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Infectious RNA Produced by in vitro Transcription of a Full-length Tobacco Rattle Virus RNA-1 cDNA

AFRC Institute of Plant Science Research (Cambridge Laboratory), Maris Lane, Trumpington, Cambridge CB2 2JB, U.K.

A near full-length cDNA clone of tobacco rattle virus (TRV) RNA-1 was constructed by joining together nine overlapping cDNA clones using restriction sites in the regions of overlap. At the 5' end of the cDNA, oligonucleotide mutagenesis was used to insert nucleotides which were missing from the cDNA placing the construct immediately on the 3' side of the P_r promoter of phage to create pTR7116. Extraneous non-viral nucleotides had been deleted from the 3' end of the TRV cDNA to create a unique SmaI site in pTR7116 in which the nucleotides CCC were provided by the viral cDNA, and GGG by the vector. As a result, pTR7116 could be linearized with SmaI and transcribed in vitro to yield RNA molecules with 5' and 3' termini identical to those of natural TRV RNA-1. These transcripts were infectious when inoculated onto leaves of tobacco and produced the subgenomic RNA species typical of an infection with TRV RNA-1.

Keywords: TRV, infectious transcripts, tobravirus

Received 22 September 1988; accepted 19 December 1988.