Fig. 2

Effects of HH Rz optimization on minireplicon reporter gene expression. a Sequences and predicted RNA secondary structures formed by HH Rz variants preceding the 5′ termini SYNV agRNA sequence. The cleavage triplets in HH1, HH2 and HH3, as well as the inactive GUG triplet in HHm are shown in bold letters, and the 5′ termini of SYNV leader are in red. Arrows indicate cleavage site, and the three stem structures (I, II and III) are shown in the HH1 Rz. b Analysis of RNA cleavage in vitro by three intact HH Rzs and a mutant version (HHm). The cleavage products were resolved on a polyacrylamide gel and visualized after ethidium bromide staining. All three HH Rzs efficiently cleaved the fused SYNV RNAs, resulting in disappearance of input RNAs and concomitant release of substrate strands and short ribozyme RNAs. c Semi-quantitative RT-PCR analysis of ribozyme cleavage efficiency in vivo. The MR agRNAs with 5′ terminus fusions to the HH Rz variants were produced in plant tissues via agroinfiltration mediated expression. The uncleaved transcripts were detected by RT-PCR after 28 cycles of amplification using primers annealing to the ribozyme and the SYNV 5′ termini sequence. Actin cDNA amplification products were used as a control. d and e Improved MR reporter expression activity by using optimized HH Rzs. N. benthamiana leaves were agroinfiltrated with MR constructs containing the indicated HH Rz variants and necessary supporting plasmids as described in Fig. 1 legends. Fluorescent foci were monitored under fluorescence microscope at 9 dpi (d) and the expression levels of GFP and RFP were evaluated by western blot analysis (e). Coomassie blue-stained Rubisco large subunit (Rub L) is shown to indicate equal protein loading