Fig. 3

Efficiency of capped and HH3 Rz-processed agRNAs in supporting MR reporter gene expression. a Visualization of cells expressing GFP and RFP in N. benthamiana leaves infiltrated with agrobacteria harbouring 35S-HH3-MR or 35St-MR along with equal mixtures carrying core protein and VSR supporting plasmids. Fluorescent reporter expression was monitored at 6 and 9 dpi with an epifluorescence microscope. b Total protein extracts prepared from infiltrated leaves at 9 dpi were analysed in Western blots using anti-GFP and anti-RFP antibodies. Coomassie blue staining of the large subunit of Rubisco (Rub L) was used as loading control. c GFP and RFP mRNAs in infiltrated leaf tissues at 9 dpi were analysed by Northern blotting using probes against GFP and RFP sequences. Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used as RNA loading control. d and e The transcript levels of MR and full-length agRNAs were analysed by qRT-PCR. Total RNA samples were isolated from N. benthamiana leaves infiltrated with 35S-HH3-MR or 35St-MR (d), or 35S-HH3-SYNV or 35St-SYNV (e), together with VSR mixtures. Two pairs of primers specific for GFP and RFP were used to amplify MR cDNAs, while three pairs of N, P and G specific primers were used to amplify SYNV full-length cDNA