Oligo dt kit

Tissue samples were homogenised in TRI reagent (Sigma-Aldrich, Dorset, UK) using a handheld homogeniser (Cole Parmer, Cambridgeshire, UK) and RNA extraction undertaken in accordance with the manufacturers’ guidelines. RNA concentrations were standardised, to allow sample normalisation, and used as a template to generate cDNA using a GoScript reverse transcription mix, Oligo (dT) kit (Promega, Southampton, UK) in accordance with the manufacturers’ guidelines.

To validate the expression changes observed from RNA-seq, we selected 10 genes that are putatively related to the JA pathway or defences and measured their expression using qPCR. For each sample, ~1μg total RNA was used to synthesise first-strand cDNA using the GoScript^™ Reverse Transcription Mix and Oligo(dT) kit (Promega, United States). The qPCRs reactions (in 10μl) were prepared according to the manufacturer’s instruction of UltraSYBR Mixture (High ROX; CWBIO, China). The qPCRs were performed on a StepOnePlus^™ Real-Time PCR System (Applied Biosystems, United States) with following PCR parameters: predenaturation at 95°C for 10min, 40cycles of denaturation at 95°C for 15s and annealing/elongation at 60°C for 1min and melting curve was carried out in the end of each PCR with the default program to validate the specificity of primers. Primer sequences of the 10 selected genes are shown in Supplementary Table 1. Tomato UBI3, a common internal reference gene for analysing gene expression changes affected by the JA-signalling pathway or biotic stresses (Fowler et al., 2009 (link)), was used as a reference gene. The expression at stage 4 from WT was used for calibration. Relative expression values were calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). Three biological replicates (samples from three plants) and two technical replicates were used.