Date Published: October 20, 2015
Publisher: Public Library of Science
Author(s): Qiang Wang, Xiaonan Ma, ShaSha Qian, Xin Zhou, Kai Sun, Xiaolan Chen, Xueping Zhou, Andrew O. Jackson, Zhenghe Li, Hui-Shan Guo.
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.
Negative-strand RNA (NSR) viruses have major impacts on public health, agriculture and ecology, and they collectively are responsible for some of our most serious human, veterinary, wildlife and plant diseases . Plant NSR viruses comprise members of the Rhabdoviridae, Bunyaviridae, Ophioviridae families, and of the unassigned Emaravirus, Tenuivirus, Varicosavirus and Dichorhavirus genera and account for many economically important crop diseases [1–3]. Most members of the plant NSR viruses are transmitted by specific arthropods (aphids, leafhoppers, thrips or mites) in which they also replicate, and many of these viruses share similarities in particle morphology, genome organization and fundemental replication strategies to their animal/human-infecting counterparts within the same families [3–7].
Recovery of infectious NSR viruses from cloned cDNAs for reverse genetic analyses is now routine for all animal NSR virus families. Although the procedures are quite inefficient, with 104 to 107 transfected cells per primary infected cell, recombinant virus particles released from primary infected cells can be passaged to permissive cell lines to obtain progeny viruses suitable for a variety of purposes [13,14,18–20]. Unfortunately, only a few insect vector cell cultures suitable for rescue of recombinant plant NSR viruses have been established [32,33]. Even these lines are difficult to maintain and to our knowledge, plasmids suitable for transient expression of multiple genes in these lines are unavailable. Moreover, introduction of multiple components into single plant cells after removal of the cell wall is inefficient and protoplast recoveries after transformation or viral transfection is low. In addition, transformation and high level expression of multiple viral proteins and RNAs in plant leaves is difficult due to the presence of the cell wall and the existence of potent plant antiviral gene silencing mechanisms [34,35]. Hence, to circumvent these problems, we turned to infiltration of N. benthamiana leaves with Agrobacterium strains harboring plasmids encoding the SYNV agRNAs and the N, P and L core proteins needed for de novo NC assembly, coupled with the use of VSRs proteins to suppress host RNA silencing. This approach has enabled in planta rescue of rSYNV from cDNAs with an infection phenotype identical to wtSYNV (Fig 1 and S1 Fig).