的传导过程，从而抑制寄主 RNA 沉默反应，促进WBD植原体顺利侵染寄主细胞。另外，研究发现植原体效应蛋白SWP16也能够促进马铃薯X病毒（PVX）侵染寄主。综上所述，本研究首次在国际上报道了植原体RNA沉默抑制子及其与寄主蛋白互作的分子机理，期望通过编辑寄主Skp1等基因，影响植原体侵染以及致病的过程，本成果不仅具有重要的理论科学意义，而且将为植原体病害防治提供了一条新途径。