Bioactivity of the chitin-binding domain (CBD) from a major grapevine chitinase against Botrytis cinerea.

During plant infection, fungal chitin synthesis at the hyphal tips is hampered by host chitinases. Our study focused on the interplay between Botrytis cinerea and a class IV chitinase, constitutively expressed in mature grapevine berries. In the initial stages of in vitro fungal growth, the fungus proteolytically removes the chitin-binding domain (CBD) of the chitinase. This removal results in a 50% reduction in the enzyme's activity and completely nullifies its botryticidal properties. We also detected the native and the cleaved forms of the chitinase in infected grapevine leaves, with a peak of protein accumulation at the early stages of infection, indicating that CBD removal observed in vitro also occurs during infection. Given the loss of antifungal activity of the cleaved form, we hypothesized that the CBD is responsible for the antifungal activity of the chitinase. Therefore, we transiently expressed the CBD in Nicotiana tabacum to evaluate its ability to enhance plant resistance to B. cinerea, observing a reduction in disease symptoms. To further validate these results, we produced transgenic Arabidopsis plants constitutively expressing the CBD. Additionally, we are heterologously expressing the CBD in Escherichia coli to microscopically characterize the effect of CBD treatment on hyphal tips and assess the antifungal activity. These findings highlight the essential role of the CBD in the chitinase's antifungal activity and its potential application in enhancing plant resistance to B. cinerea.