Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Bunyavirales are negative-sense segmented RNA viruses infecting arthropods, protozoans, plants, and animals. This study examines the phylogenetic relationships of plant viruses within this order, many of which are recently classified species. Comprehensive phylogenetic analyses of the viral RNA dependent RNA polymerase (RdRp), precursor glycoprotein (preGP), the nucleocapsid (N) proteins point toward common progenitor viruses. The RdRp of Fimoviridae and Tospoviridae show a close evolutional relationship while the preGP of Fimoviridae and Phenuiviridae show a closed relationship. The N proteins of Fimoviridae were closer to the Phasmaviridae, the Tospoviridae were close to some Phenuiviridae members and the Peribunyaviridae. The plant viral movement proteins of species within the Tospoviridae and Phenuiviridae were more closely related to each other than to members of the Fimoviridae. Interestingly, distal ends of 3′ and 5′ untranslated regions of species within the Fimoviridae shared similarity to arthropod and vertebrate infecting members of the Cruliviridae and Peribunyaviridae compared to other plant virus families. Co-phylogeny analysis of the plant infecting viruses indicates that duplication and host switching were more common than co-divergence with a host species.

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.

6 that includes all seven segments of the negative-strand RNA genome. 7 8 Abstract 32The ability to mutate the genomic nucleic acid of viruses is the most straight-forward approach 33to understanding virus genetics. Reverse genetics of RNA viruses involves the introduction of 34 mutations at the cDNA level, and then introducing cDNAs into cells to produce infectious 35 progeny virus. Reverse genetic tools are used to investigate the products of viral genes, virus-36host interactions relating to pathogenicity and immunity, and requirements for vector 37 transmission. The technology has been slow to develop for viruses with negative strand RNA 38 genomes and has been especially difficult for plant viruses with multicomponent negative strand 39RNA genomes, many of which require an insect vector for transmission to be successful. Rose 40 rosette virus (RRV; Emaravirus) is a negative-sense RNA virus with a 7-segmented genome 41 that is enclosed by a double membrane (1-4). We devised a technology for delivery of plant sap 42 inoculum which can also deliver agrobacterium containing infectious clones to rose plants. We 43 report the first reverse genetic system for a member of the Emaravirus genus, Rose rosette 44 virus (RRV). We introduced fluorescent proteins at three locations in the seven segmented 45 genome and learned that such reporters can be stably maintained during systemic infection. 46This study demonstrates that RRV can infect Arabidopsis causing significant growth alterations 47 of the plant, while causing mild to more serious disease symptoms in Nicotiana benthamiana 48and two varieties of roses. This reverse genetic system creates new opportunities for studying 49 negative strand RNA viruses infecting plants. 50 Significant Statement 51Since an infectious clone for influenza virus was developed in 1998, little progress has been 52 made in infectious clone technology for viruses with negative strand genomes. We constructed 53 an infectious clone of the seven-segmented RRV genome that is contained in a binary vector 54 and delivered by Agrobacterium. RRV has emerged as a serious threat to cultivated roses, 55 causing millions of dollars in losses to commercial producers. This technology is a game 56 changer for investigations into Emaravirus genetics, studies of molecular virus-host interactions, 57 as well as for rose breeders who can use the infectious clone for rapid germplasm screening to 58 identify useful resistance for breeding programs. 59