Identification of sequence elements of tombusvirus-associated defective interfering RNAs required for symptom modulation.

Defective interfering (DI) RNAs of tombusviruses are short, non-coding, symptom-modulating RNAs originating from the viral genome. The presence of homologous DI RNA in virus infection attenuates the otherwise lethal viral symptoms. Nicotiana benthamiana plants infected with tomato bushy stunt tombusvirus pepper isolate (TBSV-P) show severe symptoms, which culminate in the death of the plant. In contrast, plants co-inoculated with TBSV-P and TBSV-P-derived DI RNA display attenuated symptoms. However, co-inoculation of TBSV-P with heterologous DI RNA, originating from Carnation Italian ringspot tombusvirus results in development of apical necrotic symptoms. To localize the symptom-determining factors on DI RNA genome, chimeras of protective and non-protective DI RNAs have been constructed. All chimeras were biologically active and accumulated to a high level in the presence of helper virus. We identified a 5' proximal sequence element of the DI RNA as the most important symptom determinant region. However, our results demonstrated that the symptom modulating ability of this region is also influenced by the sequence composition of whole DI RNAs.

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants.

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.