拟南芥DCL4介导、不依赖DRB4的新抗病毒RNA沉默分子机制研究

RNA silencing is an important antiviral mechanism in plants. In Arabidopsis Dicer-like (DCL), dsRNA-binding proteins (DRBs), and Argonaute proteins (AGOs) are important proteins in RNA silencing signaling pathway. Our preliminary data shows a novel DCL4-initiated antiviral RNA silencing pathway which is independent of DRB4 protein in Arabidopsis while the molecular details of this novel RNA silencing pathway is still unclear. This specific objective of the proposed research is to identify DRB proteins that function together with DCL4 in the absence of DRB4 and whether AGO1, AGO2, AGO7 participate in the novel DCL4-initiated antiviral RNA silencing pathway which is independent of DRB4 protein. To achieve the objective the proposal employs molecular biology methods as well as molecular genetics research process to determine the following questions: ① whether DRB1 protein interact with DCL4 protein as well as whether it can recruit Turnip crinkle virus （TCV）in vivo; ② what kind of vsiRNA could be recruited by AGO1, AGO2 and AGO7 respectively in Aribidopsis thaliana which is absent of DCL2 and DRB4; ③ the viral small interference RNA triggered by virus induced gene silencing vector CPB-CC-PDS in Aribidopsis thaliana which is in absent of DCL2, DRB4 as well as DRB1, AGO1, AGO2 or AGO7 respectively; ④ the viral small interference RNA triggered by virus induced gene silencing vector CPB-CC-PDS in Aribidopsis thaliana whose DRB1, AGO1, AGO2 or AGO7 has been complemented through gene transformation respectively while remain in absent of DCL2 and DRB4.The proposed research is innovative because it aspires to unravel a unique antiviral defense mechanism in plants that is initiated by DCL4 while independent of DRB4 protein. Our proposed studies are significant because, upon their completion, we will have learned the molecular mechanism of antiviral silencing pathway initiated by DCL4 while independent of DRB4 protein. It will not only deepen our fundamental understanding of the regulation of RNA silencing, but also provide for a more in-depth grasp of the complex host defense mechanisms directed against viral pathogens.

RNA沉默是植物重要的抗病毒防御机制，DCL、DRB和AGO都是RNA沉默信号途径中的重要蛋白。在前期研究中首次发现拟南芥存在一条DCL4介导、但不依赖于DRB4的抗病毒RNA沉默途径，但其分子机制尚不明确。本项目以TCV病毒诱导沉默载体和拟南芥为研究材料，采用分子生物学和遗传学相结合的方法，首先研究DRB1是否与DCL4相互作用及在体内能否结合TCV，其次，研究同时缺少DCL2和DRB4时DRB1、AGO1、AGO2或AGO7蛋白分别被互补前后产生vsiRNA的变化，第三，研究同时缺少DCL2和DRB4时AGO1、AGO2、AGO7所结合的vsiRNA，从而明确参与该抗病毒RNA沉默途径的DRB蛋白及上述3个AGO蛋白是否参与该途径。研究结果将有助于解析这条新发现的抗病毒RNA沉默分子机制，加深对病毒-寄主之间复杂的相互作用分子机制的认识，亦为探索更有效的病毒防治新策略提供理论依据。

RNA沉默是植物重要的抗病毒防御机制，DCL、DRB和AGO都是RNA沉默信号途径中的重要蛋白。为解析DCL4介导、但不依赖于DRB4的抗病毒RNA沉默新途径的分子机制，本项目基本按计划执行，并且在跟踪文献的基础上增加对新报道的DRB7.1和7.2进行研究。首先，建立利用VIGS载体CPB1B研究拟南芥靶标基因是否参与抗TCV沉默的高效体系。VIGS载体CPB1B插入小于100bp的靶标片段时能高效同时沉默PDS及目的基因，并且白化现象可指示RNA沉默是否发生。其次，利用已建立的高效VIGS载体研究拟南芥DRB以及AGO1、AGO2和AGO7是否参与由DCL4介导、但不依赖于DRB4的抗病毒RNA沉默新途径，发现Hyl1可能参与该沉默新途径，并且起负调控的作用，而DRB2、DRB3、DRB5、DRB7.1、DRB7.2等DRB可能不参与或者各DRB之间具有功能冗余的现象；而AGO1、AGO2和AGO7基因被沉默均有利于病毒复制，说明都参与DCL4介导，无需DRB4的抗病毒RNA沉默途径。第三，通过遗传杂交或CRSPR基因编辑获得DRB或AGO1、AGO2和AGO7被单独或同时敲除的拟南芥双突变体dcl2drb4。用CPB1B感染这些突变体，研究结果表明Hyl1参与抗TCV沉默新途径，并且起负调控的作用，而DRB2、DRB3、DRB5、DRB7.1可能不参与该途径或者各DRB之间具有功能冗余的现象；AGO1、AGO2和AGO7均参与上述抗病毒RNA沉默新途径，并且AGO2的作用大于AGO1和AGO7。研究结果将有助于深入了解DCL4介导但不依赖DRB4的抗病毒沉默新途径的分子机制，加深对病毒-寄主之间复杂的相互作用分子机制的认识，亦为探索更有效的病毒防治新策略提供理论和实践依据。