Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Abstract

Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)8-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNβ1,4GlcNAc)4, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.

Keywords: LysM-receptor; MTI/PTI; chitin signaling; plant immunity; receptor–ligand interaction.

Fig. 1.

Functional analysis of LysM regions in the ectodomain of CEBiP by deletion experiments. (A) Amino acid sequence of CEBiP. Sp, signal peptide; L0, LysM0; L1, LysM1; L2, LysM2; Ct, C-terminal region. LysM0 and LysM2 were divided into four parts, from L0a to L0d and L2a to L2d, for deletion experiments. The C-terminal region was also divided into four parts for similar experiments (black diamonds indicate the border of each segment). (B) Effect of the deletion of LysM regions in CEBiP on GN8-Bio binding. (a) Constructs for the deletion experiments; (b and c) Expression of the deletion mutants in the tobacco BY-2 cells was analyzed by RT-PCR or Western blotting with an anti-CEBiP antibody (α-CEBiP); (d) Affinity cross-linking of GN8-Bio to the microsomal fractions (MF) from the tobacco BY-2 cells expressing deletion mutants. GN8-Bio (0.4 µM) was mixed with the MF in the presence (+) or absence (−) of excess (40 µM) unlabeled (GlcNAc)₈ as a competitor. Cross-linking with EGS and the detection with anti-biotin antibody (α-Biotin) were performed as described in Materials and Methods. (C) Effect of the partial deletion of LysM0 and LysM2 on GN8-Bio binding. (a) Constructs for the deletion experiments; (b) affinity cross-linking of GN8-Bio to the MF from the transgenic N. benthamiana leaves expressing the deletion mutants.

Fig. 2.

Functional analysis of LysM regions of CEBiP by replacement experiments with the corresponding regions of Arabidopsis CEBiP homologs. (A) Designation of LysMs in CEBiP and the Arabidopsis homologs (LYM2/AtCEBiP and LYM1/At1g21880). Chitin oligosaccharide binding ability of each protein was also shown. (B–D) Chitin oligosaccharide binding ability of chimeric proteins analyzed by affinity labeling with GN8-Bio.

Fig. 3.

STD NMR-derived epitope mapping on (GlcNAc)₇ and (GlcNAc)₈ bound to Trx-LysM1-2. (A) Reference ¹H NMR spectrum (a) and STDD NMR spectrum (b) of mixture Trx-LysM1-2: (GlcNAc)₇ (1:100). (c) Chemical structure and epitope for binding of (GlcNAc)₇ to Trx-LysM1-2; relative STD intensities are color coded according to the scale and refers to the relative STD effects as shown in SI Appendix, Table S2. (B and C) STD quantitative analysis-derived epitope mapping on the molecular envelope of (GlcNAc)₇ and (GlcNAc)₈ with color coding from the highest (red) to lowest (yellow) observed STD effect.

Fig. 4.

Structural basis of chitin oligosaccharides binding. (A) Ribbon and surface representation of the homology model of CEBiP ectodomain. LysM0, LysM1, and LysM2 are drawn in orange, blue, and magenta, respectively. Other regions are colored gray. (B) Modeling of binding of N-acetylchitooligosaccharides to the LysM1domain of CEBiP. (C) Binding of GN8-Bio to mutant CEBiP proteins. CEBiP proteins containing the mutation for the indicated amino acid residues were expressed in N. benthamiana, from which MF for affinity labeling was prepared.

Fig. 5.

CEBiP dimerization and activation of chitin receptor complex by chitin oligosaccharides. (A) Molecular size-shift measurements of the effect of (GlcNAc)₈ on the oligomerization state of LysM1-2 (a) and LysM1-2 I₁₂₂A mutant (b), respectively. (c) Effect of (GlcNβ1,4GlcNAc)₄ on molecular size. (d) Effect of (GlcNAc)₈ after pretreatment with an equimolar amount of (GlcNβ1,4GlcNAc)₄. All measurements were conducted by light scattering using a 100-μM protein concentration and a protein:ligand ratio of 1:2. (B) Specific inhibition of (GlcNAc)₈-induced ROS generation by (GlcNβ1,4GlcNAc)₄ in rice cells. Concentration of (GlcNAc)₈ was 0.1 nM and the other oligosaccharides were 100nM. Competing oligosaccharides, (GlcNβ1,4GlcNAc)₄, (GlcNβ1,4GlcN)₄ or (GlcNA)₄ were pretreated 10 min before the addition of (GlcNAc)₈.

Fig. 6.

Hypothetical model of the activation of CEBiP-OsCERK1 complex by (GlcNAc)₈. (A) Sandwich-like model of activation of CEBiP-OsCERK1 receptor complex by (GlcNAc)₈. (B) Model of dimerization/activation inhibition by (GlcNβ1,4GlcNAc)₄.