Abstract: We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3' non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5' 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5' or 3' silencing inducer rootstocks. When non-silenced scions were grafted onto 5' silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3' region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3' silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3' region of the transgene and did not spread to the 5' region. In addition, results from crossing experiments, involving non-silenced and 3' silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5'-3' direction, not in the 3'-5' direction, along the transgene mRNA.

Abstract: 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.