甜瓜Pvr抗病基因识别番木瓜环斑病毒信号的分子机制

Planting resistant cultivar is the most economic, effective and green method to control plant viral diseases. Papaya ringspot virus (PRSV) drastically damages the production of cucurbit crops. However, the main cultivars of cucurbit crops such as watermelon and muskmelon in the market cannot resist the infection of PRSV.The applicant has found Cucumis melo germplasm PI414732, which carrys a dominant resistance gene named after Pvr and produces local hypersensitive response to PRSV, can be resistant to the infection of PRSV. However, the avirulence (Avr) gene corresponding to Pvr and the molecular mechanism of Pvr gene recognizing PRSV Avr gene remain largely unknown. In this program, we plan to identify the Avr gene of PRSV using the expression system consisting of chimeric viral vectors or transient vectors, analyze the alternative splicing of Pvr gene by RNAseq and then define the transcription model and expression dynamic of Pvr gene, and elucidate the mechanism of Pvr recognizing PRSV Avr protein through Co-IP, BiFC ,YTHS, etc. The expected results will provide theoretical basis for the engineering and deployment of Pvr gene and give novel ideas to control other potyviruses.

种植抗病品种是防控作物病毒病害最经济有效且绿色环保的方法。番木瓜环斑病毒（Papaya ringspot virus，PRSV）严重危害瓜类作物的安全生产，但目前市场上的西瓜、甜瓜等葫芦科作物主栽品种都不抗PRSV。申请者发现携带显性抗病基因Pvr的甜瓜种质PI414732受PRSV侵染产生局部过敏性反应，能有效抵抗PRSV的侵染，而且Pvr基因至少存在两种转录形式。但Pvr所识别的PRSV无毒基因以及二者的识别机制尚不清楚。本项目拟利用嵌合病毒和植物瞬时表达系统鉴定Pvr基因对应的PRSV无毒基因，通过转录组测序分析Pvr基因的可变剪切模式，明确Pvr基因的转录形式和表达动态，通过免疫共沉淀、双分子荧光互补、酵母双杂交等技术解析二者的识别机制，为Pvr基因改良与应用提供理论基础，也为其它马铃薯Y病毒属病毒的防控提供新思路。

植物利用细胞内的核苷酸结合亮氨酸富集重复受体（NLR）对来直接或间接防卫病原效应因子并激活免疫防卫反应。该项目研究了两个基因连锁的瓜类NLR免疫受体Prv1及Fom1。这两个受体以共同合作的方式来识别番木瓜环斑病毒的核包涵体a蛋白酶（NIa-ProPRSV），有趣的是，Prv1的Toll/白细胞介素1受体（TIR）结构域上存在三个连续的NIa-ProPRSV识别的切割位点，而Prv1是一类非典型的TIR-NLR蛋白，在其亮氨酸富集重复（LRR）结构域之后还有一个额外的核苷酸结合（NB-ARC）结构域。NIa-ProPRSV切割Prv1的TIR结构域会激活Prv1/Fom1所介导的免疫反应。此外, 本研究还发现将一个位于Prv1-TIR结构域中三个连续NIa-ProPRSV切割位点之后的类NIa-ProPRSV切割位点中的精氨酸（R）替换为亮氨酸（L）后可以将Prv1/Fom1所介导的抗性拓展至另外两种马铃薯Y病毒属病毒。以上结果表明Prv1模拟了病毒蛋白酶底物的分子特征并提供了一个扩展其识别范围的界面。