Fig. 4. Validation of interaction between different regulators in MYB–bHLH–WD40 complexes.

a Yeast two-hybrid assays. -W/-L: SD-Trp-Leu medium; -W-L-H-A(X-gal): SD-Trp-Leu-His-Ade+(X-a-Gal) medium. b Bimolecular fluorescence complementation assays. Scale bars are 100 μm. c Split luciferase complementation assays in Nicotiana benthamiana leaves. L: Gly/Ser linker, rbs: transcription terminator. The data represent averages of four experiments. Asterisks (*) indicate the statistical significance of the difference between the experimental and the control groups. (Student’s t test, *P < 0.05, **P < 0.01). d Yeast three-hybrid assays. MYBA and MYB4 were inserted into the vector pGADT7. TTG1 was recombined into pBridge at MCS I; bHLH3 or GL3 was recombined into pBridge at MCS II. Interactions were analyzed on SC-Leu-Trp-Met-His medium containing the indicated concentrations of 3-AT (0 and 30 mM). Transformed yeast cells were diluted 10-fold, 100-fold, and 1000-fold on the medium