Fig. 7: Mutations on novel putative AUX/IAA-binding sites in TIR1 impair auxin responses in vivo.

a Yeast two-hybrid (Y2H) interaction matrix for TIR1 wild type and TIR1-mutant versions carrying amino acid exchanges on relevant CAS-identified residues with ASK1, IAA7 and IAA12 at different auxin concentrations. b Box plots depicting root length of T1 tir1-1 mutant plants carrying TIR1p:tir1 mutant constructs. Transformed T1 seeds expressing RFP were selected by microscopy, and germinated in growth media. Five-day-old seedlings were transferred to growth medium containing either 40 nM 2,4-d or 12.5 nM IAA. Root elongation was traced on the day of transfer, and at days 3 and 5 after auxin treatment. tir1-1 mutants are resistant to auxin treatment exhibiting long roots, and auxin sensitivity in tir1-1 is restored by introducing a construct expressing wild type TIR1 under its own promoter. Numbers below the boxes correspond to the number of independent T1s (open circle’s) analyzed per construct. Solid black horizontal lines represent median, dark red dots mean values and whiskers correspond to the upper and lower ~25% (1.5*IQR) of the data points. Outliers are shown as solid black dots. Shadow rectangles represent the notch size (~95% confidence interval) of the two reference data sets (empty vector and TIR1 wild type). Conservation of mutated residues in the TIR1/AFB1-5 F-box subclade is depicted as gradient colored circles above the plots (TIR1: light pink, AFB1-4: gradient of darker pink, AFB5: deep purple).