Overcome of grape chemical barriers by the fungal pathogen Botrytis cinerea

Botrytis cinerea in contact with mature grape berries encounters an environment particularly rich in polyphenols and PR proteins, where the stilbenic phytoalexin trans-resveratrol may accumulate. To mimic conditions similar to those found in grape berries, B. cinerea was grown in vitro with grape PR 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 trans-resveratrol detoxification. The grape protein pattern changed during fungal growth; most grape proteins quickly disappeared from the culture when polyphenols and trans-resveratrol 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. The effect of laccase, resveratrol and polyphenols on fungal spore germination was also studied. Results showed that resveratrol alone initially does not inhibit the spore germination. But the inhibition was completely relieved by the presence of grape polyphenols. Instead, the pre-incubation of resveratrol with laccase completely inhibited the spore germination. In addition, we investigate the involvement of B. cinerea proteases in the degradation of grape PR proteins. An aspartyl and a tripeptidyl protease were purified from B. cinerea in vitro culture. The purified proteases activities partially degraded PR proteins. The expression analysis of tripeptidyl and aspartic protease gene families revealed that several members of these families are expressed in the presence of grape PR proteins. In conclusion, our results support that in a grape berry environment characterized by an abundance of polyphenols, B. cinerea laccase not only detoxify the trans-resveratrol but also modifies the solubility of grape proteins and this environment may facilitate the fungal protease to degrade grape PR proteins.