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.

The phytohormone (ϩ)-7-iso-jasmonoyl-L-isoleucine (JA-Ile 3 ; 1) regulates many developmental and stress responses in plants (1)(2)(3)(4). Upon attack by insects or fungal pathogens, JA-Ile (1) is biosynthesized and induces protein-protein interaction (PPI) among COI1, the F-box component of Skip/Cullin/F-box (SCF COI1 ) E3 ubiquitin ligase, and the JAZ repressors, which are ubiquitinated and then targeted for degradation by the 26S proteasome. Subsequently, a different set of transcription factors targeted by JAZs activate the expression of JA-responsive genes, leading to plant defense and growth inhibition or senescence responses (5)(6)(7). COI1 and 13 subtypes of JAZ are encoded in the genome of Arabidopsis thaliana, and 1 can induce PPI between COI1 and most JAZ subtypes to cause various physiological responses. However, the detailed physiological functions of these JAZ subtypes remain unclear, mainly due to the complexity of the JA-mediated signaling cascade, the genetic redundancy of JAZ genes, and signaling cross-talk with other phytohormones (8). In addition, functional compensation by other JAZ subtype in jaz knockout mutants cause difficulties in the functional analysis of each JAZ subtype (9). Nonetheless, a few examples of specific functions of JAZ proteins have been described, e.g. JAZ2 as a controlling factor of stomata dynamics and JAZ9 as a key regulator for defense responses against necrotrophic pathogens (8, 10 -13), indicating that specific JAZ proteins regulate distinct transcription factors and downstream responses. The balance between redundant and specific mechanisms of JA signaling likely contributes to finetuning of the numerous JA-regulated responses and plant adaptation to the environment. Accordingly, practical chemical tools for the study of JA-mediated signaling cascades such as JAZ subtype-selective ligands are needed (13).
To discover such ligands, reliable and high-throughput in vitro assay systems of the ligands for all combinations of COI1-JAZ co-receptors must be prepared. One conventional method is the yeast two-hybrid system. Although capable of screening ligands against most combinations of COI1-JAZ co-receptors, false positives and negatives could also be found, in part due to the use of live yeast cells, the performance of which is affected by factors unrelated to binding affinity such as cell-penetration efficiency of the ligands and cell growth inhibition. In vitro or semi-in vivo pulldown assays can also be used for direct analyses for PPI between phytohormone co-receptors, although they are qualitative in nature and of low-throughput performance. Herein, we disclose a fluorescence anisotropy (FA)-based in vitro binding assay system for the ligands of COI1-JAZ co-receptors. FA has been widely used for the quantitative and highthroughput screening (HTS) of PPI inducers or inhibitors (14,15), and by applying it to the assay system for COI1-JAZ co-receptors, quantitative analyses of the affinity values of endogenous JA derivatives covering almost all the combinations of COI1-JAZ co-receptors were achieved for the first time. Moreover, this system has a sufficiently good signal-to-noise ratio and is capable of mix-and-read assay, and thus is amenable to a plate reader-based assay of COI1-JAZ co-receptor agonists and antagonists.

Fluorescent anisotropy-based binding assay for ligand of COI1-JAZ co-receptors
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 conjugated peptide was composed of 27 amino acids (ϳ3,500 Da; Fig. S1), and Oregon Green (OG) was attached to the additional Cys on the N terminus of the Jas motif (OG-Cys-JAZ) (13), a binding domain of JAZ proteins with COI1. The molecular masses of these conjugated peptides were significantly smaller than those of PPI complexes ( Fig. 1b; ϳ100 kDa), and thus a dramatic increase in FA can be expected upon formation of the COI1-ligand-JAZ peptide ternary complex ( Fig. 1, b and c). Initially, in a proof-ofprinciple, positive control experiment, we observed the FA (r value) change of OG-Cys-JAZ1 and COI1-GST upon addition of coronatine (COR; 2), a functional mimic of JA-Ile and strong binder of COI1-JAZ co-receptor (16). As shown in Fig. 1d, the r value was relatively low (ϳ0.092) when OG-Cys-JAZ1 and COI1-GST were dissolved but increased by 1.9-fold upon the addition of 2. In contrast, little or no anisotropy change was observed upon addition of ent2 as the negative control. Therefore, OG-Cys-JAZ1 can monitor the formation of COI1-2-JAZ ternary complex.
We next sought to enhance the sensitivity of this assay system by examination of the structures of fluorescently labeled JAZ peptides, specifically (i) the position of the fluorophore (N or C terminus of the peptide), (ii) the linker structures between fluorophores and peptides, and (iii) the structures of the fluorophores (Figs. 2a and S2). In the case of JAZ1-Cys-OG, in which OG was introduced at the C terminus through Cys (Fig.  2b), almost no anisotropy change was observed upon addition of 2 (Fig. S3a), although 2 caused PPI between co-receptors, similar to the case of OG-Cys-JAZ1 (Fig. S3b), indicating that the fluorophore should be conjugated to the N-terminal position of the JAZ peptide. Next, we examined the effect of linker structure/length between OG and each N terminus of JAZ1 (OG-EG4, EG2-JAZ1, and OG-JAZ1) (Fig. 2b). It was found that the ratio of anisotropy change (r/r 0 Ϫ 1) depended upon the linker length (without linker (OG-JAZ1; 1.

