From the Department of Chemistry, Graduate School of Science and
Precursory Research for Embryonic Science and Technology (PREST), Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
J Biol Chem. 2019 Mar 29;294(13):5074-5081. doi: 10.1074/jbc.RA118.006639. Epub 2019 Feb 6.
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 subtypes are encoded in the genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of 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 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.
植物激素 (+)-7-异茉莉酰基-l-异亮氨酸调节植物的许多发育和应激反应，并诱导 COI1（E3 泛素连接酶的 F 框成分）与茉莉酸 ZIM 结构域（JAZ）抑制剂之间的蛋白质-蛋白质相互作用。这些相互作用导致 JAZ 降解并激活茉莉酸（JA），导致植物防御反应、生长抑制和衰老。该基因在基因组中编码了 13 个亚型，但这些 JAZ 亚型的生理功能的详细了解仍然不清楚，部分原因是基因的遗传冗余。阐明复杂的 JA 信号通路的一种策略是开发一种与所有共受体组合的配体的可靠且综合的结合测定系统。在此，我们报告了一种基于荧光各向异性的 COI1-JAZ 共受体配体筛选的结合测定系统的开发。我们的测定法首次实现了对各种内源性 JA 衍生物（如冠菌素、茉莉酸和 12-羟基茉莉酰基-l-异亮氨酸）的亲和力值和 JAZ 亚型选择性的定量分析。由于其高信噪比和方便的混合读取测定系统，我们的筛选方法可用于基于平板读数器的 COI1-JAZ 共受体激动剂和拮抗剂的测定。
