Epmotion 96

The strains were then inoculated on solid synthetic defined (SD) medium with or without 20 mg/L uracil or 20 mg/L leucine containing 2% (w/v) agar at 28 °C for 2 days. Two to three single colonies were suspended in saline solution (Otsuka Pharmaceutical Co., Ltd., Ltd., Tokyo, Japan), adjusted to 2 × 10⁶ cells/mL using a cell counter (WATSON Co., Ltd., Tokyo, Japan), serially diluted 1:10, and spotted (5 µL) on solid SD medium containing 2% (w/v) agar using an epMotion^Ⓡ 96 (Eppendorf, Hamburg, Germany). After 48 h of incubation at 28 °C, pictures of the growth of cells were taken. Assays were repeated three times.

A seed of A. thaliana was sowed in 100 µL of 0.5 × Murashige and Skoog medium in each well of a 96-well plate and grown at 20 °C under continuous light for 7 days after vernalization treatment. Each of the 11 96-well plates was incubated at a different temperature (10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 °C) for 18 h. Then, each 96-well plate was divided into three parts of 32 wells each, and each part was filled with 50% EtOH, or 16.5 mM of JA or SA stock solution, forming a final concentration of 1%, 0.33 mM, and 0.33 mM, respectively; the plates were incubated for an additional 6 h. After incubation, medium was immediately discarded using 96 channel pipette (epMotion 96 (Eppendorf, Hamburg, Germany)), and A. thaliana seedlings were homogenized twice in 90 s with 200 µL of 90 mM Tris–HCl (pH 7.6) containing 100 mM DTT using TissueLyser II (Qiagen, Hilden, Germany). DeLTa-seq libraries were created as described above with minor modifications. RT products were obtained from 5 µL of lysate as the template. Pooled RT products were divided into two aliquots and each sample was allowed to react with either of the two gene specific primer mixes, Tradict targeting genes (Additional file 3: Table S2) or SA/JA-related genes (Additional file 3: Table S4).