Ratiometric Detection of SMAX1 Degradation in N. benthamiana.
(A) Diagram of the pRATIO3212-SMAX1 ratiometric reporter system. The 35S promoter drives expression of a multicistronic transcript encoding an SMAX1-mScarlet-I fusion that is separately translated from the Venus fluorescent protein due to the *F2A peptide. Relative fluorescence after transient expression of pRATIO3212-SMAX1, -SMAX1∆RGKT, and empty pRATIO3212 in N. benthamiana. The empty pRATIO3212 denotes pRATIO3212 lacking the Gateway cassette. Leaf discs were treated with 0.02% (v/v) acetone or 10 μM KAR1, KAR2, and rac-GR24 for 12 h before measurement. n = 4 to 7 leaf discs.
(B) Relative fluorescence after transient expression of pRATIO3212-SMAX1 in N. benthamiana and overnight treatment with KAR1 concentrations ranging from 200 nM to 10 μM. n = 4 to 6 leaf discs. *P < 0.01, Mann–Whitney U test, comparisons to control treatment.