Plant virus-induced diseases cause significant losses to agricultural crop production worldwide. Reverse genetics systems of plant viruses allow gene manipulation on viral genomes, which greatly facilitates studies of viral pathogenesis and interactions with host organisms. In addition, viral infectious cDNA clones have been modified as versatile recombinant vectors for virus-mediated protein overexpression, virus-induced gene silencing, and gene editing. Since genome RNAs of plant positive-strand RNA viruses are directly translatable, recovery of these viruses has been achieved more than three decades ago by simply expressing viral genome RNA or viral genome-derived in vitro synthesized transcripts in planta. In contrast, genomes of plant negative-strand RNA (NSR) viruses are complementary to their mRNAs and cannot be translated directly. Therefore, rescue of infectious plant NSR viruses from cDNA clones strictly requires the core replication proteins together with their genome RNAs which can assemble into nucleocapsid (NC) complexes as minimal infectious units. However, it is a major challenge to deliver multiple essential components in single cells and to assemble the NC complexes in vivo. Major breakthroughs in reverse genetics systems of plant non-segmented and segmented NSR viruses were just achieved in recent 5 years through various strategies, such as agroinfiltration, minireplicon systems, insect transmission and airbrush inoculation assays. In this review, we summarized critical steps toward developing reverse genetics systems for recovery of several plant NSR viruses in plants and insects. We also highlighted important applications of these reverse genetics of NSR viruses in viral gene function analyses, investigation of virus-insect-plant interactions, and genomic studies of insect vectors and host plants.
