Syber green supermix

After 96 h of treatment, total RNA was extracted by using RNeasy Plus Mini kit (Qiagen, Stockholm, Sweden) with on column DNase digestion, following the manufacturer protocol. RNA content in each sample was quantified by using a Nanodrop^® Spectrophotometer ND-1000, followed by cDNA synthesis using Omniscript Reverse Transcription Kit (Qiagen, Sweden) containing 2 μg RNA from each sample and 0.1 μg/μL Oligo(dT)_12-18 as primer. Real-time PCR was performed in a CFX96^TM Real-Time System (Bio-Rad, Stockholm, Sweden) using SyberGreen Supermix (Bio-Rad, Stockholm, Sweden), 20 ng of cDNA per reaction in a final volume of 10 μl and primer concentration ranging from 300-900 nM (primers sequence available upon request). The data were analyzed using the 2^−ΔΔCT method. The expression level of HPRT was used to normalize the relative expression levels of the genes of interest.

To set up a Q-PCR reaction, 2 µl of 1:10 diluted tissue cDNA was used as template in a 20 µl of total reaction mixture for each cDNA sample. The reaction mixture contained 12.5 µl, SYBER Green Supermix (BIO-RAD Laboratories, Hercules, CA, USA) and 1 µl of 100 nM of each forward and reverse primer. For each sample, a parallel reaction was setup using GAPDH primers as endogenous control. The Q-PCR was carried out using the Applied Biosystems AB7500 thermal cycler, ThermoFisher Scientific, Waltham, MA, USA). The reaction steps were heat activation at 95 °C for 15 min followed by 40 cycles of denaturation at 95 °C for 30 s, primer annealing at 60 °C (unless otherwise specified) for 30 s, and primer extension at 72 °C for 30 s, followed by melting curve analysis (55 to 95 °C; in 0.5 °C increments) to verify specificity of PCR products. All PCR products were also run in ethidium bromide stained agarose gels to verify the presence of single bands. All genes were analysed as biological triplicates (n = 3) and in technical triplicates from each biological sample. GAPDH was used as an internal housekeeping control, and data were displayed as LOG2 fold change of the 2^−ΔΔCt method. The primers used in this study are in Supplementary Table-Primers.

Leaf discs treated with either JA or mock solution were sampled for RNA isolation (three or four biological replicates per treatment) and ground with silica beads for 1 min in a tissue lyser. Stem tissues were ground by hand with a ceramic mortar and pestle. Samples were supplemented with 300 µL of cell lysis buffer (2% SDS w/v, 68-mM sodium citrate, 132-mM citric acid, and 1-mM EDTA) and incubated for 5 min at room temperature prior to adding 100 μL of protein/DNA precipitation buffer (4 M NaCl, 16-mM sodium citrate, and 32-mM citric acid) followed by 10 min of incubation on ice. Samples were centrifuged for 15 min at 13,000 rpm, while the supernatant was transferred to a new tube (300 µL) and supplemented with an equal volume of ice cold isopropanol. All tubes were centrifuged for 5 min at 13,000 rpm, and the pellet was washed with 300 µL of 70% ethanol (v/v). RNA pellets were air dried for 5 min before elution in 30-µL RNAse-free water. cDNA synthesis was carried out using RevertAid H minus Reverse transcriptase (Thermo scientific). Gene expression analysis was performed by RT-qPCR in a Viia7PCR device with 5-µL reaction mix containing SyberGreen Supermix (Bio-Rad). Tomato actin was used as a reference gene and fold change in expression were calculated based on 2^(−ddCt) (Livak and Schmittgen, 2001 (link)). Primer sequences used are displayed in Supplemental Dataset S3.