Defective-interfering RNAs and elevated temperatures inhibit replication of tomato bushy stunt virus in inoculated protoplasts

Abstract

Tomato bushy stunt virus (TBSV) genomic RNA and one of its defective interfering (DI) RNAs were inoculated in various combinations to protoplasts of Nicotiana benthamiana. Ethidium bromide staining of electrophoretically separated RNAs from infected protoplasts, incorporation of [ 3H]uridine into TBSV and DI RNAs, and Northern hybridization at different times after inoculation clearly demonstrated reduced accumulation of genomic RNA in the presence of DI RNA. Accumulation of genomic RNA was very rapid between 3 and 9 hr postinfection. The presence of equimolar amounts of genomic and DI RNA in the inoculum resulted in a 65% suppression of genomic RNA accumulation. Suppression of genomic RNA was mediated by a reduction in the rate at which genomic RNA accumulated. Analysis of protoplasts inoculated with increasing ratios of DI:genomic RNA suggested that DI RNA-mediated suppression of genomic RNA synthesis results from competition for factors essential for viral replication. Incubation of protoplasts at different temperatures also had a profound effect on replication of both genomic and DI RNAs. Both replicated well at 27° but were barely detectable at 32°. Suppression of genomic RNA synthesis by DI RNA was similar at all temperatures tested. Thus, this study suggests that DI suppression of TBSV symptoms in whole plants and symptom attenuation at elevated temperatures are primarily the result of reduced viral replication.