Figure 1.

Impact of DCL RNAi on local hairpin dsRNA-mediated PTGS. A to I, Local PTGS assay. The left half of leaves of N. benthamiana (A), DCL1i (B), DCL2Ai and DCL2Bi (C and D), DCL3Ai and DCL3Bi (E and F), DCL4Ai and DCL4Bi (G and H), and DCL24i (I) plants were co-infiltrated with agrobacterium harboring p35S-GFP₇₁₄ and pRNAi-GFP₇₁₄ and the right half with p35S-GFP₇₁₄ and the pRNAi-empty vector. Without PTGS, GFP₇₁₄ expression from p35S-GFP₇₁₄ shows strong green fluorescence. Induction of PTGS by the hairpin GFP₇₁₄ dsRNA generated from pRNAi-GFP eliminates GFP₇₁₄ expression and the infiltrated lamina show red auto-fluorescence. DCL4 RNAi attenuated intracellular silencing and the level of PTGS decreased in DCL4Ai, DCL4Bi, and DCL24i plants. Some green fluorescence is still visible in the infiltrated lamina of these plants (G–I). Photographs were taken under long-wavelength UV light (top) or through a stereo-fluorescent microscope (bottom) at 4 dpa. Bar = 100 μm. The ratio in the bottom right corner of each panel indicates the number of plants out of the number of agro-infiltrated plants that developed local PTGS in two experiments. The green fluorescence intensity was measured using ImageJ software (Supplemental Fig. S4). J, Analysis of GFP₇₁₄ mRNA in DCL RNAi lines. Northern detection was performed using GFP₇₁₄-specific probes (top). Equal loading of total RNAs extracted from leaf tissues at 4 dpa is indicated by the equal amount of 18S and 28S rRNAs shown on the gel (bottom). The position and size of GFP₇₁₄ mRNA and 18S and 28S rRNA are indicated. K, Detection of local L-siRNAs. L-siRNAs were analyzed by small RNA northern blots (top). Equal loading of sRNAs extracted from leaf tissues at 4 dpa is indicated by the equal amount of 5S rRNA/tRNAs shown on gel (bottom). The position of 21, 22, and 24 nt siRNA as well as 5S rRNA/tRNAs are indicated.