Development of a Reverse Genetic System for Studying Rose Rosette Virus in Whole Plants

Abstract

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 .

Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement

“…Therefore, mixtures of agrobacterial cultures harboring the vRNA1 MR GFP plasmid and the binary plasmids designed for expression of RSV N and L proteins were used to infiltrate N. benthamiana leaves. Three viral suppressors of RNA silencing (VSRs), i.e., tomato bushy stunt virus p19, barley mosaic stripe virus γb, and tobacco etch virus Hc-Pro, were also included in the infection mixtures because these VSRs have been proven to be beneficial for rescue of other plant NSV MRs (Ganesan et al, 2013;Wang et al, 2015;Gao et al, 2019;Feng et al, 2020a;Verchot et al, 2020). Despite repeated attempts and modifications of the protocol, no GFP expression was observed throughout the infiltrated leaf patches ( Figure 1C, leftmost panel), indicating that active RNPs were not reconstituted in vivo.…”

“…Owing to the low efficiencies of RNP assembly and additional technical barriers, genetic manipulation of plantinfecting NSVs has been extremely challenging (Jackson and Li, 2016). Fortunately, several technical breakthroughs have recently been made with the rhabdoviruses Sonchus yellow net virus (SYNV) (Wang et al, 2015;Ma and Li, 2020) and barley yellow striate mosaic virus (Gao et al, 2019), tomato spotted wilt tospovirus (TSWV) (Feng et al, 2020a), and rose rosette emaravirus (Verchot et al, 2020) that permit recombinant studies.…”

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

“…TSWV and RSV are the representative viruses for Orthotospovirus and Tenuivirus, respectively (6,7). RRV and European mountain ash ringspot-associated virus (EMAraV) are important members in the genus Emaravirus (8,9).…”

“…Recently we established the first reverse-genetics system of segmented plant NSR viruses for TSWV (48). Soon after, the reverse-genetics system for RRV was also established, allowing for studies of emaravirus gene function and disease pathology in whole plants (9). For 3 genera of segmented NSR viruses infecting plants, reverse-genetics model has been established for 2 of the genera, Orthotospovirus and Emaravirus (9,48,49).…”

Intercellular movement of plant RNA viruses: Targeting replication complexes to the plasmodesma for both accuracy and efficiency

“…Because of the lack of reverse genetic engineering tools, the link between the replication and cell‐to‐cell movement of negative‐sense ssRNA (‐ssRNA) viruses is largely unknown at present. Several infectious clones of ‐ssRNA viruses, including Sonchus yellow net rhabdovirus (SYNV; genus Cytorhabdovirus ), Tomato spotted wilt virus (TSWV; genus Orthtospovirus ), and Rose rosette virus (RRV; genus Emaravirus ), 137‐139 have been successfully constructed, representing a milestone in the studying of the replication and movement of ‐ssRNA viruses. Plant DNA viruses and a few ‐RNA viruses, including geminiviruses and nucleorhabdoviruses, replicate exclusively in the nucleus; how these viruses link replication and cell‐to‐cell movement is another engaging subject for exploration.…”