Figure 3. EFR^F761H/Y836F and EFR^F761H/SSAA recover receptor complex activation.

(A) In infection assays, GUS activity was high in the positive control efr-1 line. GUS activity level was reduced in the EFR^WT and EFR^F761H complementation lines, but much less so in the EFR^Y836F and EFR^SSAA complementation lines. By contrast, EFR^F761H/Y836F and EFR^F761H/SSAA complementation lines displayed substantially repressed GUS activity. Each experiment was repeated three times with similar results. Outliers are indicated by an additional asterisk and included in statistical analysis. Statistical test: Kruskal-Wallis test (P=5.704*10^–7), Dunn’s post-hoc test with Benjamin-Hochberg correction (P ≤ 0.05) Groups with like lowercase letter designations are not statistically different. (B) In IP kinase assays, ligand-induced interaction of EFR^WT and EFR^F761H with BAK1 increased transphosphorylation of BIK1^D202N, but this was abolished for EFR^Y836F and EFR^SSAA. Both EFR^F761H/Y836F and EFR^F761H/SSAA showed partially restored BIK1^D202N trans-phosphorylation as well as BAK1 S612 phosphorylation (across four replicates for EFR^F761H/SSAA and in two out of four replicates for EFR^F761H/Y836F). Samples were also probed for MAPK phosphorylation for effective ligand treatment. Treatment: 100 nM elf18 for 10 min. (C) Quantification of BIK1^D202N band intensity observed in autoradiographs from the four independent replicates performed. Dotted red line indicates unchanged band intensity in mock vs. elf18 treatment.

Figure 3—figure supplement 1. Multiple immune signaling branches are partially restored in EFR^F761H/Y836F and EFR^F761H/SSAA.

Stable transgenic complementation lines in the Arabidopsis efr-1 background were generated and physiological experiments conducted in the T3 generation (except for EFR^{F761H /Y836F}#5, which is a double insertion line in T2 generation). (A) In the oxidative burst assay, EFR F761H restored oxidative burst in EFR^F761H/Y836F and EFR^F761H/SSAA complementation lines. Two independent experiments were merged into one graph as WT controls showed comparable total oxidative burst. A third independent experiment was performed with similar results. Outliers are indicated by an additional asterisk and included in statistical analysis. Statistical test: Kruskal-Wallis test (p<2.2*10^–16), Dunn’s post-hoc test with Benjamin-Hochberg correction (p ≤ 0.05) Groups with like letter designations are not statistically different. Like oxidative burst assays, EFR F761H restored SGI (B) and MAPK activation (C) in EFR^F761H/Y836F and EFR^F761H/SSAA complementation lines. For SGI assays, four independent experiments wtih 5 nM elf18 treatment are shown. Outliers are indicated by an additional asterisk and included in statistical analysis. Statistical test: Kruskal-Wallis test (p<2.2 *10^–16), Dunn’s post-hoc test with Benjamin-Hochberg correction (p ≤ 0.05) Groups with like letter designations are not statistically different. For MAPK activation assays, a representative experiment is shown. Similar results were obtained in three more experiments.