Extended Data Fig. 2: Endodermis-expressed FLS2 and SGN3 activate both common and pathway-specific transcriptional responses.

(a) Experimental setups for RNAseq analysis comparing CIF2 and flg22 responses in root endodermis. Transcriptional data for CIF2 responses are from Fujita et al.²⁰. WT.CIF2, sgn3.CIF2: wild-type with functional SGN3 and sgn3 treated with 1 µM CIF2 vs H₂O control. FLS2.flg22, fls2.flg22: pCASP1::FLS2-GFP fls2 and fls2 treated with 1 µM flg22 vs H₂O control. Fold change (log₂FC) were calculated by comparing peptide (1 µM flg22 or 1 µM CIF2) to H₂O treatment at each time point (30, 120, 480 mins). (b) Heatmaps of genes regulated by FLS2 in whole seedlings – significantly upregulated genes in flg22-treated wild-type (left) (*, P < 0.001)³⁵ – are also strongly induced by CIF2 (WT.CIF2) and flg22 (FLS2.flg22) in the endodermis (right) (*P < 0.05). Colour code: fold change degree (log₂FC). (c) Heatmaps of fold change for reporter genes. Candidates are predicted to be preferentially CIF2 (green) or flg22 (yellow) responsive, or equally responsive (blue) in the endodermis (*P < 0.05). Reporter line analyses for selected genes are shown on the right (circles), with colour indicating observed pathway specificity (details see Supplementary Table 1). Corresponding heatmap of fold-change in myb36 compared to Wild-type (*P < 0.001, NA in grey)³⁰ shown on the left. Colour code: fold change degree (log₂FC). (d) Gene ontology (GO) enrichment analysis showing enriched GO-terms unique to FLS2 and SGN3 pathways upon stimulation in the endodermis. Colour: GO-enrichment significance. Dot size: gene ratio (the gene counts in a GO category differentially regulated by a pathway divided by the total number of genes in that GO category). (e) Workflow to determine the pathway specificity of top 10% most responsive genes and least responsive (P < 0.05, ranked by Log₂FC) at 30 min. Genes that are significantly upregulated by one pathway but not the other are considered specific, genes that are significantly upregulated by both are considered common (Cut-off: Log₂FC > 1 and P < 0.05).