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

Abstract

Edited by Joseph M. Jez 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-Lisoleucine. 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.

Extended JAZ degron sequence for plant hormone binding in jasmonate co-receptor of tomato SlCOI1-SlJAZ

“…To examine the effect of the exo-degron sequence quantitatively, we designed and synthesized fluorescein-tagged SlJAZ1-11/13 degron short peptides (SlJAZP1-11/13) of 27 amino acids (Figure 3a and Figure S4 -S5) based on previous work on Arabidopsis COI1-JAZ. 22 The pull-down assay using the GST-SlCOI1 and Fl-SlJAZ degron peptides in the presence of increasing concentrations of JA-Ile yielded very similar results to those obtained using full-length SlJAZs (Figures 2b-d). Therefore, the affinity of full-length JAZ was confirmed to depend on the sequence in these short peptides.…”

“…[19][20][21] Small differences in the Arabidopsis JAZ degron sequences (identity 37.0% in Figure 1a) are considered to account for the difference in affinity (KD) between JAZ and COI1-JA-Ile. 22 However, the 12 JAZs of Solanum lycopersicum have a remarkably wellconserved JAZ degron sequence (V/L)P(Q/I)AR(R/K) (identity 46.6% in Figure 1a), suggesting their affinities (KD) for the SlCOI1-JA-Ile complex are very similar (Figure 1a). Here, we report the first comprehensive study of the affinity of the SlJAZ-SlCOI1 co-receptor for JA-Ile, and demonstrate that the perception of JA-Ile depends on the extended JAZ degron sequence (V/L)P(Q/I)AR(R/K)XSLX.…”

Recent Advances in Plant Chemical Biology of Jasmonates

“…Thus, chemical control of individual TF or JAZs in the upstream pathway could be an efficient strategy. Progress in the development of novel chemical tools will advance the tuning and regulation of jasmonate responses [85].…”

Heterologous production of fungal natural products: Reconstitution of biosynthetic gene clusters in model host <i>Aspergillus oryzae</i>

“… 2 ) Recently, this toxin was rediscovered as an agonist of jasmonoyl- l -isoleucine, a genuine bioactive form of plant hormone jasmonic acid, and has played an important role in the recent progress of jasmonic acid research, as in the discovery of the coi1 mutant and the identification of the jasmonic acid receptor. 3 ) Although the actual role of phytotoxins is still difficult to uncover, elucidation of the function of phytotoxins is an important issue for understanding the defense response between plants and phytopathogenic microbes.…”