Analysis of the nucleocapsid protein gene from Tomato spotted wilt virus as target and inducer for posttranscriptional gene silencing

Nicotiana benthamiana was transformed with the nucleocapsid protein (N) gene of Tomato spotted wilt virus (TSWV). Out of 17 transgenic lines regenerated, 6 showed virus resistance against TSWV and recombinant Potato virus X containing the N gene (PVX.N) and accumulated relatively low steady-state levels of mRNA of the N transgene, suggesting that the resistance was manifested by a posttranscriptional gene silencing. The targets for RNA degradation in the resistant lines were mainly concentrated in the 3′ portion. Non-transgenic N. benthamiana infected with PVX.N showed resistance to TSWV. On the other hand, no resistance to TSWV was observed in plants infected with PVX containing a sequence non-homologous to TSWV. These results suggest that the protection was RNA-mediated. Both 5′ and 3′ portions of the N gene were shown to be efficient inducers for the RNA-mediated cross-protection. When a non-silenced line was inoculated with recombinant PVX containing various regions of the N gene, virus-induced gene silencing (VIGS) of the N transgene mRNA was observed. Furthermore, it was shown that the 5′ portion of the N gene induced VIGS more efficiently than the 3′ portion.