Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Transgenic expression of the P69 gene caused pleiotropic developmental defects. Three independent lines at rosette stage were shown. P69c displayed the strongest phenotype, followed by P69b and P69d, which was correlated to the level of P69 expression in these lines as detected by Northern blot hybridizations (see Figures 3A and 4B). Although siliques were small, all transformants were fertile.
• Supplemental Figure 2 - Suppression of PTGS by p69 in Agrobacterium coinfiltration experiments. This assay was carried out as described (Guo and Ding, 2002) by infiltrating 35S-GFP to the leaves of the GFP-expressing N. benthamiana line 16c to induce GFP RNA silencing in the presence or absence of viral suppressor. Constructs 35S-P69 (P69) and 35S-ΔP69 (-ΔP69) that respectively direct translation of the full-length and the first 45 codons of p69 were described in Methods. A blank vector plasmid (vector) or a plasmid carrying 35S-Cmv2b (2b; Guo and Ding, 2002) was also coinfiltrated as negative and positive controls. P69 expression led to higher levels of GFP mRNA and lower levels of GFP siRNAs in the coinfiltrated leaves both at 3 and 6 postinfiltration (lanes 3 and 8) than in the leaves coinfiltrated with 35S-ΔP69 (lanes 4 and 9). However, p69 was a weaker suppressor than 2b in this assay, possibly because N. benthamiana is a nonhost to TYMV, unlike CMV.
• Supplemental Figure 3 - (A) Comparison of the accumulation levels of miR156 and miR171 in wild-type C24 and lines P69c, P69d, and GFP142 (G). All of the three transgenic lines were in the C24 background. (B) Comparison of the accumulation levels of miR156, miR157, miR158, miR167, and miR171 in line L1 and its RdRP mutant sgs2 (Mourrain et al., 2000). The experiments were carried out as described in Figure 7A.
• Supplemental Figure 4 - The effect of TYMV infection on PTGS induced by an IR-RNA transgene targeting GFP. Both high (top panel) and low (bottom panel) molecular weight RNAs were extracted from plants carrying both the 35S-GFP (G) and 35S-IRGFP (IRG) transgenes before and 20 days after TYMV infection (+TYMV) and hybridized by GFP-specific probes. The position of a 21-nt RNA marker is indicated by a dot.
• Supplemental Figure 5 - Detection of miR167 in inflorescence, leaf, and stem tissues of wt C-24, P69c, or P69d plants.