Ligand screening system for jasmonate co-receptor
nary complexes, and thus the differences in the r values of these peptides should be derived from the microenvironmental mobility of the fluorophore in the corresponding ternary complexes. We also designed two peptides bearing fluorescein (Fl) and tetramethylrhodamine (TMR) instead of the original OG (Fl-JAZ1 and TMR-JAZ1; Fig. 2b). As shown in Fig. 2d, 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/r 0 Ϫ 1) in Fl-JAZ1 and OG-JAZ1 were higher than that in TMR-JAZ1. The Fl-JAZ1 with GST-COI1 also showed no change in r values upon addition of ent2 as the negative control, and thus Fl-JAZ1 as well as original OG-N-Cys-JAZ1 specifically detected the agonist-induced PPI between COI1 and JAZ1. Overall, we found that Fl-JAZ1 as well as the original OG-N-Cys-JAZ1 worked well as novel FA-based sensors for COI1-JAZ co-receptor formation. In the following experiments, we used Fl-JAZ1 as it is less expensive to acquire.

Quantitative binding analyses of jasmonates with COI1-JAZ co-receptors
We next applied the FA-based sensing system to the quantitative analyses of the binding affinities of various natural jasmonate derivatives bearing COI1-JAZ co-receptors. From the saturation binding curve, the apparent K d value of 2 with Fl-JAZ1 and GST-COI1 was calculated to be 5.0 nM (Fig. 3a). A sample of synthetic JA-Ile (3) containing (ϩ)-7-iso-JA-L-Ile (1) and (Ϫ)-JA-L-Ile pure diastereomeric isomers in a 95:5 ratio (Fig. 3b) and purified bioactive stereoisomer (ϩ)-7-iso-JA-L-Ile (1) also increased the r value in a dose-dependent manner. The K d value of 1 was 29 nM, which is significantly lower than that of the mixture, 3 ((Ϫ)-JA-L-Ile; 824 nM), and these results are consistent with the previous report (4). In contrast, little or no change was observed upon addition of either jasmonic acid (4) or 12-hydroxy-JA-L-Ile (5) (Figs. 3b and S5), which is consistent with previous reports, although the K d values of 2 and 3 are almost a single order of magnitude lower than the reported values (48 nM for 2 and 18 M for 3).
Thirteen JAZ gene subtypes are coded in Arabidopsis; the Jas motifs of JAZ5 and -6 are identical, and JAZ7, -8, and -13 are expected to have no or weak affinity with COI1 because they lack a binding sequence (8). Thus, we prepared nine short peptides corresponding to the remaining subtypes (Fl-JAZs; Fig.  S4) with Fl-JAZ13 as a negative control. Next, 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. As shown in Fig. 3c and with the except of Fl-JAZ13, 2 induced an increase in FA in the presence of GST-COI1 and the corresponding Fl-JAZ. The affinity values of 2 are summarized in Table 1 (see also Fig. S5), and those corresponding to JAZ1, -3, -6, and -10 are consistent with previous reports (7,(17)(18)(19). Although no affinity values of 2 have been reported for JAZ2, -4, -9, -11, and -12 with COI1, pulldown experiments demonstrated that 2 indeed caused PPI for these COI1-JAZ combinations, similar to the case of JAZ1 (Fig. S6). In the cases of 1, 3, and 5, the anisotropy was also increased in most combinations of COI1-JAZ co-receptors except for JAZ13. In contrast, little change was observed for 4 against all co-receptors (Table 1 and Fig. S5). Affinity values for 1, 3, 4, and 5 with any COI1-JAZ co-receptors were previously unreported, and so our results are the first

