Figure 4. Mutations at Auxin, Aux/IAA and InsP₆ Binding Sites Impair Auxin-Dependent TIR1-Aux/IAA Interaction And Compromise TIR1 Function In Vivo.

a. Side view of auxin (green) and IAA7 peptide (yellow) (left), auxin and InsP₆ (rainbow) (middle), and InsP₆ (right) binding sites in TIR1 (grey). Selected ligand-interacting residues in TIR1 are shown in mostly white stick representation. b. Yeast-two hybrid ASK1, and auxin-induced IAA7 interactions with wild type (WT) TIR1 or TIR1 carrying mutations on ligand-binding sites. c. Five-day-old seedlings grown on MS media were transferred to media containing either 40 or 80 nM 2,4-D. Root elongation was measured after additional 4 days and expressed as a proportion of growth in the absence of auxin (scale bars represent STDEV). Auxin inhibits root growth elongation in wild type plants (Col-0) but tir1-1, as well as the tir1-1afb2–3 auxin-receptor mutants are resistant to auxin treatment. Auxin resistance in tir1-1 mutants is reverted by introducing TIR1 wild type fused to the β-glucuronidase (GUS) reporter gene, under the TIR1 promoter. Interrupted lines indicate that unlike TIR1p:TIR1:GUS, versions of TIR1 that carry mutations in ligand binding sites are unable to restore auxin sensitivity in tir1-1.