拟南芥光信号转录因子FAR1与EDS1互作调控植物免疫的机理研究

Light has a profound influence on plant immunity, and full activation of the defense response to pathogens is often dependent on photoreceptors; however, the underlying mechanism is elusive. Our previous study demonstrate that loss of two key transcription factors of the phytochrome A signaling pathway, FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) and FAR-RED IMPAIRED RESPONSE 1 (FAR1), caused the over-accumulation of salicylic acid (SA), activated defense gene expression, and resulted in constitutive expression of defense mechanisms. These defects of the fhy3 far1 mutant were largely restored by mutation of PHYTOALEXIN DEFICIENT 4 or SALICYLIC-ACID-INDUCTION DEFICIENT 2, thus revealing that FHY3 and FAR1 negatively regulate plant immunity. Recently, our yeast two-hybrid and bimolecular fluorescence complementation experiments showed that FAR1 interacts with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Transcriptional activity analysis demonstrated that EDS1 functioned as a transcriptional corepressor. These findings establish the molecular linkage between light signaling and plant immunity. In this study, we propose to further analyze the molecular relevance of the FAR1-EDS1 interaction, and to elucidate the mechanism of FAR1 in regulating plant immune. Furthermore, we will test whether FAR1 help EDS1 to transfer between the cytoplasm and nucleus to regulate plant immune responses, and reveal the molecular mechanism of nuclear EDS1 in driving transcriptional reprogramming during plant defense. We believe that this study will provide experimental evidence and gain new insight into the functional relationship between light signaling and plant immune.

光对植物免疫具有重要影响，免疫应答的全面激活往往依赖于光受体，而人们对其调控机制的认识却知之甚少。我们过去的研究发现光敏信号转录因子FHY3/FAR1功能缺失引起水杨酸累积、诱导免疫基因表达、引发植物体产生组成型免疫应答；而免疫信号因子突变体pad4和sid2能够恢复fhy3far1的上述性状，表明FHY3/FAR1与植物免疫间的密切关联。近阶段，我们通过体外酵母双杂交和体内双分子荧光互补实验证实FAR1能够与免疫信号关键因子EDS1相互作用；而且转录功能分析显示EDS1能部分抑制FHY3和FAR1的转录活性，这为研究光信号调控植物免疫建立了重要“桥梁”。本项目拟深入分析FAR1与EDS1互作的生物学意义；研究FAR1调控植物免疫的分子机制；分析FAR1对EDS1蛋白定位及其生理功能的影响，探究EDS1调节核内基因表达的分子机理，为揭示光信号与植物免疫的相互关联提供实验证据和理论基础。

植物的生长发育与免疫反应是植物体内两种拮抗的生物学过程，维持二者间的动态平衡是植物生存的关键，但其具体的调控机制仍然鲜有报道。光对植物的生长与植物免疫具有重要影响。本项目研究发现，植物光敏信号转录因子FHY3/FAR1能够响应病原菌信号，通过与免疫信号因子EDS1直接相互作用，对EDS1蛋白核-质分布进行调控，促进EDS1蛋白的核内积累，参与植物免疫应答的调控。同时，EDS1蛋白能够部分抑制转录因子FHY3/FAR1对下游靶基因的结合与激活，重塑FHY3/FAR1的转录模块，进而平衡植物的生长与免疫应答。首先通过体外酵母双杂交实验（H2Y）、体内双分子荧光互补（BiFC）、免疫共沉淀（Co-IP）以及荧光素互补实验（LCI），表明FAR1能够与EDS1直接相互作用。接着，对二者互作的生物学意义进行研究。利用“农杆菌—烟草瞬时表达体系”，亚细胞定位以及蛋白免疫印迹实验显示，FHY3/FAR1能够促进EDS1-GFP融合蛋白的核内积累；同时，对FHY3/FAR1下游靶基因的转录活性分析显示，EDS1能够抑制FHY3/FAR1下游靶基因HEMB1的表达，染色质免疫共沉淀实验（ChIP）也是显示EDS1可抑制FHY3/FAR1对HEMB1的结合，表明EDS1可抑制转录因子FHY3/FAR1的活性。此外，对EDS1下游基因SID2的表达分析显示，FAR1可抑制EDS1对下游基因的激活。转录组数据显示，fhy3/far1内受病原菌上调表达基因中有86%是依赖于EDS1抗病信号通路所诱导表达的，而且eds1和fhy3/far1的转录组对比分析也显示FHY3/FAR1可大规模抑制EDS1诱导基因的表达，表明二者对下游基因的修饰存在拮抗功能。我们推测FAR1与EDS1的互作可能作为植物体维持自身生长发育与免疫反应平衡的关键节点。本研究为揭示植物平衡生长与免疫的精细作用机理奠定了坚实的工作基础，对优化植物的生存具有指导意义。