FIG 7.

Mutating the conserved lysine impairs GFP replicon production in the 2IRGFP plants by Rep. (A) Diagram depicting the production of circular extrachromosomal IR-GFP molecules (ECMs) from the 2IRGFP cassette by Rep activity on IR and, subsequently, GFP expression from these ECMs driven by the 35S promoter. (B) UV images revealing GFP accumulation due to activity of WT Rep and the K-to-A variants. Images were taken at 4 days after agroinfiltration. Similar results were obtained in three independent replicates; in each experiment, every construct was infiltrated in two leaves of two different plants (n = 4). One representative leaf is shown per construct. (C) Immunoblot of a total protein extract from agroinfiltrated 2IRGFP leaves to determine the Rep-RFP protein levels (anti-RFP). To confirm equal protein loading, the membranes were stained with Ponceau S. (D) Real-time PCR quantification of ECMs in total DNA extracted from 2IRGFP agroinfiltrated leaf areas. Normalized ECM values on the y axis indicate the ECM copy number normalized to the internal control (25S rRNA gene) to correct for differences in template DNA.