Coelenterazine

ORco agonists, repellents, and the 50 VOCs analyzed in the current study are presented in Table S1. Carvacrol (CRV), linalyl acetate (LA), (2E,4E)-2,4-octadienal (OCT), and (1S)-3-carene (CAR) were purchased from Sigma Aldrich; isopropyl cinnamate (IPC) from Alfa Aesar; cumin alcohol (CA) from Acros Organics; N-(4-ethylphenyl)-2-{[4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl]thio}acetamide (ORco Receptor Agonist Molecule 2, ORcoRAM2) from Asinex Corporation and Vitas M Chemical Ltd; N-(4-ethylphenyl)-2-{[4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl]thio}acetamide (VUAA1) from Innovapharm Ltd; N,N-diethyl-3-methylbenzamide (DEET) from Sigma-Aldrich; and coelenterazine from Biosynth. Initial stock solutions for ORcoRAM2 and VUAA1 were prepared as needed in dimethyl sulfoxide (DMSO) and stored at −20 °C, whereas initial stocks of VOCs and coelenterazine were prepared in ethanol as needed and stored at −20 °C. For the insect cell–based screening assay, working solutions were prepared in modified Ringer's buffer (25 mM NaCl, 190 mM KCl, 3 mM CaCl₂, 3 mM MgCl₂, 20 mM Hepes, 22.5 mM glucose, pH 6.5; 35), so that the final DMSO concentrations did not exceed the range of 0.2% to 0.35%.

Seedlings were initially grown on 1/2 MS plates for 3 days before being transferred to 96-well plates (655075, Greiner Bio-One) in 100 μL liquid MS for 5 days. The evening before calcium measurements the liquid MS was replaced with 100 μL 20 μM coelenterazine (EC14031, Carbosynth) and the seedlings incubated in the dark overnight. The following morning the coelenterazine solution was replaced with 100 μL water and rested for a minimum of 30 min in the dark. Readings were taken in a VARIOSKAN MUTIPLATE READER (ThermoFisher) before and after adding 50 μL of 3×concentrated elicitor solution or mock. For each well readings were normalised to the average RLU value before elicitor addition (L₀).