Infectious in vitro transcripts from cloned cDNA of beet necrotic yellow vein virus RNA3 and RNA4and their functional study

The full-length double-stranded cDNAs of beet necrotie yellow vein virus RNA3 and RNA 4were synthesized by using oligo(dT) 15 as well as RNA3 and RNA4 specific primers, and cloned downstream of the bacteriophage Sp6 RNA polymerase promoter of the transcription vector pGEM3Zf(+). The in vitro "run-off" transcription products obtained in the presence of Sp6 RNA polymerase and template DNA have high biological activities. In the 2 transcription systems, the transcription and capping in 2 separate reactions are more efficient. The simultaneous transcription and capping are not so efficient, but the transcripts are more infectious. Although there are a number of nonviral nucleotide sequences at the 5- and 3'-ends of RNA3 and RNA4 transcripts, the biological activities of both transcripts were not affected. The mechanical coinoculation of sugarbeet roots with the infectious transcripts of the prepared RNA3 and RNA4 and BNYW Rgl isolate has confirmed that RNA3 is the main cause of sugarbeet rhizomania.