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 |

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 Å crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-HisAsp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc) 2 for activity and proceeds through a tetrahedral oxyanion intermediate.Colletotrichum lindemuthianum is a plant fungal pathogen found extensively in tropical and subtropical regions. Colletotrichum species are the causative agent of anthracnose that affects economically important crop species (1). Furthermore, Colletotrichum sp. have recently been reported to cause subcutaneous and systemic infections among immunosuppressed patients (2). Colletotrichum sp. are facultative biotrophs. Before the fungal hyphae can successfully penetrate and gain access to host tissue, the fungus has to first evade plant antimicrobial hydrolases such as chitinases and -(1,3)glucanases (3, 4). The chitinases degrade fungal chitin, an insoluble linear polymer of -(1-4)-linked N-acetylglucosamine (GlcNAc). 1 The breakdown products may act as elicitors of active defense responses within the plant (5-7). Studies of cell wall composition of invasive fungal hyphae suggest that exposed fungal chitin polymers are partially de-N-acetylated during infection and initial growth within the host (8). Chitosan, the de-N-acetylated product, is a poor substrate for chitinases, which require the presence of N-acetyl moieties for recognition and catalysis (9). Thus, conversion of chitin to chitosan during plant extracellular colonization may protect pathogenic fungal hyphae from being lysed by secreted plant chitinases. The enzyme responsible for chitin modification is a developmentally regulated, secreted, chitin deacetylase (CDA) (10). C. lindemuthianum chitin deacetylase (ClCDA) is a member of the family 4 carbohydrate esterases (CE-4s) as defined by the CAZY database [http://afmb.cnrs-mrs.fr/∼cazy/CAZY (11)], which include several members that share the primary structure assigned as the "NodB homology domain" (12). Rh...

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