Interference with brome mosaic virus replication by targeting the minus strand promoter

Sense and antisense strategies for interfering with the replication of brome mosaic virus (BMV) were examined. The effects of 200 nucleotide-long sense and antisense transcripts, corresponding to the viral 3' end (-) strand promoter, on the accumulation of progeny viral RNAs were studied by co-inoculation with wildtype BMV RNAs. Progeny accumulation in barley protoplasts transfected with either sense or antisense transcripts of the (-) strand promoter and BMV RNAs-1 and -2 was decreased by more than 90%, and by 60 to 80% when RNA-3 was also present. This trans interference was concentration-dependent, and reduced both (+) and (-) strand progeny accumulation to a similar extent. The appearance of complementary (-) strands indicated that sense interfering transcripts could serve as templates for (-) strand synthesis, and the use of deletion mutants revealed that the observed interference was in part mediated by this template activity. The reproducibility of the protoplast assay used here allows rapid evaluation of interference strategies and comparisons to be made of alternative approaches to engineered resistance. The results presented here suggest that targeting viral (-) strand promoters with sense and antisense transcripts may be an effective method for engineering plant resistance to viral infection.