Post-transcriptional gene silencing and virus resistance in Nicotiana benthamiana expressing a Grapevine virus A minireplicon

Grapevine virus A (GVA) is closely associated with the economically important rugose-wood disease of grapevine. In an attempt to develop GVA resistance, we made a GFP-tagged GVA-minireplicon and utilized it as a tool to consistently activate RNA silencing. Launching the GVA-minireplicon by agroinfiltration delivery resulted in a strong RNA silencing response. In light of this finding, we produced transgenic Nicotiana benthamiana plants expressing the GVA-minireplicon, which displayed phenotypes that could be attributed to reproducibly and consistently activate post-transcriptional gene silencing (PTGS). These included: (i) low accumulation of the minireplicon-derived transgene; (ii) low GFP expression that was increased upon agroinfiltration delivery of viral suppressors of silencing; and (iii) resistance against GVA infection, which was found in 60%, and in 90-95%, of T1 and T2 progenies, respectively. A grafting assay revealed that non-silenced scions exhibited GVA resistance when they were grafted onto silenced rootstocks, suggesting transmission of RNA silencing from silenced rootstocks to non-silenced scions. Despite being extremely resistant to GVA infection, the transgenic plants were susceptible to the closely related vitivirus, GVB. Furthermore, infection of the silenced plants with GVB or Potato virus Y (PVY) resulted in suppression of the GVA-specific defense. From these data we conclude that GVA-minireplicon- mediated RNA silencing provides an important and efficient approach for consistent activation of PTGS that can be used for controlling grapevine viruses. However, application of this strategy for virus resistance necessitates consideration of possible infection by other viruses.