Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter. In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis. N. benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter. Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts. DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation. Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.