Fig 4. Detection of CP-RT in vitro and in vivo.

(A) Schematic representation of sgRNA4 and its UGG and 2st mutant clones under a T7 promoter. (B) SDS-PAGE of proteins translated in wheatgerm extracts from in vitro transcripts of sgRNA4 and its two mutants. Mock–no RNA added. After drying the gel, proteins radioactively labelled with [³⁵S]Met were detected using a phosphorimager. (C) Schematic representation of the AV2 RNA3 with a tag (grey rectangle) appended to the 3′ end of the CP-RT gene. (D) Detection of CP and CP-RT-myc by western immunoblotting in plants infected with AV2 or AV2-myc. Samples were collected from the inoculated leaf and the 2nd upper non-inoculated leaf at 4 and 7 dpi, and the 3rd upper non-inoculated leaf (see Fig 3B) at 14 dpi. (E) Detection of CP and CP-RT-myc by western blotting in extracts of plants infected with AV2, AV2-myc or AV2-2st-myc. Samples were collected from the 2nd upper non-inoculated leaf at 4 dpi (see Fig 3B). Positions of CP and CP-RT-myc are indicated on the left. Sizes of molecular weight markers are indicated on the right in panels B, D and E. Ponceau red staining of the large Rubisco subunit (RbcL) was used as a loading control in panels D and E.