Fig. 1

Combined strategies used in development of reverse genetics systems of plant NSR viruses. a Agrobacterium tumefaciens-mediated expression of genes in N. benthamiana leaves was utilized for simultaneous delivery of plant NSR viruses core protein genes and genomic RNAs in single cells. b The minireplicon (MR) containing a double cauliflower mosaic virus 35S (2 × 35S) promoter, exact termini of the virus, gene junctions, and reporter genes could replicate in conjunction with the ectopic expression of viral core proteins and VSRs, which could validate whether the core replicate proteins are functional in viral transcription and replication. c Full-length viral antigenomic RNA (agRNA) was cloned into the A. tumefaciens binary vector containing a 2 × 35S promoter and a ribozyme (Rz) for cleavage of the 5′ and 3′ ends of the transcribed agRNAs with exact termini. d Insect-mediated transmission of rBYSMV from N. benthamiana leaves to monocot plants. Grinding the infected N. benthamiana leaves and injecting the extracts into the thoraxes of SBPHs. The rBYSMV could be recovered in the SBPHs. After a period of incubation, the infected SBPHs transmitted recombinant viruses to barley plants. e Codon optimization removed cryptic splicing sites in the cDNA of TSWV L and M RNAs for stable expression of the large RdRp protein and genome RNAs in plants. f Inoculation by an airbrush was effective in delivering agrobacterium cultures containing RRV vectors to A. thaliana, N. benthamiana and roses. In the middle panels, combined technologies were used for developing the reverse genetics systems of SYNV, BYSMV, TSWV, and RRV