Broad-spectrum protection against tombusviruses elicited by defective interfering RNAs in transgenic plants

We have designed a DNA cassette to transcribe defective interfering (DI) RN As of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenic Nicotiana benthamiana plants from tombusvirus infect ions. To produce RNAs with authentic 5' and 3' termini identical to those o f the native B10 DI RNA,the DI RNA sequences were flanked by ribozymes (RzD I). When RzDI RNAs transcribed in vitro were mixed with parental TBSV trans cripts and inoculated into protoplasts or plants, they became amplified, re duced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F-1 and F-2 RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformed N . benthamiana plants and transformed negative controls died. Although infec tion of transgenic RzDI plants initially induced moderate to severe symptom s, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tomb us-like virus and to unrelated viruses.