Progression of geminivirus-induced transgene silencing is associated with transgene methylation.

•  The association of viral-induced gene silencing (VIGS) elicited by a DNA virus with DNA methylation of the silenced transgene was studied. •  35S-Green fluorescent protein (GFP) transgenic Nicotiana benthamiana were treated with an inhibitor of DNA methylation, 5-azacytidine (5-Aza-C), and VIGS of the transgene was observed upon inoculation with tomato golden mosaic virus carrying the GFP coding sequence. •  The onset of VIGS of the 35S-GFP transgene occurred 14-16 d after inoculation in both control and 5-Aza-C-treated plants. At this stage, the silencing was observed in localized regions. Silencing was uniform by 30 d after inoculation in plants that had methylated GFP-DNA, whereas plants that continued to display the same phenotype as seen at 14-15 d after inoculation had hypomethylated GFP-DNA. Viral expression of GFP persisted in pockets throughout the life of infected plants. •  This is the first demonstration of a correlation between post transcriptional gene silencing induced by a DNA virus, and transgene methylation. The results suggest that, while DNA methylation is not necessary for the initiation of silencing, the progression of silencing is affected by inhibition of DNA methylation.