Key Laboratory of Plant Immunity, Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, People's Republic of China.
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Jiangsu Technical Service Center of Diagnosis and Detection for Plant Virus Diseases, Nanjing, People's Republic of China.
J Virol. 2021 Jun 24;95(14):e0058921. doi: 10.1128/JVI.00589-21.
Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in humans and livestock and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing an RSV genomic RNA3 enhanced green fluorescent protein (eGFP) reporter [MR3], a nucleocapsid (NP), and a codon usage-optimized RNA-dependent RNA polymerase (RdRp). Using this mini-replicon system, we determined that RSV NP and RdRp are indispensable for the eGFP expression from MR3. The expression of eGFP from MR3 can be significantly enhanced in the presence of four viral suppressors of RNA silencing (VSRs), NSs, and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV coding sequence acts as a element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that the mini-replicon system described here will allow studies of the RSV replication, transcription, cell-to-cell movement, and host machinery underpinning RSV infection in plants. Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into three genera: , and . Reverse-genetics systems have been established for members of the genera and . However, there is still no reverse-genetics system available for . Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damage to the rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plants.
负链 RNA (NSR) 病毒包括感染动物和植物的病毒，这些病毒经常导致人类、家畜和农业作物严重疾病。水稻条纹病毒 (RSV) 是一种具有四个负链/反义 RNA 节段的植物 NSR 病毒，是许多亚洲国家最具破坏性的水稻病原体之一。由于缺乏可靠的反向遗传学技术，RSV 基因功能及其与宿主植物相互作用的分子研究受到严重阻碍。为了克服这一障碍，我们开发了一种基于 mini-replicon 的 RSV 基因功能分析反向遗传学系统，用于 Nicotiana benthamiana。我们首先开发了一个表达 RSV 基因组 RNA3 增强型绿色荧光蛋白 (eGFP) 报告基因 [MR3]、核衣壳 (NP) 和密码子使用优化的 RNA 依赖性 RNA 聚合酶 (RdRp) 的 mini-replicon 系统。使用该 mini-replicon 系统，我们确定 RSV NP 和 RdRp 对于从 MR3 表达 eGFP 是必不可少的。在存在四个 RNA 沉默抑制子 (VSRs)、NSs 和 P19-HcPro-γb 的情况下，MR3 表达的 eGFP 可以显著增强。此外，RSV 的运动蛋白 NSvc4 促进了 eGFP 在细胞间的运输。我们还在 Nicotiana benthamiana 中开发了一种基于反基因组 RNA3 的 replicon。然而，我们发现 RSV 编码序列充当 元件，调节病毒 RNA 表达。最后，我们制作了代表 RSV 所有四个基因组 RNA 的 mini-replicons。这是第一个用于单子叶植物感染的 tenuivirus 的基于 mini-replicon 的反向遗传学系统。我们相信，这里描述的 mini-replicon 系统将允许研究 RSV 在植物中的复制、转录、细胞间运动和宿主机制。 感染植物的分段负链 RNA (NSR) 病毒分为三个属： 、 和 。已经建立了属 和 的成员的反向遗传学系统。然而，仍然没有用于 的反向遗传学系统。水稻条纹病毒 (RSV) 是一种感染单子叶植物的 tenuivirus，具有四个负链/反义 RNA 节段。它是亚洲国家最具破坏性的水稻病原体之一，对水稻产业造成了重大损失。由于缺乏可靠的反向遗传学系统，RSV 基因功能的分子特征以及 RSV 感染植物的宿主机制极其困难。为了克服这一障碍，我们在 Nicotiana benthamiana 中开发了一种基于 mini-replicon 的 RSV 反向遗传学系统。这是第一个用于 tenuivirus 的基于 mini-replicon 的反向遗传学系统。我们认为，该系统将为研究人员提供一个新的工作平台，以阐明决定分段 tenuivirus 在植物中感染的分子机制。
