麦长管蚜唾液蛋白效应子鉴定及其作用机制

The effectors from aphid saliva are key factor to activate or suppress plant defense responses. The aim to understand the species and function of effectors in wheat aphid in the project, Sitobion avenae was chose as test target. Firstly, the candidate secreted salivary effectors are over-expressed in wheat leaves using type Ⅲ secretion system (T3SS). Observation of leaf symptom, histological staining and RT-qPCR are performed to detect the effects of candidate effectors over-expression on wheat defense responses. The effects of over-expressed effectors on aphid development, host choice and feeding behavior are also detected to determine the effectors involving in the interactions with wheat. The aphid effectors are silenced using artificial diet-mediated RNAi and then detect the effects of gene silencing on wheat defense to further confirm the roles of effectors. Aphid feeding induced cDNA library of wheat leaves are constructed and then Yeast Two-Hybrid System are performed to discover the target proteins of effectors in wheat, and further reveal the roles of effectors on aphid-wheat interactions. Subcellular localization of the effector proteins and wheat target proteins are also determined. Bimolecular fluorescence complementation(BiFC) are performed to further confirm the interaction of the effectors with their host targets in plant. This research will provide insight into the aphid-wheat interactions and new strategy for aphid biological control.

蚜虫唾液中的效应子蛋白是诱导/抑制植物防御反应的重要因子。为解析麦蚜唾液效应子种类及作用机制，本研究以麦长管蚜为对象，采用麦类作物的效应子筛选方法（即细菌Ⅲ型分泌系统T3SS），在小麦叶片中过表达麦长管蚜唾液分泌蛋白候选效应子，通过表型观察、组织染色及RT-qPCR检测对小麦防御反应，并结合室内生测及蚜虫取食刺吸电位EPG技术检测效应子过表达对麦长管蚜生命参数、选择及取食行为的影响，筛选鉴定效应子种类；通过人工饲料介导的RNAi技术沉默效应子，利用反向遗传学方法进一步验证效应子作用；构建麦长管蚜取食诱导的小麦cDNA文库，利用酵母双杂交技术筛选效应子在小麦叶片内的靶标蛋白，并对效应子及其靶标蛋白进行亚细胞定位，通过BiFC进一步验证其互作位点。明确蚜虫唾液中效应子及其作用机制，对蚜虫-小麦互作关系的解析及新型蚜虫绿色防控技术研发具有重要的理论和实践意义。

蚜虫在取食过程中可向植物细胞内分泌唾液，唾液中的效应子蛋白在调节植物免疫中具有重要作用。荻草谷网蚜（在我国其学名一直误用为“麦长管蚜”）为我国小麦生产上的重要害虫，解析荻草谷网蚜效应子功能，为阐明麦蚜-小麦互作机制及抗虫品种培育具有重要意义。本研究首先通过蛋白质组学鉴定到114个荻草谷网蚜唾液蛋白；根据同源序列选择Sm10, SmC002, SmGST1, SmCSP4作为候选效应子开展相关功能研究，通过酵母分泌系统发现，4个候选效应子基因重组转化酵母菌株YTK12均可分泌蔗糖酶，能在只含棉籽糖的YPRAA培养基正常生长，同时与2,3,5-氯化三苯基四氮唑发生红色显色反应，验证了其分泌功能。通过荧光假单胞杆菌介导的细菌Ⅲ型分泌系统，成功将Sm10、SmC002、SmGST1和SmCSP4传递到小麦叶片细胞中实现瞬时表达，苯胺蓝染色结果表明， Sm10与SmC002可显著降低小麦叶片中胼胝质的积累，而SmGST1和SmCSP4均可显著增加小麦叶片胼胝质的积累。进一步经RT-qPCR和LC-MS/MS检测相关防御基因表达量及植物激素含量变化，结果表明4种唾液蛋白均可显著提高小麦叶片内水杨酸（SA）途径防御基因PR-1和PAL表达量及SA含量，但降低了莉酸（JA）防御途径相关基因LOX、FAD表达量。通过室内生测研究发现，取食Sm10及SmC002过表达的小麦叶片，蚜虫产仔蚜量均显著升高。相反，取食SmGST1和SmCSP4过表达小麦叶片后，蚜虫存活率和产仔蚜量均显著下降。表明唾液蛋白Sm10和SmC002具有提高荻草谷网蚜适应性的作用，而SmGST1和SmCSP4则降低了蚜虫在小麦上的适应性。以SmCSP4作为研究对象，进一步通过酵母双杂交、双分子荧光互补（BIFC）、免疫共沉淀（Co-IP）等方法发现SmCSP4可与小麦转录因子TaWRKY76互作，利用酵母单杂交、双荧光素酶报告基因检测进一步表明TaWRKY76可激活水杨酸降解酶DMR6基因表达，而SmCSP4可抑制Ta WRKY76激活活性，进而引起小麦植株内水杨酸含量增加，提高寄主抗蚜性，阐明了荻草谷网蚜效应子蛋白SmCSP4激发小麦免疫分子机制。研究结果对蚜虫-小麦互作关系的解析及新型蚜虫绿色防控技术研发具有重要的理论和实践意义。