A two-hybrid-receptor assay demonstrates heteromer formation as switch-on for plant immune receptors

Abstract

Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a "two-hybrid-receptor assay" is of more general use for studying heteromeric receptor complexes.

Figure 1.

Heteromeric complex formation of FLS with BAK1 switches on flagellin-dependent transmembrane signaling. A, Model for the flg22-dependent heteromeric receptor complex. Schematic representation of FLS2 (blue), BAK1 (red), and the chimeric receptor constructs Ftm-B and Btm-F. Ftm-B comprises the extracellular and the transmembrane domains of FLS2 (blue) and the cytoplasmic domain of BAK1 (red). The receptor chimera Btm-F represents the reciprocal construct with the cytoplasmic part of BAK1 replaced by that of FLS2. The cytoplasmic domain of FLS2 was C-terminally tagged with a GFP. B and C, Functional comparison of native FLS2 and BAK1 (B) with the two hybrid receptors Ftm-B and Btm-F (C). The experiments show luciferase activity in Arabidopsis fls2 bak1-4 double mutant protoplasts cotransformed with pFRK1::Luciferase as a reporter and the receptor constructs indicated. At 0 h (dashed line) protoplasts were treated with 100 nm of flg22 (black diamonds) or 100 nm of the inactive analog flg22^Atum (white circles). Light emission of the protoplasts was measured with a luminometer. Values represent averages and sds of three replicates. Data shown are representative for at least three independent repetitions of the experiments with all constructs. D and E, Dose-response relationship for flg22-dependent induction of pFRK1::Luciferase in protoplasts expressing the receptor constructs indicated. Values represent increase in luciferase activity after 5 h of treatment as percentage of the increase observed with FLS2 plus BAK1 treated with saturating doses of greater than or equal to 10 nm flg22. Comparison of (half-maximal stimulation values in D and E shows that cells coexpressing Ftm-B plus Btm-F responded at least as sensitive to flg22 as cells coexpressing FLS2 plus BAK1. A combination of Ftm-B with the kinase dead version Btm-F_KD was not functional, even when treated with 1,000 nm flg22. LU, Light units.

Figure 2.

Ligand-induced receptor complexes FLS2/BAK1 and Ftm-B/Btm-F. Coimmunoprecipitation assays with receptor constructs expressed in N. benthamiana leaves. Detergent-solubilized extracts from leaves, treated for 2 min with 100 nm flg22 as indicated, were immunoprecipitated via the GFP tag present at the C terminus of FLS2 and Btm-F. Western blots were developed with antibodies against the myc tag on BAK1 and Ftm-B (top) or the GFP tag present on FLS2 and Btm-F (bottom).

Figure 3.

Functionality of the chimeric EFR-BAK1 receptors Etm-B and Btm-E. A, Oxidative burst measurements in N. benthamiana leaves expressing Etm-B, Btm-E, a combination of Etm-B plus Btm-E, or EFR. Leaf pieces were treated with 100 nm elf18 or 100 nm of the inactive analog elf18G5, and ROS production was measured as light units (LU) by a luminometer for 20 min. Data show average and sd for three replicates. Experiments shown are representative for at least five independent repetitions of the experiments with all constructs. B and C, Elf18-induced complexes Etm-B/Btm-E and EFR/BAK1. Coimmunoprecipitation assays with receptor constructs expressed in N. benthamiana leaves. Detergent-solubilized extracts from leaves, treated for 2 min with 100 nm elf18 as indicated, were immunoprecipitated via the GFP tag present at the C terminus of EFR and Btm-E. Western blots were developed with antibodies against the myc tag on BAK1 and Etm-B (top) or the GFP tag present on EFR and Btm-E (bottom).