Total RNA was isolated as described in Verdonk et al. (2003) and treated with DNase (Ambion). cDNA was synthesized from 4 µg of total RNA using M-MuLV Reverse Transcriptase (Fermentas) in a 20 µl reaction. Quantitative reverse transcription–PCR (qRT–PCR) was performed in CFX96™ Optics Module (BIO-RAD) using iQ™ SYBR® Green Supermix (BIO-RAD). PCRs of 20 µl contained 0.25 µM of each primer and 1 µl of cDNA. The cycling program was set to 5 min at 50°C, 2 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C, followed by a melting curve analysis. Three biological replicates with two technical qRT–PCR replicates (i.e. individual plants) were analyzed per treatment. Actin was used as a reference gene. The normalized expression (NE) data were calculated with the 2−ΔΔCt method. NE values were scaled to the lowest average NE within the plot, which was set to 1. Transcript levels of the JA marker gene WIPI-II, the SA marker gene PR-P6 (Alba et al. 2015 (link)) and the ET-responsive marker gene SlERF1b (Nambeesan et al. 2012 (link)) were analyzed. The gene-specifc primers used for the qRT–PCRs are shown in Supplementary Methods S1.