A rationally designed JAZ subtype-selective agonist of jasmonate perception

Abstract

The phytohormone 7-iso-(+)-jasmonoyl-L-isoleucine (JA-Ile) mediates plant defense responses against herbivore and pathogen attack, and thus increases plant resistance against foreign invaders. However, JA-Ile also causes growth inhibition; and therefore JA-Ile is not a practical chemical regulator of plant defense responses. Here, we describe the rational design and synthesis of a small molecule agonist that can upregulate defense-related gene expression and promote pathogen resistance at concentrations that do not cause growth inhibition in Arabidopsis. By stabilizing interactions between COI1 and JAZ9 and JAZ10 but no other JAZ isoforms, the agonist leads to formation of JA-Ile co-receptors that selectively activate the JAZ9-EIN3/EIL1-ORA59 signaling pathway. The design of a JA-Ile agonist with high selectivity for specific protein subtypes may help promote the development of chemical regulators that do not cause a tradeoff between growth and defense.

Fig. 1

One stereoisomer of COR is a potential JAZ subtype-selective agonist. a Chemical structures of jasmonate derivatives and coronatine diastereomers. b, c Schematic diagram of ligand-induced protein-protein interactions (PPIs) between COI1-JAZ co-receptors; b naturally occurring ligands (i.e., coronatine) can interact with all co-receptors, whereas c the stereochemical isomer (used in this study) can interact with only some co-receptors. d, e Pull down assay of purified GST-COI1 (5 nM) with recombinant proteins expressed in E. coli, including d MBP-JAZ1 (full length, approximately 40 nM), and e MBP-JAZ3 (full length, approximately 40 nM), in the presence of COR derivatives (100 nM). GST-COI1 bound to MBP-JAZ proteins was pulled down with amylose resin and analyzed by immunoblotting. Goat HRP-conjugated anti-GST antibody was used to detect GST-COI1 (black triangles). Rat anti-MBP antibody and goat HRP-conjugated rat-IgG antibody were used to visualize MBP-JAZ protein levels as the input materials (white triangles)

Fig. 2

In vitro COI1-JAZs binding assay with epitope-conjugated JAZ short peptide. a Schematic of in vitro co-immunoprecipitation assay system for agonists of COI1-JAZ co-receptors using epitope-conjugated JAZ degron peptide and GST-COI1. An anti-fluorescein antibody was used to purify the ternary complex, and a GST-tag was used for the immunoblot assay with an anti-GST antibody. b Molecular design of the Oregon green (OG)-conjugated Jas motif of JAZ1. c Pull-down assay of purified GST-COI1 (5 nM) with each OG-conjugated JAZ peptide (10 nM) in the presence of stereoisomers of (+)-COR (3, ent3, 6, or ent6, 100 nM). d Pull-down assay of purified GST-COI1 (5 nM) with OG-conjugated JAZ13 peptide (10 nM) in the presence of 3, ent3, 6, or ent6 (100 nM). e Pull-down assay of purified GST-COI1 (5 nM) with OG-conjugated JAZ1 peptide (10 nM) in the presence of 3, ent3, 1 or 2 (100 nM); Goat HRP-conjugated anti-GST antibody was used to detect GST-COI1 in c–e

Fig. 3

Rational design of JAZ subtype-selective agonist by an in silico docking study. a The obtained average structure of MD simulation of COI1-3-JAZ1. The hydrogen bond between the ketone group of 3 with A204^JAZ1 or R496^COI1 is indicated by a yellow dotted line. b The obtained average structure of COI1/JAZ9 complexed with ent6, which was constructed by in silico docking analyses and MD simulation. The hydrogen bond between the ketone group of ent6 with R496^COI1 is shown as yellow dotted line. c The obtained average structure of COI1/JAZ10 complexed with ent6, which was constructed by in silico docking analyses and MD simulation. The hydrogen bond between the ketone group of ent6 with A170^JAZ10 (corresponding to A204^JAZ1) or R496^COI1 is indicated by a yellow dotted line. d Synthesis scheme for compounds 7–9 from ent6 as a starting material and corresponding oxime molecules. e Pull-down assay of purified GST-COI1 (5 nM) with all OG-conjugated JAZ peptides (10 nM) in the presence of ent6, 7, 8, or 9 (500 nM). HRP-conjugated anti-GST antibody was used to detect GST-COI1. f Evaluation of GUS activity in the roots of 4-day-old 35S:JAZ1-GUS, 35S:JAZ9-GUS, and 35S:JAZ10-GUS plants. Seedlings were pretreated for 30 min with or without ligand (3, ent6, or 8, 1 µM), and stained with 5-bromo-4-chloro-3-indolyl glucuronide; the experiments were repeated three times with similar results. Scale bar, 1 mm. g Quantification of GUS activity in 20 roots of 4-d-old 35S:JAZ1-GUS, 35S:JAZ9-GUS, and 35S:JAZ10-GUS plants (n = 4). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01). Seedlings were pretreated as described above. Three independent replicates were measured, and values represent mean ± s.d. (Supplementary Methods)

