■柑橘褪绿矮化相关病毒(CCDaV)是一种与卷叶相关的经济上重要的柑橘病毒,变形,以及在中国发现的萎黄病。植物已经进化出RNA沉默来防御病毒感染;然而,CCDaV抑制柑橘RNA沉默的机制尚不清楚.
■使用pCHF3载体在烟草benthamiana16c中异位表达由CCDaV编码的六种蛋白质以鉴定RNA沉默抑制活性。
■CCDaV编码的V2蛋白抑制了GFPRNA引发的局部RNA沉默和系统性RNA沉默,但不妨碍N.benthamiana16c中RNA沉默信号的短距离移动。GFP荧光观察表明,V2蛋白抑制RNA沉默的能力弱于番茄丛生特技病毒P19。缺失分析表明,推定的核定位信号(NLS,25-54aa)与V2蛋白的RNA沉默抑制活性有关。此外,V2蛋白不能阻断dsRNA触发的RNA沉默。亚细胞定位实验表明,V2蛋白定位于本氏芽孢杆菌的细胞核。
■总的来说,这项研究的结果表明,CCDaV-V2具有沉默抑制活性。这是第一个报道的由Citlodavirus编码的RNA沉默抑制剂,对于揭示CCDaV感染的分子机制具有重要意义。
UNASSIGNED: Citrus chlorotic dwarf-associated virus (CCDaV) is an economically important citrus virus associated with leaf curling, deformation, and chlorosis found in
China. Plants have evolved RNA silencing to defend against viral infections; however, the mechanism by which CCDaV suppresses RNA silencing in citrus remains unknown.
UNASSIGNED: Six proteins encoded by CCDaV were ectopically expressed in Nicotiana benthamiana 16c using the pCHF3 vector to identify RNA-silencing suppression activities.
UNASSIGNED: V2 protein encoded by CCDaV suppressed local RNA silencing and systemic RNA silencing triggered by GFP RNA, but did not impede short-distance movement of the RNA silencing signal in N. benthamiana 16c. GFP fluorescence observations showed that the ability of V2 protein to suppress RNA silencing was weaker than tomato bushy stunt virus P19. Deletion analysis showed that the putative nuclear localization signal (NLS, 25-54 aa) was involved in the RNA silencing suppression activity of V2 protein. Furthermore, V2 protein cannot block dsRNA-triggered RNA silencing. The subcellular localization assay suggested that V2 protein was localized to nucleus of N. benthamiana.
UNASSIGNED: Overall, the results of this study demonstrate that CCDaV-V2 acts as an activity of silencing suppression. This is the first reported RNA-silencing suppressor encoded by Citlodavirus and will be valuable in revealing the molecular mechanism of CCDaV infection.