RT-qPCR validation of mRNA sequencing and time course analysis
To validate mRNA-sequencing results and to investigate the temporal dynamics of PA-IR, twelve genes were chosen for RT-qPCR analysis in a time-course experiment. These genes were selected based on differential expression after PA treatment in the mRNA-sequencing dataset, and because they covered most pathways or groups of functionally related genes identified in the mRNA-sequencing data (see Supplementary Table S1 ).
Gene expression was measured at three time points (6, 24 and 72 hours after PA treatment) in root and shoot to study the spatiotemporal evolution of the plant response to PA (Figure 4) . There was significant agreement between mRNA-seq and RT-qPCR data at 24 hours (Spearman’s ρ = 0.57, P = 0.003; Supplementary Figure S5 ), which supports the validity of our mRNA-sequencing results.
For most genes assayed via RT-qPCR, PA-treated shoot samples showed the highest differential expression after 6 and 24 hours. Root samples showed a smaller fold change at 6 hours, followed by greater differences after 24 and 72 hours. This hints at a temporal difference in gene expression changes after PA treatment, with (directly treated) shoots showing a more rapid response than (systemic) roots.