Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation

Previous analysis of potato spindle tuber viroid (PSTVd) RNA-infected tobac co plants has suggested that an RNA-DNA interaction could trigger de novo m ethylation of PSTVd transgene sequences, Using the genomic sequencing techn ique, the methylation pattern associated with the RNA-directed DNA methylat ion process has been characterized. Three different PSTVd transgene constru cts all showed a similar pattern of methylation, Most of the cytosines at s ymmetrical as well as non-symmetrical positions appeared to be methylated i n both DNA strands of the viroid sequences. Heavy methylation was mostly re stricted to the viroid cDNA sequences. Flanking DNA regions immediately adj acent to the viroid cDNA displayed a lower but significant level of cytosin e methylation, The observation that the heavy methylation was essentially c o-extensive with the length of the PSTVd cDNA sequences provided evidence t hat a direct RNA-DNA interaction can act as a strong and highly specific si gnal for de novo DNA methylation. These data also confirmed that de novo me thylation was not limited to canonical CpG and CpNpG sites, but can also in volve all the cytosine residues located in the genomic region where the RNA -DNA interaction takes place.