Development of infectious transcripts from full-length and GFP-tagged cDNA clones of Pepper mottle virus and stable systemic expression of GFP in tobacco and pepper.

A full-length cDNA clone (pSP6PepMoV-Vb1) of the genomic RNA of a Korean isolate of Pepper mottle virus (PepMoV-Vb1) was constructed downstream of a bacteriophage SP6 RNA polymerase promoter in the plasmid. In vitro RNA transcripts generated from pSP6PepMoV-Vb1 corresponded to PepMoV-Vb1 RNA (9641nt) with an extra guanosine residue at the 5' terminus and a 15-nt, poly (A) tract at the 3' end. The RNAs synthesized from the pSP6PepMoV-Vb1 clone, by in vitro run-off transcription in the presence of the 5' cap analog m(7)GpppG, were highly infectious in Nicotiana benthamiana and Capsicum annuum cv. Early Calwonder. Visible symptoms appeared at 4-5 days post-inoculation, at essentially the same time as occurred on these host plant species inoculated with wild-type PepMoV-Vb. Symptoms induced by progeny virus of the transcripts were indistinguishable from wild-type PepMoV-Vb on their experimental and natural hosts. The gene encoding the green fluorescent protein (GFP), turboGFP, was inserted between the coding regions for NIb and CP in the pSP6PepMoV-Vb1 clone. RNA transcripts of the resulting GFP-tagged clone, designated SP6PepMoV-Vb1/GFP, were highly infectious and symptoms were not different from those induced by either transcripts of pSP6PepMoV-Vb1 or wild-type PepMoV-Vb. However, GFP expression could be detected earlier than virus-induced symptom in plants infected by SP6PepMoV-Vb1/GFP. This study is the first report of the construction of a biologically active, full-length cDNA copy of the Pepper mottle virus RNA genome and the stable expression of a foreign gene within the modified virus.