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. 2008 May 13;105(19):7100-5.
doi: 10.1073/pnas.0802332105. Epub 2008 May 5.

COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine

Leron Katsir  1 , Anthony L Schilmiller, Paul E Staswick, Sheng Yang He, Gregg A Howe
Affiliations

COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine

Leron Katsir et al. Proc Natl Acad Sci U S A. .

Abstract

Jasmonate (JA) is a lipid-derived hormone that regulates diverse aspects of plant immunity and development. An amino acid-conjugated form of JA, jasmonoyl-isoleucine (JA-Ile), stimulates binding of the F-box protein coronatine-insensitive 1 (COI1) to, and subsequent ubiquitin-dependent degradation of, jasmonate ZIM domain (JAZ) proteins that repress transcription of JA-responsive genes. The virulence factor coronatine (COR), which is produced by plant pathogenic strains of Pseudomonas syringae, suppresses host defense responses by activating JA signaling in a COI1-dependent manner. Although previous data indicate that COR acts as a molecular mimic of JA-Ile, the mechanism by which JA-Ile and COR are perceived by plant cells remains unknown. Here, we show that interaction of tomato COI1 with divergent members of the JAZ family is highly specific for JA-Ile and structurally related JA conjugates and that COR is approximately 1,000-fold more active than JA-Ile in promoting this interaction in vitro. JA-Ile competes for binding of COR to COI1-JAZ complexes, demonstrating that COR and JA-Ile are recognized by the same receptor. Binding of COR to the COI1-JAZ complex requires COI1 and is severely impaired by a point mutation in the putative ligand-binding pocket of COI1. Finally, we show that the C-terminal region of JAZ3 containing the highly conserved Jas motif is necessary and sufficient for hormone-induced COI1-JAZ interaction. These findings demonstrate that COI1 is a critical component of the JA receptor and that COR exerts its virulence effects by functioning as a potent agonist of this receptor system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Specificity of JA–amino acid conjugates in promoting COI1–JAZ interaction. Pull-down assays were performed with recombinant JAZ1–His (a) or JAZ3–His (b) and extracts from 35S-COI1–Myc plants. Assays were supplemented with various JA–amino acid conjugates at the indicated concentration and incubated for 30 min at 4°C. Protein bound to JAZ1–His or JAZ3–His was analyzed by immunoblotting for the presence of COI1–Myc. The Coomassie Blue-stained blot in each panel shows the recovery of JAZ–His by the Ni affinity resin.
Fig. 2.
Fig. 2.
Coronatine promotes formation of COI1–JAZ complexes. (a) Molecular structures of JA–Ile and COR. (b) JAZ1–His-containing pull-down assays supplemented with buffer (indicated by 0) or various concentrations of COR were processed as described in the legend to Fig. 1. The Coomassie blue-stained blot shows recovery of JAZ1–His. (c) Pull-down reactions containing extracts from 35S-COI1–Myc plants and JAZ3–His (filled circles) or JAZ1–His (open circles) were incubated with [3H]COR in the presence of increasing concentrations of unlabeled COR. Radioactivity recovered with JAZ–His is indicated (CPM). (d) Pull-down reactions containing 35S-COI1–Myc leaf extract, JAZ3–His, and [3H]COR were incubated in the absence (indicated by 0) or presence of the indicated amount of unlabeled COR, JA (jasmonic acid), or JA–Ile. (e) Pull-down reactions containing 35S-COI1–Myc leaf extract, JAZ3–His, and increasing concentrations of [3H]COR were used to construct a saturation curve for specific binding. Shown is a Scatchard plot of the saturation-binding data for a representative experiment. Error bars in c and d denote the SD of triplicate assays.
Fig. 3.
Fig. 3.
COI1 is an essential component of the jasmonate receptor. (a) Pull-down reactions containing JAZ3–His, [3H]COR, and crude leaf extract from the indicated tomato genotype (or an equivalent volume of buffer; indicated by Mock) were incubated in the presence (+) or absence (−) of 1,000-fold unlabeled COR. The amount of radioactivity recovered in the JAZ3–His complex is shown. Error bars denote the SD of triplicate assays. (b) Northern blot analysis of proteinase inhibitor II transcript accumulation in wild-type and jai1-3 plants treated in a closed container for 10 h with various amounts of vaporous MeJA. Blots were hybridized to an eIF4A cDNA as a loading control. (c) Specific binding of [3H]COR in pull-down assays containing JAZ3–His and leaf extract from either WT (closed circles) or jai1-3 (open circles) plants.
Fig. 4.
Fig. 4.
The C-terminal region of JAZ3 is required for COI1 interaction and specific binding of COR to the COI1–JAZ3 complex. (a) Schematic diagram of full-length and truncated JAZ3 constructs. The positions of the conserved ZIM (gray box) and Jas (black box) motifs are shown. The N-terminal MBP and C-terminal His6 fusions are not shown. (b) Pull-down assays containing 35S-COI1–Myc leaf extract and the indicated JAZ3 fusion protein were incubated in the presence (+) or absence (−) of 1 μM JA–Ile. Reactions were processed as described in the legend to Fig. 1. (c) Pull-down reactions containing 35S-COI1–Myc leaf extract, [3H]COR, and the indicated JAZ3 fusion protein were incubated in the presence (+) or absence (−) of unlabeled COR. The amount of radioactivity recovered in the JAZ3–His complex is shown.
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