Development of the HTRF assay to evaluate the auxin-induced binding between TIR1 and IAA7.

Auxin plays diverse roles in plant growth and development, including sensing environmental changes. Quantifying the interaction between auxin coreceptors provides the molecular basis for cells to sense and adapt to environmental cues. Although several assays are available, a more high-throughput method is necessary to efficiently evaluate the auxin-induced binding of coreceptors. We developed a homogeneous time-resolved fluorescence (HTRF) assay to quantitatively measure the binding between the Arabidopsis thaliana TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and indole-3-acetic acid 7 (IAA7) auxin coreceptor proteins. The HTRF assay provides a rapid analysis with sensitivity similar to the enzyme-linked immunosorbent assay. We demonstrated its effectiveness by analyzing the potency of several auxin analogs to induce binding between TIR1 and IAA7. We also found that a mild increase in temperature impairs the binding activity of TIR1 to IAA7. This method provides a rapid and robust tool to evaluate the auxin-induced binding between TIR1 and Aux/IAA auxin coreceptors. A similar strategy may also be applicable to study other plant hormone heterodimer coreceptors.