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. 2024 Jul 19;12:RP92110. doi: 10.7554/eLife.92110

Figure 5. Related EFR kinases from LRR-RK XIIa in the Arabidopsis genus can function independent of their calatytic activity.

(A) Phylogenetic analysis of LRR-RK subfamily XIIa. Selected LRR-RK XIIa kinase domains are labeled and highlighted with purple points. The EFR-like clade contains all Arabidopsis XIIa kinases except FLS2 and XIIa2 and also selected XIIa kinases from Arabidopsis lyrata and Brassica rapa. (B, C) The ectodomain of EFR was fused to the transmembrane and intracellular domain of selected LRR-RK XIIa members to create elf18-responsive chimeras for testing the immune signaling function and catalytic dependency of the related kinase domains. The chimeras were transiently expressed in N. benthamiana and tested in oxidative burst assays. All Arabidopsis LRR-RK XIIa members induced an oxidative burst except XIIa2, the closest FLS2 related kinase in the subfamily. Catalytic dependency of the kinase domains appears to vary from kinase to kinase, with catalytically dead versions of EFR, FEXL1 and XIIa5 inducing a WT-like oxidative burst and XPS1 and XIIa6 displaying a reduced oxidative burst. FLS2 kinase dead exhibited a diminished oxidative burst. Experiments were repeated three times with similar results.

Figure 5.

Figure 5—figure supplement 1. XIIa5D839N exhibits largely XIIa5WT-like characteristics.

Figure 5—figure supplement 1.

(A, B) Catalytic site mutation of XIIa5 exhibited the least delayed onset of oxidative burst. In A, quantification of the time to reach the half maximum of the oxidative burst is shown. The underlying data are the same as used for total oxidative burst in the main figure. In B, the actual oxidative burst curves are presented as average of six individual plants transiently expressing the indicated chimeric protein. Error bars represent standard error of the mean (n=6). (C) A catalytic site mutation of XIIa5 did not negatively affect BIK1 trans-phosphorylation or BAK1 autophosphorylation. Experiments were performed as described in Figure 1. (D) Quantification of band intensities on autoradiographs from three independent experiments are shown. BRI1D1009N and EFRD849N displayed results similar to Figure 1B and C. In contrast to EFRD849N, for which BIK1 and BAK1 relative band intensities slightly decreased compared to wild type EFR, BIK1D202N and BAK1 relative band intensities were wild-type-like for XIIa5D839N.
Figure 5—figure supplement 1—source data 1. Raw data for autoradiography shown in Figure 5—figure supplement 1C.
Figure 5—figure supplement 2. Protein accumulation of EFR-XIIa chimeras in N. benthamiana.

Figure 5—figure supplement 2.

All constructs exhibited detectable protein accumulation in transiently transformed N. benthamiana leaves. Similar protein accumulation was observed in 3 replicates for (A). Protein accumulation for constructs in (B) was tested once.
Figure 5—figure supplement 2—source data 1. Raw data for autoradiography shown in Figure 5—figure supplement 2.