Development of a mini-replicon-based reverse-genetics system for rice stripe tenuivirus

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human 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 RSV genomic RNA3 eGFP reporter (MR3(-)eGFP), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRpopt), respectively. Using this mini-replicon system we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of 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 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 monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants.IMPORTANCEPlant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus. The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus, respectively. 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 damages to 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 plant.