Resistance to multiple viruses in transgenic tobacco expressing fused, tandem repeat, virus-derived double-stranded...

Transgenic tobacco plants expressing fused, tandem, inverted-repeat, double-stranded RNAs derived either from the three viruses [potato virus Y (PVY), potato virus A (PVA), and potato leafroll virus (PLRV)] or the five viruses [PVY, PVA, PLRV as well as tobacco rattle virus (TRV), and potato mop-top virus (PMTV)] were generated in this study to examine whether resistance could be achieved against these three viruses or five viruses, respectively, in the same plant. The transgenic lines were engineered to produce 600- or 1000-bp inverted hairpin transcripts with an intron, in two orientations each, which were processed to silencing-inducing RNAs (siRNAs). Fewer lines were regenerated from the transformants with either 1000-bp inverted hairpin transcripts, or a sense-intron-antisense orientation versus antisense-intron-sense orientation. Resistances to PVA and two strains of PVY (-O and -N) were achieved in plants from most of lines examined, as well as resistance to co-infection by a mixture of PVY-O and PVA, applied to the plants by either rub inoculation or using aphids. This was regardless of the orientation of the inserted sequences for the 600-bp insert lines, but only for one orientation of the 1000-bp insert lines. The lines containing the 1000-bp inserts also showed resistance to infection by TRV inoculated by rub inoculation and PMTV inoculated by grafting. However, all the lines showed only low-to-moderate (15-43%) resistance to infection by PLRV transmitted by aphids. The resistances to the various viruses correlated with the levels of accumulation of siRNAs, indicating that the multiple resistances were achieved by RNA silencing.