Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

Abstract

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus

“…Our previous studies showed that combined expression of the N, P, and L core protein in a single plasmid, when supplemented by additional amounts of L proteins (NPL + L), significantly improved SYNV rescue efficiency [16]. In our current experiments, the more desirable core protein mixture (NPL + L) also resulted in an increased infection rate in 35St-SYNV infiltrated plants (~ 20%).…”

Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing

“…We hypothesize that the P protein of cytorhabdoviruses has evolved to target multiple steps of the RNA silencing pathway, linked to their relative weak local RSS activity. Hence, further detailed studies should be conducted to probe the mechanism underlying the RSS activity of this protein, preferably using approaches that resemble the natural context of the virus-plant pathosystem, like the use of a reverse genetics system similar to the one recently developed for SYNV (Wang et al, 2015). leaves expressing mGFP5-ER were co-agroinfiltrated with mGFP5-ER plus either ADV P or P6.…”

The family Rhabdoviridae: mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins

“…The recent development of the first-ever reverse genetics system for a plant rhabdovirus provided a major technical breakthrough and a guide for the future study of other plant negative-sense ssRNA viruses (Ganesan et al, 2013; Wang et al, 2015; Jackson and Li, 2016). It allowed the recovery of infectious recombinant virus (rSYNV) from agroinfiltrated plants and the generation of rSYNV stably expressing a green fluorescent protein (GFP) reporter.…”

“…It allowed the recovery of infectious recombinant virus (rSYNV) from agroinfiltrated plants and the generation of rSYNV stably expressing a green fluorescent protein (GFP) reporter. Deletion analyses of rSYNV-GFP demonstrated the involvement of the sc4 (P3) protein in cell-to-cell movement and the importance of the G protein in virion morphogenesis (Wang et al, 2015). …”