Fig. 4

JA responses and microarray analyses in 3/ent6/8-treated Arabidopsis seedlings. a WT Arabidopsis seedlings grown for 6 days on 1/2 MS medium containing 3, ent6, or 8 (1 µM). Scale bar, 10 mm. b Quantification of root length or fresh weight of the aerial part in the ligand-treated seedlings shown in a (n = 18). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01). c Quantification of accumulated anthocyanins in the ligand-treated seedlings shown in a. (n = 15). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01). d Venn diagram indicating the number of genes up-regulated at least 2.5 times, in response to different treatments (p < 0.05, FDR followed by Tukey’s HSD as post hoc test). e Heat map illustrating changes in gene expression in response to different treatments. Genes with at least a 2.5-fold increase in expression by 8 (p < 0.05, FDR followed by Tukey’s HSD as post hoc test). f Go enrichment analysis of Fig. 4d. g–k Analysis of JA-responsive gene expression by quantitative RT-PCR (qRT-PCR) in 6-d-old WT Arabidopsis seedlings with or without ligands (3, ent6, or 8, 1 µM) treatment for 2 h (g, i, k) or for 8 h (h, j) (n = 3). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.01)

Fig. 5

Effects of 3 and 8 on plant growth and defense of adult Arabidopsis. a–d The effects of the repetitive treatment of the compounds (3 or 8 at 50 µM, 5 times per week from 1-week-old to 5-week-old plants) in the aerial part of WT Arabidopsis adult plants grown for 5-week-old plants (see Supplementary Methods). Scale bar, 2 cm. The representative images of plants treated repetitively with 50 µM of each compound (a), fresh weights of aerial parts of the ligand-treated adult plants (b), and gene expression level of the aerial parts of the ligand-treated adult plants (c; PDF1.2, d: ORA59) (n = 7). Significant differences were evaluated by one-way ANOVA/HSD post hoc test (p < 0.05). Experiments were repeated three times with similar results. e–g Wild-type Col-0 plants were treated with mock solution, 3 or 8 (50 µM) (n = 11–20) and infected with A. brassicicola. Representative leaves of plants infected with A. brassicicola are shown in e. Scale bar, 10 mm. Quantification of fungal spores (f) and lesion area (g) was undertaken 6 days after inoculation; the results are depicted using box-plots; horizontal lines are medians, boxes show the interquartile range and error bars show the full data range. Outliers are indicated as circles. Asterisks above columns indicate significant differences compared to the mock-treated values evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.05). Experiments were repeated three times with similar results. h–i jaz9 and jaz10 mutant plants were treated with mock solution, 3 or 8 (50 µM) (n = 14–20) and infected with A. brassicicola. Quantification of fungal spores was undertaken 7 days after inoculation; the results are depicted using box-plots as described in f. Asterisks above columns indicate significant differences compared to the mock-treated values analyzed by one-way ANOVA/Tukey HSD post hoc test (p < 0.05)

Fig. 6

Selectively activate JAZ9-EIN3/EIL1-ORA59 signaling pathway by 8. a–c Analysis of JA-responsive gene expression by qRT-PCR in 6-d-old WT (Col-0), jaz9-1, or jaz10-1 Arabidopsis seedlings with or without ligands (3 or 8, 1 µM) treatment (PDF1.2 (a), ORA59 (b), or VSP1 (c)). Results shown are the mean with s.d. (n = 4). Significant differences were evaluated by one-way ANOVA/Tukey HSD post hoc test (p < 0.05). d, e Predicted signaling pathways involving the role of 3 (d) or 8 (e) for MYC-branch and ERF1/ORA59-branch gene expression