Fig. 1
From: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
SYNV reverse genetics systems using a truncated 35S promoter and 5′ hammerhead ribozyme to process viral antigenomic (ag) RNAs. a Schematic representation of the SYNV full-length agRNA and a minireplicon (MR) derivative (SYNV-MRGFP-RFP) containing GFP and RFP genes replacing the N and P ORFs, respectively. The grey illustrations depict the cauliflower mosaic virus 35S promoter-based transcription plasmids (35S-HH1) for posttranscriptional processing of agRNAs with a hammerhead ribozyme variant (HH1) to yield precise uncapped 5′ termini, and a truncated promoter (35St) for synthesis of SYNV agRNAs with capped precise 5′ termini. Both constructs employed a hepatitis delta virus (HDV) antigenomic ribozyme followed by a NOS terminator to release the exact agRNA 3′ termini. b Illustration of 5′ terminus processing by HH1 Rz (upper panel) and 35St (lower panel). The sequences of the HH1 Rz and the 35St promoter are shown in black, and the 5′ termini of SYNV leader are shown in red. Arrows indicate the HH1 Rz cleavage site in 35S-HH1, or the transcription start site of the 35St. c Comparison of reporter gene expression mediated by 35S-HH1-MR and 35St-MR reporters in agroinfiltrated leaves. Agroinfiltration mixtures for delivery of the 35S-HH1-MR or 35St-MR transcription plasmids were infiltrated into N. benthamiana leaves. In the left panels, Agrobacteria mixtures harbour plasmids for expression of the N, P, and L core proteins (N + P + L) and viral suppressors of RNA silencing, and the right panels illustrate results of parallel experiments in which the core protein plasmids were omitted from the Agrobacteria mixtures (No N + P + L). Expression of fluorescent reporters in infiltrated leaf tissues at 6 days postinfiltration (dpi) was monitored by fluorescence microscopy under GFP (upper panels) and RFP (lower panels) channels. d Protein gel blots of GFP and RFP recovered from agroinfiltrated leaf extracts assessed by the GFP- and RFP-specific antibodies. The protein blots were stained with Ponceau S to verify similar levels of the large subunit of Rubisco (Rub L)