柑橘衰退病毒编码的RNA沉默抑制子p20与柑橘蛋白CuCOR15的互作机制

Citrus tristeza virus (CTV) is the most serious viral pathogen of citrus plants. The p20 protein encoded by CTV is an important viral suppressor of RNA silencing. Previously, our studies revealed the direct interaction between a citrus unshiu cold-regulated 15 kDa protein (CuCOR15) and the p20 protein, which could reduce the suppression activity of p20. In this project, we will perform on subcellar localization of the two proteins during their interaction, study the functions of their interaction on the regulation for host gene expression, identify host components joining into the complex formed by citrus protein CuCOR15 and CTV protein p20, which eventually reveal the roles of the interaction between p20 and CuCOR15 in suppressing RNA silencing. To understand the functions of CuCOR15 on CTV pathogenicity, Nicotiana benthamiana plants over- or down-expressing CuCOR15 will be created and the effect of the expression levels of CuCOR15 on the RNA silencing efficiency of p20 and the CTV replication will be evaluated. After the project is fulfilled, for the first time, the mechanism for suppression RNA silencing of the CTV p20 protein will be revealed at protein-protein interaction level. The obtained results will provide valuable information for further understanding the molecular mechanism of clostero-RNA virus infection in plants. In practice view, it can contribut a fundamental base for discovering new host genes to be resistant to the virus and exploring gene inferring strategies for the control of plant virus diseases. Therefore, the performance of the project is theoretically and practically important.

柑橘衰退病毒（CTV）是严重危害柑橘的病原，CTV编码的p20是重要的RNA沉默抑制子，前期研究发现柑橘的脱水素类蛋白CuCOR15可与p20直接互作并影响p20的沉默抑制活性。本项目将研究这两种蛋白互作的亚细胞定位特点和对寄主基因表达调控作用，鉴定参与二者互作复合体形成的寄主蛋白组分，明确二者互作在RNA沉默中的作用；构建超表达和沉默CuCOR15的本氏烟植株，分析该蛋白表达水平对CTV致病性的影响，解析该蛋白在p20抑制RNA沉默和寄主抵抗CTV侵染中的作用。项目的完成将首次从蛋白质互作特点上解析CTV的p20在介导抑制RNA沉默中的作用机制，在理论上可为进一步揭示长线型RNA病毒侵染植物的分子机制提供重要信息，在实践上可为发掘新的抗病毒基因资源、探索基因干扰策略控制病毒病提供参考依据并奠定基础，具有重要的理论意义和潜在应用价值。

柑橘衰退病毒（CTV）是严重危害柑橘的病原，CTV编码的p20,p23和CP是重要的RNA沉默抑制子，明确这些蛋白的作用机理，可以为抗病毒资源挖掘及改良提供重要参考信息。目前已完成原计划各项研究任务，取得新的研究成果。明确了CTV编码p20 的RNA沉默抑制效果以及该蛋白与寄主蛋白CuCOR15和 NbSGS3的互作特点；揭示了这些蛋白的亚细胞共定位特点及其与p20 的RNA沉默抑制作用的关系；比较分析发现p20 和 CuCOR15 的瞬时表达对烟植株RNA 沉默途径中关键基因表达水平具有不同影响；构建了CuCOR15超表达和基因编辑本氏烟；发现p20可激发寄主植物的细胞自噬，导致NbSGS3降解，从而抑制寄主植物的RNA沉默作用；CuCOR15通过与NbSGS3和p20互作，降低NbSGS3的降解，抑制病毒复制，在抗CTV中发挥作用。项目实施过程中在研究内容上有所拓宽，分析了CTV编码蛋白p23与柑橘FK506结合蛋白FKBP17-2的互作特性，发现二者互作可能与该病毒编码CP的胞内转运相关；明确了p20 与寄主蛋白 PP2C 的互作及共定位特点及p20磷酸化与其沉默抑制活性的关系。