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 Ile 122 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 chitinelicitor receptor kinase-1.plant immunity | MTI/PTI | chitin signaling | receptor-ligand interaction |

Receptor Kinases in Plant-Pathogen Interactions: More Than Pattern Recognition

“…T. thus proposed a "cross-linking" model in which the two ends of long chitin oligomers each bind a LysM-ECD to form a dimer. In a separate NMR study assisted by synthetic chemistry, N-acetyl groups of alternating sugars were found to be located on two opposite sides of the chitin chain and directly bound by the LysM-ECD (Hayafune et al, 2014). Depleting N-acetyl groups from one side of the chitin chain did not affect chitin binding to a CEBiP-ECD, but abolished dimerization.…”

“…Liu et al, 2012;Hayafune et al, 2014). Two competing models have been proposed for chitin-induced dimerization of LysM-ECDs.…”

“…Depleting N-acetyl groups from one side of the chitin chain did not affect chitin binding to a CEBiP-ECD, but abolished dimerization. This led to the proposal of a "sandwich" model in which two CEBiP-ECDs bind the chitin chain, one on each side (Hayafune et al, 2014). However, this does not explain why shorter chitin oligomers do not induce dimerization.…”

“…However, this does not explain why shorter chitin oligomers do not induce dimerization. Whether CEBiP possesses significantly different affinities toward short and long chitin oligomers remains controversial, in part because different fragments of CEBiP-ECD were used in different studies (Hayafune et al, 2014;S. Liu et al, 2016).…”

Chitin and chitin-related compounds in plant–fungal interactions

“…In the situation of defence responses to pathogenic fungi, LysM RLK1 (Wan et al 2008) or CERK1 (Miya et al 2007)/CERK1 (as in Arabidopsis ) or CERK1/CEBiP receptor dimers (as in rice, Shimizu et al 2010) needs longer chito-oligomers, like octamers to activate its response through heterodimerisation (Hayafune et al 2014; Liu et al 2015). The perception of shorter oligosaccharides such as chitotetraose could instead rely on monomeric receptors (Miyata et al 2014; Shinya et al 2015) as into each receptor’s LysM domain, half of this oligomer molecule (C4) fits (Liu et al 2012; Shinya et al 2015).…”