Yeast two-hybrid and three-hybrid assays

The yeast two-hybrid screens were performed as described previously (Zhang et al., 2012). To reduce autoactivation, 100 mM 3-AT (3-amino-1,2,4-triazole) was added to the selection medium. We detected no auto-activation of BD-REV in the presence of 3-AT. A yeast three-hybrid assay was performed according to the manufacturer's instructions; the yeast strain AH109 was transformed with pairs of plasmids (pB-REV-DRN/DRNL and pA-ZPR3 or pB-REV-DRN/DRNLΔAP and pA-ZPR3).

Transformed colonies were selected on synthetic complete medium lacking Leu and Trp. Three independent clones with four respective replicates were used in each experiment. Cell cultures were placed under the indicated light conditions and incubated at 30°C until the OD₆₀₀ was between 0.5 and 0.8 with the conditional expression of the bridge proteins. The relative β-galactosidase activities were calculated as described previously (Ding et al., 2015). At least three independent experiments were performed and the result of one representative experiment is shown.