A DNA target of 30 bp is sufficient for RNA-directed DNA methylation

In higher plants, RNA-DNA interactions can trigger de nova methylation of g enomic sequences via a process that is termed RNA-directed DNA methylation (RdDM), In potato spindle tuber viroid (PSTVd)-infected tobacco plants, thi s process can potentially lead to methylation of all C residues at symmetri cal and nonsymmetrical sites within chromosomal inserts that consist of mul timers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we fou nd that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de nova cytosine methylation is not limited to the canonical CpG, CpNpG sit es. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methy lated in nearly all tobacco leaf cells, With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation w as significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two differ ent 60-bp constructs further suggested that the sequence identity of the ta rget may influence the methylation mechanism. Finally, a link between viroi d pathogenicity and PSTVd RNA-directed methylation of host sequences is pro posed.