C4 interacts with several leucine-rich repeat (LRR)-RLKs in vivo. (A) Co-immunoprecipitation of BAM1-, CLV1-, BRI1-, BRL3-, NIK1-, FLS2-, and BAK1-RFP with C4-GFP following transient expression in N. benthamiana. (B) Co-immunoprecipitation of BAM1-, PSKR1- and PERK1-RFP with C4-GFP following transient expression in N. benthamiana. IP: immunopurification, IB: immunoblotting. Three independent biological replicates were performed with similar results; one replicate is shown. (C) Fӧrster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) analysis of the RLKs-RFP/C4-GFP interaction following transient expression in N. benthamiana leaves. Box plots denote distribution of eight measurements ± SD. FE: FRET Efficiency. Asterisks indicate a statistically significant difference according to Student’s t-test (**** p value < 0.0001; *** p value < 0,005; * p value < 0.05; n.d, no difference). Colored markers above and below whiskers indicate outliers. Three independent biological replicates were performed with similar results. One replicate is shown.