Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA

Tobacco plants were transformed with constructs in which the transgene was a cDNA of replicating potato virus X (PVX) RNA. The constructs, referred to here as amplicons, were the intact genome of PVX and PVX constructs modified to carry the beta-glucuronidase (GUS) reporter gene either as an additional gene or as a replacement for the coat protein gene (PVX/GUS/CP and PVX/GUS respectively). Transformed plants carrying these constructs displayed several phenotypes that we attribute to post-transcriptional gene silencing. These phenotypes include the absence of viral symptoms, low accumulation of transgene-derived RNA, extreme strain-specific resistance against PVX, low and non-uniform GUS expression (in the PVX/GUS and PVX/GUS/CP plants) and suppression of transiently expressed RNA sharing homology with the transgene. Importantly, the amplicon-mediated gene silencing was exhibited in all lines tested. There was no evidence of gene silencing in seven lines expressing a PVX RNA that was unable to replicate. From these data we conclude that the replicating viral RNA is a potent trigger of gene silencing. Moreover, amplicon-mediated gene silencing provides an important new strategy for the consistent activation of gene silencing in transgenic plants.