Role of the chitin-binding domain (CBD) of a grapevine class IV chitinase against Botrytis cinerea

During plant infection, fungal chitin deposition at the hyphal tip is counteracted by host chitinases. We studied the interaction between Botrytis cinerea and a class IV chitinase, that is constitutively expressed in mature grapevine berries. Early during fungal growth, the fungus proteolytically removes the chitin-binding domain (CBD). This removal results in a 50% reduction in the enzymatic activity of the chitinase and a complete loss of its botryticidal activity. Despite this, the pattern of chitin degradation by the native and the cleaved chitinase remains similar. We also detected the expression of this class IV chitinase in grapevine leaves during B. cinerea infection. Analysis of chitinase activity in leaf samples at various infection stages revealed that while this chitinase is not the most active among all chitinases, it is the most abundant at the protein level, with its expression correlating with infection progression. MALDI-TOF analysis of specific bands after SDS-PAGE confirmed the presence of both the native and CBD-deprived forms of the chitinase in the infected leaves, indicating that CBD removal observed in vitro also occurs during infection. Given the relatively low activity of this chitinase, as shown by zymogram, we further investigated the role of the CBD against B. cinerea. We transiently expressed the CBD in Nicotiana tabacum and heterologously expressed it in Pichia pastoris to assess its function. These findings highlight the significant role of the CBD in the chitinase's antifungal activity and its potential application in enhancing plant resistance to fungal infections.