DETAILED DYNAMICS OF THE INITIAL PHASES OF DE NOVO RNA-DIRECTED DNA METHYLATION IN PLANT CELLS

De novo establishment of repressive cytosine marks by RNA-directed DNA methylation is essential for control of chromatin activity in plants. Here we show the first detailed description of the initial phases of this process made possible by our unique system based on tobacco BY-2 cell lines. We found that these cells respond very synchronously to the induced expression of an RNA hairpin guiding the methylation machinery to the CaMV 35S promoter. High level of promoter-specific siRNAs triggered rapid methylation, observed already twelve hours after the induction. This methylation was independent of the prior presence of methylcytosines at the target locus. Methylation dynamics and targeting accuracy varied depending on the cytosine sequence context; CHH peaked at about 80% in two days and then declined, whereas CG and CHG methylation needed more time, but after ten days CHG reached practically 100%. Pol IV was not involved in these initial phases, as deduced from the sRNA profile. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was fully suppressed in two days.