Infectious DNA clone technology and inoculation strategy for Rose Rosette Virus that includes all seven segments of the negative-strand RNA genome

Abstract

Rose rosette virus (RRV) is an Emaravirus, a negative-sense RNA virus with a 7-segmented genome that is enclosed by a double membrane. While the genome sequences of many emaraviruses are reported, there is negligible information concerning virus replication and movement in host plants. Computational methods determined that RNA1 encoded the RNA dependent RNA polymerase (RdRp), RNA2 encoded glycoprotein precursor, and the RNA3 encoded the nucleocapsid (N), all share significant homologies with similar proteins of the Orthobunyavirus family. The RRV terminal UTR sequences are complementary and share significant identity with the UTR sequences of Bunyamwera virus. We report a minireplicon system and a full length infectious clone of RRV, which are the first for any emaravirus species. The photoreversible fluorescent iLOV protein was used to replace the RNA5 open reading frame (R5-iLOV). We demonstrate that agro-infiltration of Nicotiana benthamiana leaves to deliver RNA1, RNA3, and R5-iLOV cDNAs led to iLOV expression. A mutation was introduced into the RdRp active site and iLOV expression was eliminated. Delivery of four segments or seven segments of the RRV infectious clone produced systemic infection in N. benthamiana and rose plants. iLOV was also fused to the glycoprotein precursor (R2-iLOV). Using confocal microscopy, the R2-iLOV was seen in spherical bodies along membrane strands inside N. benthamiana epidermal cells. This new technology will enable future research to functionally characterize the RRV proteins, to study the virus-host interactions governing local and systemic infection, and examine the subcellular functions of the Gc. IMPORTANCERRV has emerged as a severe threat to cultivated roses, causing millions of dollars in losses to commercial producers. The majority of the viral gene products have not been researched or characterized until now. We constructed a minireplicon system and an infectious clone of the seven-segmented RRV genome that is contained in a binary vector and delivered by Agrobacterium. This technology has been slow to develop for viruses with negative-strand RNA genomes. It has been especially tricky for plant viruses with multicomponent negative-strand RNA genomes. We report the first reverse genetic system for a member of the genus Emaravirus, Rose rosette virus (RRV). We introduced the iLOV fluorescent protein as a fusion to the Gc protein and as a replacement for the open reading frame in genome segment 5. This game-changing reverse genetic system creates new opportunities for studying negative-strand RNA viruses in plants.