Figure 4.

Mutation of TGMV Rep Ser-97 reduces viral replication. Protoplasts prepared from tobacco suspension cells (NT1) were transfected with DNA (10 µg) corresponding to an AL4-knockout TGMV-A replicon (lanes 1–3) or AL4-knockout replicons containing the Rep S97D (lanes 4–6), S97N (lanes 7–9), or R94G (lanes 10–12) mutation, all of which impair SnRK1 phosphorylation of Ser-97. Total DNA was extracted at 3 d post transfection, cut with XhoI, and digested with DpnI to cut Dam-methylated input plasmid DNA but not nascent viral DNA. The DNA was analyzed on a DNA gel blot using a radiolabeled TGMV-A probe (top left gel). A longer exposure (10-fold; lanes 13–15) shows weak signals from the Rep S97D mutant. The bottom gel shows ethidium bromide-stained, digested DNA before transfer. wt, Wild type.