Figure 3

RDR6-independent intracellular RNA silencing.

(a and b) The RDR6 gene does not affect replication of TCV-GFPΔCP. The RDR6 mRNA were analysed 12 days post-inoculation (dpi) by quantitative (q) RT-PCR, in triplicate, using total RNAs extracted from mock- or TCV-GFPΔCP-inoculated leaves of wild-type N. benthamiana (Nb) and NbRDR6i (RDR6i), transgenic GFP 16c (GFP16c) and GFP16c/RDR6i plants. Virus-induced intracellular RNA silencing targets TCV-GFPΔCP. TCV-GFP RNA was analysed by qRT-PCR in triplicate, using total RNAs extracted from mock- or TCV-GFPΔCP-inoculated GFP16c and GFP16c/RDR6i leaves at 12 dpi. N. benthamiana housekeeping GAPDH (a) and EF1α (b) transcripts were used as internal controls. Relative RNA levels of RDR6 or TCV were obtained by normalising against the baseline expression levels of GAPDH (a) and EF1α (b) mRNA, respectively and showed similar tendencies between the two internal controls. Student's t-tests were carried out to evaluate whether there would be any statistical significance in RNA levels between different biological samples (Table S1). (c and d) Fluorescence microscopic examination of gfp expression in a single epidermal cell in TCV-GFPΔCP-inoculated Nb (c) or NbRDR6i (d) leaves 6 dpi using a Zeiss Axiophot microscope through a green filter. Bar = 100 µm.