Botrytis cinerea cleaves a grapevine chitinase reducing its enzymatic activity and its detrimental effects on fungal growth

Chitin represents the main fibrillary component of the cell wall in filamentous fungi. During plant infection, chitin apposition to the fungal cell wall is counteracted by host chitinases. Following Botrytis cinerea infection, a chitinase IV is highly expressed in grapevine leaves and is also abundantly contained in grapevine berries. The fungus decreases the molecular size of this protein (Favaron et al., 2009, Journal of Plant Pathology, 91, 579-588) by its proteolytic activity (Marcato et al., 2017, Physiological and Molecular Plant Pathology, 99, 7-15). The cleavage of the chitinase occurs early during the in vitro fungal growth. The N-terminal sequencing of the cleaved chitinase shows that the fragment removed is the chitin-binding domain (CBD). Without the CBD, the chitinase decreases its activity by about 50%. To investigate the possible effects of the native and cleaved chitinase on B. cinerea, the two purified proteins were administered at 100 µg/ml to the fungal spores providing the proper conditions to avoid the chitinase cleavage. The native chitinase significantly decreased the conidia germination rate and the length of the germination tube while the cleaved chitinase did not. Protease inhibition assays provided evidence that metalloprotease activity is involved in the chitinase cleavage. To ascertain whether the native and cleaved chitinase can differently affect the expression of genes involved in B. cinerea cell wall modeling, we analyzed the expression of five fungal chitin synthase (Chs) and four chitin deacetylase (Cda) genes. By quantitative PCR we observed that only one Chs gene decreased its expression in presence of the native chitinase and three Cda genes were slightly down-regulated by both native and cleaved chitinase. In conclusion, the removal of the CBD by B. cinerea proteases appears as a mechanism preserving fungal growth from plant chitinase activity as highlighted also with other fungi (Jashni et al. 2015, Frontiers of plant science, 6, 1-7). Further experiments will better clarify the type of B. cinerea protease activity capable to disarm the plant chitinases.