A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1–JAZ co-receptor in plants

The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine regulates many developmental and stress responses in plants and induces protein–protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activate jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescence anisotropy–based in vitro binding assay system to screen for the ligands of the COI1–JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid, and 12-hydroxyjasmonoyl-l-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader–based assays of both agonists and antagonists of COI1–JAZ co-receptors.