Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, a Key Laboratory of Beijing Municipality, China Agricultural University, Beijing, 100193, China.
Department of Plant Pathology, China Agricultural University, Beijing, 100193, China.
Mol Plant Pathol. 2018 Feb;19(2):355-368. doi: 10.1111/mpp.12525. Epub 2017 Feb 28.
Grapevine leafroll-associated virus 2 (GLRaV-2) p24 has been reported to be an RNA silencing suppressor (RSS). However, the mechanisms underlying p24's suppression of RNA silencing are unknown. Using Agrobacterium infiltration-mediated RNA silencing assays, we showed that GLRaV-2 p24 is a strong RSS triggered by positive-sense green fluorescent protein (GFP) RNA, and that silencing suppression by p24 effectively blocks the accumulation of small interfering RNAs. Deletion analyses showed that the region of amino acids 1-188, which contains all predicted α-helices and β-strands, is required for the RSS activity of p24. Hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, previously shown to be critical for p24 self-interaction, are also crucial for silencing suppression, and western blotting results suggested that a lack of self-interaction ability results in decreased p24 accumulation in plants. The mutants showed greatly weakened or a lack of RSS activity. Substitution with two basic residues at positions 2 or 86, putatively involved in RNA binding, totally abolished the RSS activity of p24, suggesting that p24 uses an RNA-binding strategy to suppress RNA silencing. Our results also showed that W54 in the WG/GW-like motif (W54/G55) is crucial for the RSS activity of p24, whereas p24 does not physically interact with AGO1 of Nicotiana benthamiana. Furthermore, p24 did not promote AGO1 degradation, but significantly up-regulated AGO1 mRNA expression, and this effect was correlated with the RSS activity of p24, indicating that p24 may interfere with microRNA-directed processes. The presented results contribute to our understanding of viral suppression of RNA silencing and the molecular mechanisms underlying GLRaV-2 infection.
葡萄扇叶病毒 2（GLRaV-2）p24 已被报道为 RNA 沉默抑制子（RSS）。然而，p24 抑制 RNA 沉默的机制尚不清楚。通过农杆菌浸润介导的 RNA 沉默测定，我们表明 GLRaV-2 p24 是由正链绿色荧光蛋白（GFP）RNA 触发的强 RSS，并且 p24 的沉默抑制有效地阻止了小干扰 RNA 的积累。缺失分析表明，包含所有预测α-螺旋和β-链的氨基酸 1-188 区域是 p24 RSS 活性所必需的。先前显示对 p24 自我相互作用至关重要的疏水性残基 I35/F38/V85/V89/W149 和 V162/L169/L170 对于沉默抑制也至关重要，Western blot 结果表明缺乏自我相互作用能力导致 p24 在植物中的积累减少。突变体显示 RSS 活性大大减弱或丧失。推测涉及 RNA 结合的位置 2 或 86 处的两个碱性残基的取代完全消除了 p24 的 RSS 活性，表明 p24 使用 RNA 结合策略来抑制 RNA 沉默。我们的结果还表明，WG/GW 样基序（W54/G55）中的 W54 对 p24 的 RSS 活性至关重要，而 p24 与 Nicotiana benthamiana 的 AGO1 没有物理相互作用。此外，p24 不会促进 AGO1 降解，但会显著上调 AGO1 mRNA 表达，并且这种效应与 p24 的 RSS 活性相关，表明 p24 可能干扰 microRNA 指导的过程。所呈现的结果有助于我们理解病毒对 RNA 沉默的抑制作用以及 GLRaV-2 感染的分子机制。
