Background Botrytis cinerea secretes a high number of proteins that are predicted to have numerous O-glycosylation sites, frequently grouped in highly O-glycosylated regions, and analysis of mutants affected in O-glycosylation has shown, in B. cinerea and in other phytopathogenic fungi, that this process is important for fungal biology and virulence.ResultsWe report here the purification of glycoproteins from the culture medium, for a wild-type strain of B. cinerea and for three mutants affected in the first step of O-glycosylation, and the identification of components in the purified protein samples. Overall, 158 proteins were identified belonging to a wide diversity of protein families, which possess Ser/Thr-rich regions (presumably highly O-glycosylated) twice as frequently as the whole secretome. Surprisingly, proteins predicted to be highly O-glycosylated tend to be more abundant in the secretomes of the mutants affected in O-glycosylation than in the wild type, possibly because a correct glycosylation of these proteins helps keep them in the cell wall or extracellular matrix. Overexpression of three proteins predicted to be O-glycosylated in various degrees allowed to confirm the presence of mannose 1-2 and/or 1-3 bonds, but no mannose 1-6 bonds, and resulted in an enhanced activity of the culture medium to elicit plant defenses.ConclusionsGlycosylation of secretory proteins is very prevalent in B. cinerea and affects members of diverse protein families. O-glycosylated proteins play a role in the elicitation of plant defenses.