Institut de Biologie Moléculaire des Plantes, Laboratoire Propre du CNRS (UPR 2357) Conventionné avec l'Université de Strasbourg, 12 rue de Générale Zimmer, 67084 Strasbourg, France.
Mol Plant Microbe Interact. 2013 Feb;26(2):168-81. doi: 10.1094/MPMI-06-12-0142-R.
The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.
已研究了甜菜坏死黄脉病毒（BNYVV）和甜菜土传花叶病毒（BSBMV）富含半胱氨酸的 p14 蛋白对 RNA 沉默抑制的特性。通过沉默的 Nicotiana benthamiana 16C 的病毒感染、用绿色荧光蛋白（GFP）农杆菌浸润的 Nicotiana benthamiana 和用补充了病毒 RNA 沉默抑制子（VSR）构建体的 GFP-FG 发夹触发物，或在用沉默抑制缺陷型 BNYVV 病毒互补的情况下，可明显看出抑制 RNA 沉默活性。在农杆菌浸润试验中的小干扰 RNA（siRNA）的Northern blot 分析显示，siRNA 的量减少，尤其是二级 siRNA，表明贝尼病毒 VSR 作用于 siRNA 产生的下游。通过对表达与增强型 GFP 报告基因融合的功能性 p14 蛋白的感染原生质体进行共焦激光扫描显微镜成像，我们表明贝尼病毒 p14 独立于其他病毒因子在核仁中和细胞质中积累。定点突变显示在 VSR 功能和 p14 蛋白固有稳定性中嵌入的核仁定位信号在 C4 锌指结构域中的重要性。相反，RNA 沉默抑制似乎独立于该蛋白的核仁定位，并且建立了 BNYVV VSR 表达与长距离运动之间的相关性。