Ligand screening system for jasmonate co-receptor
quantitative analyses of the affinity of endogenous JA derivatives with most combinations of COI1-JAZ co-receptors. Interestingly, the measured affinity values for 1, 2, and 5 against each JAZ subtype were found to be weakly correlated ( Fig. S7; correlation factor is 0. 55 for 2 and 1, 0.72 for 2 and 5, and 0.72   for 1 and 5), indicating that the JAZ-subtype selectivity of these jasmonates is similar but not identical to each other.
Additionally, as expected, an obvious FA change could be observed with Fl-JAZ1 and GST-COI1 upon addition of 2 in a dose-dependent manner with the conventionally used plate     (7,17,18). b ND, the value could not be determined due to the low signal change. c Although no value has been reported in the literature, COR-induced PPI was confirmed by another method. d PPI induction has not been reported.

Ligand screening system for jasmonate co-receptor
reader equipment (Fig. S8). Therefore, our FA-based binding assay system is amenable to the plate reader-based HTS of COI1-JAZ co-receptor agonists.

Anisotropy-based binding assay systems for antagonist of COI1-JAZ co-receptors
Our FA-based binding assay systems can also evaluate antagonists for COI1-JAZ by monitoring the disassembly of the ternary complex. Previously 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 thus a proof-of-principle experiment could be carried out using COR-MO as a positive control. Initially, the COI1-(ϩ)-7-iso-JA-L-Ile (1)-Fl-JAZ1 ternary complex was prepared in a similar manner to that described above (Fig. 4b). The r value of this ternary complex was found to decrease upon addition of 6 in a dose-dependent manner (Fig. 4b), indicating that 6 disrupted the formation of the COI1-1-Fl-JAZ1 ternary complex and released the small peptide Fl-JAZ1 into solution. The half-maximum inhibitory concentration (IC 50 ) of 6 was 1.92 M, which was consistent with the literature value (IC 50 ) of 0.43-1.53 M. An inhibitory effect of 6 on COI1-1-Fl-JAZ1 was further confirmed by our HTS system with plate reader equipment (Fig. 4c). These results clearly exemplify the utility of our HTS system for the screening of the COI1-JAZ antagonists.

Discussion
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). Our quantitative analyses of 2 and bioactive hormone (ϩ)-7-iso-JA-L-Ile (1) for the co-receptors suggested that their JAZ-subtype selectivities were obviously different; that is, affinity values of 1 and 2 for each JAZ subtype were weakly correlated ( Fig. S7; the correlation factor is 0.55), providing a possible explanation for the differences in bioactivities of 1 and 2, at least in vitro.
Although several synthetic jasmonates derivatives have been developed, their structure-activity relationship profiles have been mainly collected from whole-plant bioassays (24) and are therefore reflective of many different processes. In contrast, our binding assay system for the quantitative assessment of ligandsubtype selectivity will provide new insights into not only the mode of action of these derivatives but also the biological role of each JAZ subtype.
Other phytohormones such as auxin, gibberellin, and abscisic acid also act as PPI inducers for the corresponding co-receptors, of which there are various subtypes with high genetic redundancy (25)(26)(27). Napier and co-workers et al. (28) previously reported a pioneering surface plasmon resonance (SPR)based binding assay system of auxin co-receptors that yielded definitive, quantitative structure-activity relationship profiles by using biotin-conjugated degron peptides with F-box protein counterparts, including TIR1 and AFB5. Alternatively, the FAbased binding assay has been used in HTS analyses of PPI modulators, similar to SPR-based methods (the PPI inhibitor database reports fluorescence polarization or anisotropy assays in the top ranking 51% of their cases) (29). One advantage of the FA-based assay is that it is "mix-and-read": all the components of the assay can be mixed prior to compound administration and read out directly, allowing for the rapid HTS assay of various ligands simultaneously. Also, fluorescent probes are used in rather low concentrations (50 -100 nM in this case, depending on the signal-to-noise ratio or ligand affinity), and its miniaturization into sample uncoated microplates, which saves biological materials (proteins or peptides) and compounds by simple reduction of the assay volume, is relatively straightforward and results in costs per well that are lower than for SPR or other biomolecule immobilization-based PPI detection methods (AlphaScreen or biolayer interferometry).
Recently, Littleson et al. (30) have reported a scalable and flexible synthetic strategy with coronatine and successfully created a huge library (ϳ120) of its derivatives, although their JAZsubtype selectivity has yet to discussed. We developed a versatile binding assay method for the chemical screening of both agonist and antagonist ligands of COI1-JAZ co-receptors. Our design strategy of the FA-based JAZ sensors is simple and may be applicable to agronomically relevant plant species in addition to Arabidopsis thaliana (e.g. rice; three COI1 and 15 JAZ are coded in its genome, and all Jas motifs in JAZ have already been identified) (31). Therefore, the library of coronatine derivatives combined with our screening method should constitute a Ligand screening system for jasmonate co-receptor useful chemical tool for the elucidation of complex, JA-related signaling cascades as well as molecularly targeted agrochemicals in plants in the future (13).

