Arabidopsis thaliana pattern recognition receptors for bacterial elongation factor Tu and flagellin can be combined to form functional chimeric receptors

Abstract

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.

FIGURE 1.

Schematic representation of receptor constructs used in this work. EFR (open bars, At5g2480) and FLS2 (gray bars, AT5G46330) encode receptor kinases with a signal peptide (SP), an N terminus conserved in LRR receptors (LRRNT; LRRNT_2 pfam08263), a LRR-domain with 21 repeats in EFR or 28 repeats in FLS2, respectively, an outer juxtamembrane domain (oJM), a transmembrane domain (TM), an inner juxtamembrane domain (iJM) and a serine/threonine protein kinase domain (Kinase, S_TKc). All constructs were C-terminally fused in-frame to a GFP tag.

FIGURE 2.

Functionality of intact and truncated forms of EFR after transient expression in leaves of N. benthamiana. A, Western blot with extracts from leaves expressing the different forms of EFR stained with anti-GFP antibodies. B, ethylene biosynthesis in transformed leaf pieces treated for 3 h in the absence or presence of 100 nm elf18. Values show mean ± S.D. (error bars) of n = 3 replicates. C, oxidative burst in leaf pieces of transformed plants treated with 100 nm elf18. Data show mean ± S.D. in Luminol-dependent light emission (RLU, relative light units) of n = 4 replicates. D, binding of elf-¹²⁵I in the presence (nonspecific binding) and absence (total binding) of 1 μm unlabeled elf18. Measurements were done in duplicate with bars indicating the mean of the two values.

FIGURE 3.

Binding of elf-¹²⁵I to EFR and chimeric receptors. A, Western blot with extracts from leaves expressing EFR or the different chimeric receptors stained with anti-GFP antibodies. B, binding of elf-¹²⁵I in the absence (total binding, filled bars) or presence of 1 μm elf18 (nonspecific binding, open bars). Data shown represent two replicates with bars indicating the mean. C, competition of specific binding with different concentrations of unlabeled elf18.

FIGURE 4.

Functionality of chimeric receptors after expression in N. benthamiana leaves. A, ethylene biosynthesis in leaf tissues expressing EFR or chimeric forms of EFR and FLS2 after treatment with 100 nm elf18. B, time courses of oxidative burst after treatment with 100 nm elf18. C, maxima in oxidative bursts after treatment with different concentrations of elf18. Values represent mean ± S.D. (error bars) of n = 4 replicates.

FIGURE 5.

Functionality of chimeric receptors after transient expression in mesophyll protoplasts from efr × fls2 mutants of A.thaliana. Luciferase activity in protoplasts treated with different concentrations of elf18 is shown.