Artificial defective interfering RNAs derived from brome mosaic virus

RNAs) greatly reduce the accumulation of their helper virus in vivo, but are rarely associated with plant positive-strand RNA viruses. Deletion mutants pRNA-2 M/S and pRNA-2 E/S, derived from brome mosaic virus (BMV) genomic RNA-2, replicated in a manner dependent on BMV RNA-1 and-2, and effectively interfered with their accumulation in barley proto-plasts. Based on their mode of replication, these mutant RNAs have been termed parasitic RNAs (pRNAs). When present with RNA-1 and-2 at low inoculum amounts, pRNA-2 M/S and pRNA-2 E/S reduced the l vel of replication of RNA-2, the parental RNA, by 37 % and 64 %, respectively. Greater amounts of pRNA in the inoculum completely eliminated the replication of both RNA-I and-2. Mutations that prevented translation of truncated proteins from the pRNAs did not affect interference, but those that reduced pRNA replication decreased their ability to interfere with genomic RNA replication. At a molar pRNA: genomic RNA inoculum ratio of 1.5: 1, pRNA-2 E/S reduced the accumulation of all helper virus RNAs by> 60%. This occurred in the presence of wild-type RNA-3 or ASGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter neces-sary for coat protein expression, demonstrating that the interference mediated by the pRNAs was not effected by encapsidation. These data indicate that the expression of pRNAs that function as artificial DI-RNAs in transgenic plants may be an approach for inducing resistance to virus infection which is applic-able to a wide range of plant viruses