Chitin-Binding Protein ofVerticillium nonalfalfaeDisguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity

Abstract

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, 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 and to interfere with chitin-triggered host immunity.

Comparative Genomic Data Provide New Insight on the Evolution of Pathogenicity in Sporothrix Species

“…CBM48, similar to CBM20, is mostly involved in starch biosynthesis ( Wilkens et al, 2018 ). CBM50, also known as LysM domains, plays an important role in inhibiting host defenses ( Volk et al, 2019 ). Peptidases provide alternative carbon sources for glucose starving cells through degrading proteins ( Handtke et al, 2018 ), and promote various progresses, such as cell growth and differentiation, apoptosis, cell cycle, and signaling ( Ng et al, 2009 ).…”

Characterization of the CAZy Repertoire from the Marine-Derived Fungus Stemphylium lucomagnoense in Relation to Saline Conditions

“…CBM50 is a module of approximately 50 residues, generally grafted to various enzymes that cleave chitin or peptidoglycan, including families GH18, 19, 23-25, and 73. When not associated with other enzymatic modules, CBM50, like CBM18, is involved in the recognition or binding of chitin and prevents hydrolysis of the fungal cell wall by plant chitinases, therefore interfering with chitin-triggered host immunity [25].…”

Verticillium longisporum Elicits Media-Dependent Secretome Responses With Capacity to Distinguish Between Plant-Related Environments

“…Well known examples of fungal effectors include the Avr4 and Ecp6 effectors from the leaf mold fungus Cladosporum fulvum that bind to chitin oligosaccharides via a carbohydrate-binding module or LysM domain, respectively ( van Esse et al, 2007 ; de Jonge et al, 2010 ; Lo Presti et al, 2015 ; Volk et al, 2019 ). Similarly, a chitin scavenging function has also been assigned to Cp1 in V. dahliae strain XH-8.…”

“…CP1 knockout mutants were affected in cotton virulence ( Zhang et al, 2017 ). This chitin protection leads to the suppression of the pathogen-associated molecular patterns-triggered immunity of the plant and shields the fungal cell wall from plant chitinases that hydrolyze chitin ( van Esse et al, 2007 ; de Jonge et al, 2010 ; Lo Presti et al, 2015 ; Volk et al, 2019 ). Other fungal effectors such as metalloproteases possess enzymatic activity and are able to truncate plant chitinases that attack the fungal cell wall ( Naumann et al, 2011 ; Han et al, 2019 ).…”