Cytosine methylation is associated with RNA silencing in silenced plants but not with systemic and transitive RNA silencing through grafting

RNA silencing is often associated with methylation of the target gene. The DNA methylation level of transgenes was investigated in post-transcriptionally silenced or non-silenced Nicotiana benthamiana carrying either the 5′ region (200 or 400 bp) or the entire region of the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Higher levels of transgene cytosine methylation were observed in both symmetrical (CpG, CpNpG) and non-symmetrical (CpHpH) contexts (CpG > CpNpG > CpHpH) in silenced lines, but there was very lower levels or no transgene methylation in non-silenced lines. RNA silencing was induced in non-silenced scions from silenced rootstocks and spread to the 3′ region of the transgene mRNA (Haque et al., Plant Mol. Biol. 2007; 63: 35–47). In this system, transgene methylation levels were analyzed in scions at different time intervals after being grafted onto silenced or non-silenced rootstocks to investigate if transgene methylation was associated with induction or transitivity of RNA silencing. We observed that, there was no change of transgene methylation level in the initial target or in extended regions in scions. These results showed that transgene methylation was associated with RNA silencing in individual transformants, but it was not associated with systemic RNA silencing and/or transitive RNA silencing through grafting.