Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc) ₆ reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein

Abstract

Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc) ₆] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc) ₆ binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc) ₆ molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc) ₆ positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc) ₆ enabled the elucidation of the precise topography and amino acid composition of Avr4’s ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4’s stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family.

Author summary

Microbes mobilize an array of secreted effectors to manipulate their hosts during infections, whereas in response, hosts utilize cognate immune receptors to perceive effectors and mount a defense. To date, the structural basis of effector function and recognition by immune receptors are still poorly understood. Here we present the crystal structure in complex with chitohexaose of CfAvr4, a CBM14 lectin and the founding member of a fungal effector family that binds and protects chitin in fungal cell-walls from chitinases. This is the first structure of a CBM14 protein to be co-crystalized with its ligand that further reveals how Avr4 effectors function. Specifically, by leveraging structural and functional data, we elucidate the molecular basis for ligand-binding by CfAvr4 and show that two effector molecules are brought together through the ligand to form a sandwich structure that laminates two chitohexaose molecules within the dimeric assembly. We further show that recognition of CfAvr4 by the cognate Cf-4 immune receptor is not mediated through residues directly interacting with chitohexaose, thereby structurally uncoupling the ligand-binding function of Avr4 from recognition by Cf-4 and challenging early postulations that the broad recognition of Avr4 effectors by Cf-4 stems from perceiving residues implicated in binding their ligand.