Abstract
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.
Highlights
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
Developed a rationally designed antagonist, coronatine O-methyloxime (COR-MO; 6; Fig. 4a), of COI1–JAZ co-receptor based on a crystallographic structure of the ternary complex [20], and a proof-of-principle experiment could be carried out using COR-MO as a positive control
Coronatine [2] has been considered as a structural and biological mimic of JA-Ile [1], but several researchers have claimed it to have a broader range of functions that includes rapid chlorosis and plant defense responses in both COI1–JAZ-dependent and -independent manners [21,22,23]
Summary
Short (27-amino-acid) peptide fragments composed of Jas motifs of a JAZ protein were previously found to be sufficient for co-receptor formation, and so we designed a fluorophoreconjugated JAZ peptide for PPI detection of COI1–JAZ co-receptors according to the previously reported crystal structure of COI1–1–JAZ1 complex [7]. The r values of all these peptides bearing GST-COI1 increased upon addition of 2 in a dose-dependent manner, and the ratios of anisotropy changes (r/r0 Ϫ 1) in Fl-JAZ1 and OG-JAZ1 were higher than that in TMR-JAZ1. We prepared nine short peptides corresponding to the remaining subtypes (Fl-JAZs; Fig. S4) with Fl-JAZ13 as a negative control We applied this sensing system to evaluate the binding affinities of 2, 1, 3, 4, and 5 for all combinations of COI1 and 10 JAZ subtypes. No affinity values of 2 have been reported for JAZ2, -4, -9, -11, and -12 with COI1, pulldown experiments demonstrated that 2 caused PPI for these COI1–JAZ combinations, similar to the case of JAZ1 (Fig. S6). Affinity values for 1, 3, 4, and 5 with any COI1–JAZ co-receptors were previously unreported, and so our results are the first
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