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

• DOI: 10.1371/journal.ppat.1005223
• Publication Date: 2015-10-20T18:10:00Z
• Authors: Qiang Wang; Xiaonan Ma; ShaSha Qian; Xin Zhou; Kai Sun; Xiaolan Chen; Xueping Zhou; Andrew O. Jackson; Zhenghe Li
• Keywords: DNA, Complementary; QH301-705.5; Immunology; Immunoblotting; Plant Biology; Microbiology; Fluorescence; Plant Viruses; Sonchus; 03 medical and health sciences; Complementary; Virology; Rhabdoviridae Infections; Genetics; 2.2 Factors relating to the physical environment; Aetiology; Biology (General); Plant Diseases; 580; 2. Zero hunger; Microscopy; 0303 health sciences; Reverse Transcriptase Polymerase Chain Reaction; DNA; Plant; Biological Sciences; Medical microbiology; RC581-607; 3. Good health; Emerging Infectious Diseases; Infectious Diseases; Good Health and Well Being; Microscopy, Fluorescence; Medical Microbiology; RNA, Plant; RNA; Immunologic diseases. Allergy; Rhabdoviridae; Infection; Research Article

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.