The tRNA-like domain present at the 3' end of each of the three genomic RNAs of brome mosaic virus (BMV) encompasses the (-)-strand promoter essential for replication. The replicative competence of two BMV RNA-2 transcripts bearing mutations delta 5' and 5'AGA in the tRNA-like domain (previously shown by in vitro assays to be deficient in tyrosylation) was evaluated in barley protoplasts. Transfection of protoplasts with low (2 micrograms) amounts of delta 5'RNA-2, together with transcripts of wild-type RNA-1 and -3, not only incapacitated the replication of RNA-2 but also significantly interfered in trans with the synthesis and accumulation of the other viral RNAs. In contrast, RNA-2 mutants bearing either 5'AGA or M4 (a mutation yielding enhanced minus-strand replication activity in vitro) were inhibitory to viral replication only when present at a relatively high level (12 micrograms). Coinoculation of protoplasts with high levels (12 micrograms) of each of the three RNA-2 mutants and transcripts corresponding to wild-type RNA-1, -2 and -3 (2 micrograms each) revealed that the mutants were capable of competing in trans, resulting in greatly reduced accumulation of the viral RNA and suggesting that their expression from constitutive promoters in transgenic plants may provide protection against viral infection.