CBM18 containing lectin-like protein of Verticillium nonalfalfae binds chitin and protects hyphae from degradation by xylem hydrolases

Verticillium nonalfalfae is a soil-borne hemibiotrophic ascomycete infecting various plant species, including hop (Humulus lupulus L.). During the infection of hop, the fungus produces a 38.6 kDa cysteine rich lectin-like protein (VnaCBP8.213) harboring six carbohydrate binding CBM18 domains. Its gene expression increases with disease progression, reaching the highest abundance in stems of susceptible hop. We examined here genetic diversity of VnaCBP8.213 in various isolates of Verticillium spp. and biochemically characterized recombinant VnaCBP8.213 to determine its biological role.

The VnaCBP8.213 gene was PCR-amplified and its sequence variation was studied. It was detected in all V. nonalfalfae and V. alfalfae isolates, but was not present in V. dahliae, V. tricorpus and V. isaacii. Deletion of the VnaCBP8.213 gene did not affect the fungal growth, conidiation or pathogenicity of V. nonalfalfae in susceptible hop.

E. coli expressed and purified VnaCBP8.213, infiltrated into Nicotiana benthamiana leaves, did not trigger a visible HR response. The nonspecific subcellular localization pattern of mRFP fusion protein in N. benthamiana leaves led to the hypothesis that VnaCBP8.213 could function as an apoplastic effector involved in the evasion of chitin-triggered plant immune responses. A carbohydrate sedimentation test confirmed that the recombinant protein VnaCBP8.213 specifically binds to chitin beads and crab shell chitin, but not to cellulose and xylan. Carbohydrate binding specificity and affinity will be determined by surface plasmon resonance (SPR). Furthermore, the addition of 3 µM VnaCBP8.213 caused an aggregation of fungal hyphae, shielding them from degradation by xylem sap hydrolases. To confirm the immunosuppressive activity of VnaCBP8.213, the inhibition of a chitin oligomer triggered immune response in tobacco BY-2 suspension cells will be examined.

This work benefitted from interactions promoted by COST Action FA 1208 https://cost-sustain.org. The authors acknowledge financial support from the Slovenian Research Agency, core funding P4-0077, fellowship 342257, and project J4-8220.