By integrating co-fractionation with coronatine-induced transcriptional responses, we identified NATA1, a polyamine N-acetyltransferase, as a coronatine-associated host protein binder. Biochemical assays confirmed direct coronatine–NATA1 interaction, whereas the close homolog NATA2 did not bind coronatine. Coronatine did not strongly inhibit NATA1 catalytic turnover under our assay conditions but consistently depressed NATA1 dimer formation in vitro and in planta. In silico docking together with targeted mutational analyses identified a coronatine‑sensitive interface on NATA1, revealing residue‑specific coupling between ligand binding, assembly state, and acetyl‑CoA engagement. Collectively, these findings establish PROMIS as a robust platform for infection‑state PMI discovery and uncover a coronatine‑responsive regulatory node that links small‑molecule signaling to host polyamine acetylation. The presented dataset comprises microscale thermophoresis (MST) binding data characterizing interactions between purified NATA1, NATA1 amino‑acid substitution point mutants, and NATA2 with small‑molecule ligands, including coronatine, jasmonoyl‑isoleucine, and acetyl‑CoA. In addition, the dataset includes MST‑based analyses of NATA1 dimerization in the absence and presence of coronatine, as well as enzymatic activity measurements of wild‑type NATA1 and NATA1 point mutants under coronatine‑treated and untreated conditions.

Protein purification 

Frozen bacterial cells from terrific broth autoinduction were used for subsequent protein purification. The soluble fraction was purified using a 1 mL HisTrap (Cytiva) column connected to NGC Quest 10 (Bio-Rad), previously equilibrated with the buffer containing 25 mM Tris–HCl pH 8.0, 300 mM NaCl, 5% (v/v) glycerol, 10 mM imidazole. The column was washed with 10 mL of the buffer, and the recombinant protein was eluted with a linear gradient of imidazole (10–300 mM). The fractions containing the protein of interest were collected and concentrated and desalted using ultra-centrifugal filters. The purified protein was stored in 0.1 M Tris-HCl buffer pH 7.5 at 4 °C for use in future experiments.

Site-directed mutagenesis 

Site-directed mutagenesis of NATA1 gene was performed using Q5 site-directed mutagenesis kit (New England Biolabs) according to the manufacturer’s protocol. Mutagenic primers were designed using NEBasechanger online tool (NEB) and are listed in Supplementary Table 1. The plasmid pET28a-NATA1 served as the template for PCR amplification. Following sequence verification of the resulting mutant constructs, recombinant protein expression and purification were carried out under the same conditions as those described above.  

Microscale thermophoresis (MST) 

Ligands (coronatine, JA-Ile, and acetyl-CoA) were serially diluted in MST buffer (50 mM potassium phosphate, pH 6.5; 7.6 mM (NH₄)₂SO₄; 1.7 mM MgCl₂; 1.7 mM NaCl; 0.05% Tween-20) using a 16-step, twofold dilution series. RED-labeled His-tagged NATA1 and its variants (625 nM) were mixed with each ligand (6 µL per sample) and loaded into premium capillaries (NanoTemper Technologies). Measurements were performed on a Monolith instrument (NanoTemper Technologies). Data were analyzed using standard MST traces, and normalized fluorescence (Fnorm) was plotted against ligand concentration to calculate dissociation constants (Kd). To enable cross-experiment comparison, fraction bound values were computed to minimize dependence on amplitude and initial fluorescence. Data analysis was conducted using Monolith Analysis software. For dimerization assays, NATA1 and variants were labeled with RED-NHS dye (NanoTemper Technologies) following the manufacturer’s protocol. Labeled NATA1s (50 nM) were incubated with unlabeled NATA1s (starting at 200 µM) in the presence or absence of 100 µM coronatine, 100 µM JA-Ile, or 500 µM putrescine. Each experiment was performed in triplicate.

Enzymatic assays 

NATA1 enzymatic activity was measured using a Multiskan Skyhigh Microplate Spectrophotometer (Thermo Fischer Scientific). The reaction mixture contained 50 mM Tris-HCl (pH 6.8), 20 mM NaCl, 1 mM acetyl-CoA, 2 μM purified NATA1 or variant protein, and varying concentrations of putrescine as the substrate. To assess the effect of coronatine on enzymatic activity, 100 μM coronatine was included in the reaction mixture. Reactions were incubated at 30 °C for 10 min and terminated by the addition of an equal volume of quenching buffer consisting of 6 M guanidine hydrochloride and 0.1 M Tris-HCl (pH 8.0). The production of free coenzyme A was quantified by adding Ellman’s reagent [0.2 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 0.1 M Tris-HCl (pH 8.0), and 1 mM EDTA], and measuring the absorbance at 412 nm. All measurements were performed in technical triplicates, and the data were plotted using GraphPad Prism 10 (GraphPad software).