The role of grape polyphenols on trans-resveratrol activity against Botrytis cinerea and of fungal laccase on the solubility of putative grape PR proteins

The necrotrophic fungus Botrytis cinerea is the causal agent of grey mould disease on grapevine. In contact with mature grape berries, the fungus encounters an environment particularly rich in polyphenols and proteins, where the stilbenic phytoalexin trans-resveratrol may accumulate. Some grape proteins are structurally and functionally related to plant pathogenesis-related proteins. To mimic conditions similar to those found in grape berries, B. cinerea was grown in vitro with proteins and polyphenols extracted from mature grapes, and with trans-resveratrol. Results showed that in the presence of highly toxic amounts of trans-resveratrol, grape polyphenols allowed total recovery of fungal growth, and proteins allowed partial recovery. These resveratrol-polyphenol or resveratrol-protein combinations also induced a strong release into the medium of laccase activity, which is likely to be involved in transresveratrol detoxification. The protein pattern changed during fungal growth; most grape proteins quickly disappeared from the culture when polyphenols and transresveratrol were present together. Similar protein patterns were obtained in vitro by incubating grape proteins with grape polyphenols and/or trans-resveratrol with a purified B. cinerea laccase. Under these conditions, most proteins became insoluble. The grape protein pattern obtained from grape berries infected by B. cinerea strongly resembled that obtained in vitro by incubating grape proteins and polyphenols with fungal laccase. It seems that B. cinerea, through laccase secretion and activity and by exploiting the berry polyphenols, easily neutralizes the toxicity of grape stilbenic phytoalexins and makes the grape pathogenesis-related proteins insoluble.