General materials and methods
All chemical reagents and solvents were obtained from commercial suppliers (Wako Pure Chemical Industries Co. Ltd., Nacalai Tesque Co. Ltd., Watanabe Chemical Industries Co. Ltd., Thermo Fisher Scientific K.K., and GE Healthcare) and used without further purification. Ultraviolet (UV)-visible spectra were recorded on a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). Fluorescence spectra and anisotropy were recorded on a FP-8500 (Jasco, Tokyo, Japan). Plate reader assays were recorded on a SpectraMax-M5 (Molecular Devices, LLC). SDS-PAGE and Western blotting were carried out using a Mini-Protean III electrophoresis apparatus (Bio-Rad). Chemiluminescence signals were detected with a LAS 4000 imaging system (Fujifilm, Tokyo, Japan). Reversed-phase or straight-phase high-performance LC (HPLC) was carried out on a PU-4180 Plus with UV-4075 and MD-4010 detectors (Jasco). UV detection was performed at 220 nm. MALDI-TOF MS analyses were performed on a 4800 Plus MALDI-TOF/TOF Analyzer (AB Sciex, Framingham, MA).

Fluorescence anisotropy titration experiments
All chemicals were dissolved in ethanol to generate 10 mM stock solutions and diluted with 20% ethanol aqueous for preparation of 100 M stock solutions. All fluorescent peptides were dissolved in sterilized water to generate stock solutions. The concentrationsofthesepeptidesweredeterminedbytheirabsorbance (at 495 nm for Oregon Green and 494 nm for fluorescein) in 0.1 N NaOH aqueous using a molar extinction coefficient of 76,000 M Ϫ1 cm Ϫ1 for Oregon Green (35) and 75,000 M Ϫ1 cm Ϫ1 for fluorescein (36). The concentration of rhodamine-conjugated peptide was determined by the absorbance at 543 nm in methanol using a molar coefficient of 92,000 M Ϫ1 cm Ϫ1 (37). The concentration of COI1-GST was estimated by the Bradford assay and the efficiency of pulldown experiments with 2 and OG-N-Cys-JAZ1 (13), allowing stock solutions of known concentration to be prepared. Fluorescence anisotropy titration experiments were performed at 25°C in 50 mM Tris-HCl buffer (pH 7.8, 100 mM NaCl, 20 mM 2-mercaptoethanol, 10% glycerol, 0.1% Tween20, 100 nM inositol 1,2,4,5,6-pentakisphosphate) using a quartz cell (50 l). The ligand was added dropwise to the solution containing COI1-GST (100 nM) and each fluorescent JAZ peptide (100 nM), and anisotropy intensities were measured ( ex / em ϭ 485 nm/522.5 nm for Oregon Green, ex / em ϭ 485 nm/516.5 nm for fluorescein, ex / em ϭ 543 nm/573.5 nm for tetramethylrhodamine). Fluorescence anisotropy values (r) were calculated using the following equation: r ϭ (I VV Ϫ G ϫ I VH )/(I VV ϩ 2G ϫ I VH ) where I VV and I VH are the fluorescence intensities observed through polarizers parallel and perpendicular to the polarization of the exciting light, respectively, and G is a correction factor to account for instrumental differences in detecting emitted compounds. An average value of three independent measurements was plotted for each point. Anisotropy titration curves were analyzed with nonlinear curve-fitting analysis to evaluate apparent K a and K d values.

Ligand screening system for jasmonate co-receptor Fluorescence anisotropy measurements with plate reader equipment
Fluorescence anisotropy was measured at 25°C in the same buffer for fluorescence anisotropy titration experiments (pH 7.8) using a 96-well black clear-bottomed plate (100 l/well). The ligand was added dropwise to the solution containing COI1-GST (50 nM) and each fluorescent JAZ peptide (50 nM), and anisotropy values were measured ( ex / em ϭ 485 nm/516 nm for fluorescein).