Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs

Transcripts were produced in vitro by run-off transcription from full-length cDNA of RNA1 and RNA2 of grapevine fanleaf nepovirus (GFLV; isolate F13) cloned downstream from a bacteriophage RNA polymerase promoter. These transcripts, which possess a 5' terminal cap structure and a non-viral G residue instead of the naturally occurring genome-linked viral protein (VPg), are infectious to Chenopodium quinoa protoplasts when inoculated by electroporation. Synthetic RNA1 alone replicated in protoplasts. Inoculation of C. quinoa plants with synthetic RNA1 plus RNA2 produced symptoms similar to, but weaker, than those observed in plants infected with natural GFLV 6 to 8 days post-inoculation. Co-inoculated RNA1 and RNA2 were able to replicate and spread systemically in plants but RNA1 alone produced no symptoms and was not detected in non-inoculated leaves, suggesting that virus spread requires RNA2. Analysis of the genomic RNAs in plants infected with transcripts showed that the non-viral G at their 5' ends was not retained in the progeny.