The choice of homology region in the Tomato spotted wilt virus genome is crucial to induce resistance using exogenously applied double-stranded RNAs.

Tomato spotted wilt virus, (TSWV), a negative-sense RNA virus belonging to the Bunyaviridae family, is a devastating plant pathogen, causing huge crop losses worldwide. Indeed, due to its wide host range and the emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved in transgenic plants expressing parts of the viral genome or artificial miRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents a very attractive and promising alternative, already shown to effectively protect plants against different positive-sense RNA viruses and viroids. In the present study, the efficacy of external application of dsRNAs against the TSWV nucleocapsid (N) or the movement protein (NSm) coding genes in protecting plants against TSWV infection was evaluated. DsRNA molecules corresponding to portions of the viral genome were synthesized in vitro using a PCR approach followed by T7 RNA polymerase transcription. Almost all Nicotiana benthamiana plants (~90%) treated with TSWV N-derived dsRNAs remained asymptomatic and free of virus until 40 days post inoculation (dpi), while NSm-derived dsRNAs showed a lower efficacy (~30%). Preliminary data suggest that movement and stability of dsRNAs may be important factors for effectively induce resistance and that the different dynamic in systemic spread of dsRNA molecules, possibly linked to their primary sequence, could be responsible for the different efficacy of dsRNAs in protecting plants against viral infection. These results indicate that the choice of the viral target sequence in designing dsRNA-based vaccines against plant viruses is crucial to obtain virus resistance through exogenous application of dsRNAs.