Development of a Mini-Replicon-Based Reverse-Genetics System for Rice Stripe Tenuivirus

文献类型： 外文期刊

第一作者： Feng, Mingfeng

作者： Feng, Mingfeng;Li, Luyao;Cheng, Ruixiang;Yuan, Yulong;Dong, Yongxin;Chen, Minglong;Guo, Rong;Yao, Min;Xu, Yi;Ding, Xin Shun;Tao, Xiaorong;Zhou, Yijun;Wu, Jianxiang;Zhou, Xueping;Zhou, Xueping

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关键词： rice stripe tenuivirus; reverse-genetics system; mini-replicon; negative-sense/ambisense RNA virus

期刊名称：JOURNAL OF VIROLOGY （ 影响因子：5.103； 五年影响因子：5.078 ）

ISSN： 0022-538X

年卷期： 2021 年 95 卷 14 期

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收录情况： SCI

摘要： 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((-)eGFP)], a nucleocapsid (NP), and a codon usage-optimized RNA-dependent RNA polymerase (RdRp(opt)). Using this mini-replicon system, we determined that RSV NP and RdRp(opt) are indispensable for the eGFP expression from MR3((-)eGFP). The expression of eGFP from MR3((-)eGFP) can be significantly enhanced in the presence of four viral suppressors of RNA silencing (VSRs), NSs, and P19-HcPro-gamma b. 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 NS3 coding sequence acts as a cis 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 monocotinfecting 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. IMPORTANCE Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into three genera: Orthotospovirus, Tenuivirus, and Emaravirus. Reverse-genetics systems have been established for members of the genera Orthotospovirus and Emaravirus. However, there is still no reverse-genetics system available for Tenuivirus. 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-repliconbased 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.

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