Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3

Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3
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水稻矮化病毒非结构蛋白 p5 作为 RNA 沉默的病毒抑制因子并与非结构蛋白 p3 相互作用

DOI:
10.1007/s00705-015-2560-6
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发表时间:
2015-08
影响因子:
2.7
通讯作者:
Zujian Wu
Zujian Wu
中科院分区:
医学4区
文献类型:
--
作者:
Peng Zou;Liang Yang;Jianguo Wu;Zujian Wu

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水稻草矮病毒(Rice grassy stunt virus,RGSV)是细病毒属(Tenuivirus)的成员,在东南亚国家引起严重的水稻病害。在这项研究中,一个绿色荧光蛋白(GFP)为基础的瞬时表达试验进行了显示,p5,编码的RNA 5在病毒的意义上,是一种病毒抑制RNA沉默(VSR)。使用基于Gal 4的酵母双杂交(Y2 H)实验研究了p5与除pC 1和pC 2之外的所有RGSV蛋白之间的蛋白质-蛋白质相互作用(PPI)。结果表明,p5与自身相互作用,并与RNA 3上编码的p3在病毒意义上相互作用。双分子荧光互补(BiFC)法检测p5-p5和p5-p3相互作用,亚细胞共定位和免疫共沉淀(Co-IP)法检测p5-p3相互作用。使用Y2 H系统,我们证明了p5-p3相互作用需要p5的N-末端(氨基酸残基1至99)和C-末端(氨基酸残基94至191)结构域。此外,p5和p3均能增强马铃薯X病毒(PVX)对本氏烟草的致病性。当p5和p3共同表达时,观察到PVX致病性和积累的显著增强。我们的数据还表明,RGSV p3不作为VSR的功能,它不影响p5的VSR活性或p5在烟草细胞中的亚细胞定位模式。
Rice grassy stunt virus (RGSV), a member of the genusTenuivirus, causes serious rice disease in Southeast Asian countries. In this study, a green fluorescent protein (GFP)-based transient expression assay was conducted to show that p5, encoded on RNA5 in the viral sense, is a viral suppressor of RNA silencing (VSR). Protein-protein interactions (PPIs) between p5 and all RGSV proteins except pC1 and pC2 were investigated using Gal4-based yeast two-hybrid (Y2H) experiments. The results demonstrated that p5 interacts with itself and with p3 encoded on RNA3 in the viral sense. p5-p5 and p5-p3 interactions were detected by bimolecular fluorescence complementation (BiFC) assay, and the p5-p3 interaction was confirmed by subcellular co-localization and co-immunoprecipitation (Co-IP) assays. Using the Y2H system, we demonstrated that the p5-p3 interaction requires both the N-terminal (amino acid residues 1 to 99) and C-terminal (amino acid residues 94 to 191) domains of p5. In addition, either p5 or p3 could enhance the pathogenicity of potato virus X (PVX) inNicotiana benthamianaplants. A much more significant enhancement of PVX pathogenicity and accumulation was observed when p5 and p3 were expressed together. Our data also showed that RGSV p3 does not function as a VSR, and it had no effect on the VSR activity of p5 or the subcellular localization pattern of p5 in plant cells fromNicotiana benthamiana.
DOI: 10.1038/nprot.2007.13
发表时间: 2007-01-01
期刊: NATURE PROTOCOLS
影响因子: 14.8
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