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Supplementary Figure S1 Sasaki et al.
A
B
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Supplementary Figure S1. Analysis of 1.6x cis mutant.
A. Northern blot analysis of hairpin-derived siRNAs in 1.6x and 15.5a (T+S35S) cis mutants. A mutant defective in the DNA
methyltransferase DRM2, which contains the T+SWT transgene silencing system, is shown as a positive control for 21-, 22-,
and 24-nt hairpin-derived siRNAs. (Naumann et al., 2011). In this initial experiment, siRNAs were not observed in the two cis
mutants. Lanes 1.6x and drm2 are reproduced with permission of the Genetics Society of America (GSA). The copyright is
retained by the GSA.
B. Bisulfite sequence analysis of DNA methylation in the transgene target enhancer in the 1.6x cis mutant. No methylation
was observed. For the levels of methylation induced by the original unaltered silencer in
T+SWT plants, see Fig. 3c (T+SWT).
C. Reactivation of GFP expression in the 1.6x cis mutant (right). GFP is silenced in T+SWT plants (left).
D. Genotyping of Target and Silencer. +T/+S and -T/-S indicate presence and absence of the junction fragments containing
target (T) or silencer (S) transgenes and flanking plant DNA. Both junction fragments are present in T+S WT, 1.6x and 15.5a,
although the latter two have undergone partial deletions of the S transgene construct (Fig. 2; Fig. S2). Primers that amplify
the T construct-plant DNA and S construct-plant DNA junction fragments are shown in Table S2.
Reference: Naumann U. et al. (2011) Genetic evidence that DNA methyltransferase DRM2 has a direct catalytic role in RNAdirected DNA methylation in Arabidopsis thaliana. Genetics 187:977-979.