File:Figure1. RSS3, bHLH, and JAZ Form a Ternary Complex.jpg

(A) Yeast three-hybrid assay to verify interaction of RSS3N318, bHLH094, and JAZ9. Representative results of more than six independent transformants are shown for each combination. D21 represents the 21–amino acid deletion mimicking rss3 mutation. The control and selective medium were as described in Figure 5. Media lacking Met (-M) was used for induction of the gene encoding the 3rd protein by MET25 promoter for testing interaction between the three proteins expressed. “Empty” means the vector control. 3AT, 3-amino-1,2,4-triazole

(B) and (C) Interaction among JAZ9, RSS3N203, and bHLH094, as analyzed by BiFC-apFRET. Representative images (B) and FRET efficiencies (C) of apFRET in onion cells using YFPN-RSS3N203, bHLH094-YFPc, and ECFP-JAZ9 or nonfused ECFP are shown. Pseudocolor images represent fluorescence intensities. Bar = 10 µm. Mean ± sd; n = 3 (ECFP) and 6 (ECFP-JAZ9). Asterisks indicate P value < 0.05 (Student’s t test).