小麦FtsH2蛋白与WYMV CP互作研究

 小麦黄花叶病毒(Wheat yellow mosaic virus, WYMV)是马铃薯Y病毒科大麦黄花叶病毒属成员,主要危害冬小麦。实验室前期以WYMV外壳蛋白(coat protein,CP)为诱饵,通过酵母双杂交筛选小麦cDNA文库,发现FtsH2蛋白部分片段与WYMV CP互作。FtsH2蛋白是AAA蛋白酶家族成员,参与植物叶绿体光损伤修复和类囊体发育进程。本研究利用酵母双杂交和双分子荧光互补技术进一步对FtsH2蛋白全长与WYMV CP进行互作验证,并鉴定互作结构域;利用荧光蛋白标记技术研究FtsH2蛋白与CP的亚细胞定位。实验结果表明FtsH2蛋白全长与CP互作;两者互作的关键结构域包含FtsH2蛋白低复杂区、跨膜区及AAA结构域和WYMV CP的中段区域(61-293 aa)。FtsH2蛋白单独表达时定位在细胞质、细胞核和叶绿体;CP单独表达时定位在细胞质;两者共同表达时亚细胞定位均没有发生明显变化,且主要共定位于细胞质中。WYMV CP与小麦FtsH2蛋白的互作可能会干扰植物绿叶体的发育和功能。本研究对了解WYMV的症状形成机制具有一定意义。

Study of the interaction between wheat FtsH2 and Wheat yellow mosaic virus CP

Wheat yellow mosaic virus (WYMV) is a member of the genus of Bymovirus and family of Potyviridae, mainly bring harms to the culture of winter wheat. In our previous work, the cDNA library of wheat was screened via yeast two-hybrid system, with the WYMV CP as bait protein, and the fragment of the wheat FtsH2 was identified as interacting with CP. FtsH2, a member of AAA (ATPase associated with diverse cellular activities) protease families, involves in the repair system of photodamage and the process of thylakoid development. In the present work, yeast two-hybrid system was further taken to demonstrate the interaction between entire FtsH2 and CP or their key domains; fluorescent proteins were also utilized to study the subcellular localization of FtsH2 and CP. The results confirmed the actual interaction between entire FtsH2 and CP; the FtsH2 fragment including a low complexity region, transmembrane region and the AAA domain and the CP_61-293 fragment were the key domains for their interaction. Alone expression of FtsH2 resulted in its localization at cytoplasm, nucleus and chloroplast; alone expression of CP resulted in its localization at cytoplasm; simultaneous expression of FtsH2 and CP did not change their subcellular localization and their co-localization was mainly occurred at cytoplasm. The interaction between WYMV CP and FtsH2 protein may interfere with the development and function of chloroplast, and our research is therefore useful for studying the mechanism of symptom formation of WYMV.