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

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.Plants have the ability to detect potential pathogens through the recognition of microbe-associated molecular patterns (MAMPs; also known as pathogen-associated molecular patterns), such as flagellin, elongation factor Tu (EF-Tu), peptidoglycan, LPS, chitin, and β-glucan, which are typical molecular signatures for whole classes of microbes (1, 2). MAMP-triggered defense is the first barrier to prevent the invasion of pathogens and plays a major role in the basal resistance of plants against various pathogens. It is also well known that this defense system is strikingly similar to the innate immunity of animals (1, 3, 4).Leucine-rich repeat receptor-like kinases, flagellin-sensetive 2 (FLS2), and EF-Tu receptor, have been shown to recognize bacterial flagellin and EF-Tu, respectively, and serve as receptors for these MAMPs (5). On the other hand, two types of lysin motif (LysM) proteins, CEBiP (chitin-elicitor binding protein) and CERK1 (chitin-elicitor receptor kinase-1), were identified as the cell-surface receptor for chitin, a representative fungal molecular pattern (6–8). Knockout/-down experiments of these genes showed that both of these LysM proteins are required for chitin perception and signaling in rice, whereas CEBiP-type molecules are not involved in chitin signaling in Arabidopsis, indicating the difference between the chitin receptor systems in these model plants (8). Additionally, another LysM receptor-like kinase, LYK4, was also indicated to contribute to chitin signaling in Arabidopsis (9). In the case of rice, it was also shown that CEBiP and Oryza sativa (Os)CERK1 form a heterooligomeric receptor complex ligand dependently (10).Both CEBiP and OsCERK1 have LysMs, which have been known to bind peptidoglycan and chitin (11), in their ectodomains. In Arabidopsis, CERK1 was shown to bind chitin and trigger immune responses as a kind of “all-in-one” receptor. On the other hand, CEBiP seems to play a major role in the perception of chitin in rice, as the knockdown of CEBiP almost abolished the binding of a radio-labeled chitin oligosaccharide to the plasma membrane, whereas OsCERK1 was shown not to bind chitin (6, 12). Liu et al. recently reported that two other CEBiP homologs, OsLYP4 and -6, also bind chitin and contribute to chitin responses and disease resistance in rice (13), although it is not clear to what extent these proteins contribute as the cell surface receptor for chitin oligosaccharides.It was also shown that the perception of peptidoglycan in Arabidopsis requires CEBiP-like molecules (14). Arabidopsis homologs of CEBiP, LYM1 and LYM3, play a major role for the binding of peptidoglycan and activation of downstream defense responses through the receptor kinase, CERK1. These results showed that the receptor kinase CERK1 is required for both chitin and peptidoglycan signaling, at least in Arabidopsis. The peptidoglycan receptor system in Arabidopsis seems similar to the rice chitin receptor for the requirement of a binding protein and a receptor kinase, although the receptor complex formation by these two proteins was not confirmed.Thus, the detailed analysis of ligand recognition by these CEBiP-like molecules and succeeding formation and activation of receptor complex is critically important to understand the molecular mechanisms leading to the activation of downstream defense responses triggered by these MAMPs. Such information would also contribute to the design of novel receptor molecules suitable for future biotechnological application. We show herein the results obtained by biochemical studies on the binding site of CEBiP, epitope mapping of chitin oligosaccharides by saturation transfer difference (STD) NMR spectroscopy, and molecular modeling/docking studies combined with site-directed mutagenesis of the ectodomain of CEBiP. These results clearly indicated that two CEBiP molecules simultaneously bind to one N-acetylchitoheptaose/octaose, (GlcNAc)_7/8, through a binding site located in the central LysM region of the ectodomain, resulting in the dimerization of CEBiP. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and OsCERK1.