The invisibility cloak: Chitin binding protein ofVerticillium nonalfalfaedisguises fungus from plant chitinases

AbstractDuring fungal infections, plant cells secrete chitinases that digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune receptors results in the activation of defence signalling pathways. We report here thatVerticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents this recognition process by secreting a CBM18 (carbohydrate binding motif 18)-chitin binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by theVna8.213gene which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however,Vna8.213knockout mutants exhibit wilting symptoms similar to the wild type fungus, suggesting thatVna8.213activity is functionally redundant during fungal infection of hop. In binding assay, recombinant VnaChtBP binds chitin and chitin oligomersin vitrowith submicromolar affinity and protects fungal hyphae from degradation by plant chitinases. Using a yeast-two-hybrid assay, homology modelling and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases.