甜菜严重曲顶病毒Rep蛋白N端结构域介导基因沉默抑制并诱导VIM5表达

N-terminal domain of Rep encoded by beet severe curly top virus mediates suppression of RNA silencing and induces VIM5 expression

摘要:

双生病毒严重危害农作物产量。双生病毒编码的复制起始蛋白(replication initiator protein, Rep)是病毒复制的必需蛋白。甜菜严重曲顶病毒(beet severe curly top virus, BSCTV)属于双生病毒科曲顶病毒属，其Rep蛋白可以诱导拟南芥E3泛素化连接酶VIM5在叶片中表达，VIM5介导甲基转移酶通过泛素化途径降解，进而降低了BSCTV基因组C2-3启动子区域的甲基化水平，然而Rep蛋白中负责诱导VIM5的具体结构域并不清楚。一些双生病毒的Rep蛋白具有抑制基因沉默的功能，然而BSCTV编码的Rep蛋白是否具有沉默抑制活性尚不明确。本研究利用烟草瞬时表达系统，研究Rep蛋白的不同结构域在抑制基因沉默和诱导VIM5表达方面的功能。发现Rep蛋白N端结构域(第1?180个氨基酸)可以抑制外源GFP在16c-GFP转基因烟草注射叶片上的降解；进一步研究发现，Rep蛋白N端的第1?104个氨基酸可以显著诱导VIM5的表达，而Rep蛋白C端的结构则不能诱导VIM5的表达。本研究表明BSCTV编码的Rep蛋白的N端结构域能够抑制基因沉默同时诱导VIM5表达，有助于更加深入地理解双生病毒复制起始蛋白Rep和宿主植物的互作机制。

Abstract:

Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus Curtovirus, family Geminiviridae) induces VARIANT IN METHYLATION 5 (VIM5) expression in Arabidopsis leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for VIM5 induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type Nicotiana benthamiana plants to analyze the VSR and the VIM5-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1?180) of Rep suppressed GFP silencing in 16c-GFP transgenic N. benthamiana leaves. The minimal N-terminal fragment (amino acids 1?104) induced VIM5 expression upon co-infiltration, while C-terminal truncations lacked VIM5-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces VIM5 expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.