Figure 3.

Specific components of the RdDM pathway are required for PTGS-induced DNA methylation but not PTGS establishment. (A) GUS siRNA accumulation and GUS activity in leaves of 8-week-old plants of the indicated genotypes grown under short day conditions. RNA was extracted from a bulk of four plants for small RNA gel blots, and ethidium bromide staining is shown as loading control. Distinct boxes indicate that the samples were separated by tracks that are not relevant for this work. The original blot is presented in Supplementary Figure S3. GUS activity is expressed as fluorescent units per minute per microgram of total proteins quantified by Bradford. Averages and standard deviations correspond to 16 plants. (B) Percentage of DNA methylation at representative CHH and CHG methylation-sensitive enzymes of the 5′ and 3′ region of the GUS coding sequence in leaves of plants of the indicated genotypes grown under short day conditions. Analyses were performed at HaeIII-49, MspI-126, HaeIII-1467 and MspI-1529 (see Supplementary Figure S2). The percentage of DNA methylation was calculated as described in Figure 1D. Mean and standard deviation bars are based on two biological replicates.