Identification of glycoproteins secreted by wild-type Botrytis cinerea and by protein O-mannosyltransferase mutants

Abstract

BackgroundBotrytis 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 α1-2 and/or α1-3 bonds, but no mannose α1-6 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0254-y) contains supplementary material, which is available to authorized users.

BcSUN1, a B. cinerea SUN-Family Protein, Is Involved in Virulence

“…BcSUN1 (Bcin06g06040.1, from the B. cinerea protein database in EnsemblFungi 1 ; van Kan et al, 2016) is a highly glycosylated protein of 48 kDa, initially identified as a component of the B. cinerea early secretome (Espino et al, 2010) and also present in the glycosecretome (González et al, 2014). The alignment of its sequence with the four S. cerevisiae proteins that belong to Group-I of the SUN family (Supplementary Figure S2) showed an overall identity ranging from 28.1% for NCA3 to 38.6% for SIM1 ( Table 1 ), while sequence conservation was higher for the C -terminal region containing the SUN domain, with amino acid sequence identities ranging from 41 to 46%.…”

“…Botrytis cinerea strains used in this work were B05.10 (Quidde et al, 1999), a wild type strain, and B05.10-BcSUN1, which expresses a tagged version of the BcSUN1 protein under the control of the OliC promoter (González et al, 2014). These were kept as conidial suspensions in 15% glycerol at -80°C for long storage, and were maintained on 3% malt extract agar (MEA, Oxoid, UK) plates for routine use.…”

“…moneymaker and Phaseolus vulgaris plants were maintained in a growth chamber at 22°C, 70% humidity with a light/dark cycle of 14 h light/10 h dark. When tobacco seedlings were required, seeds were sterilized as explained before (González et al, 2014) and incubated 2 days at 4°C in darkness to break dormancy before germination on solid MS medium [0.5% Murashige and Skoog (Duchefa Biochemie, The Netherlands), 0.8% agar, pH 5.7] for 1 week.…”

“…The Bcsun1 gene encodes a protein of 471 amino acids that contains a signal peptide for secretion, as well as several Ser/Thr-rich regions that are potentially hyper- O -glycosylated (González et al, 2012). Oligosaccharides with mannose α1-2 and/or α1-3 bonds, but not mannose α1-6 bonds, were experimentally confirmed to be present in the BcSUN1 protein (González et al, 2014). In addition, the culture medium from a B. cinerea strain overexpressing BcSUN1 showed an enhanced capacity to elicit plant defenses, as compared with the wild type strain (González et al, 2014), suggesting that BcSUN1 may be recognized by the plant immune system.…”

“…BcSUN1 is a member of the β-glucosidase SUN family that has been experimentally identified as a component of the B. cinerea secretome (Espino et al, 2010; González et al, 2014). The Bcsun1 gene encodes a protein of 471 amino acids that contains a signal peptide for secretion, as well as several Ser/Thr-rich regions that are potentially hyper- O -glycosylated (González et al, 2012).…”