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

González M, Brito N, González C - BMC Microbiol. (2014)

Bottom Line: 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.Glycosylation 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.

View Article: PubMed Central - PubMed

Affiliation: U.D. Bioquímica y Biología Molecular, Universidad de La Laguna, 38206, La Laguna (Tenerife), Spain. mario_hztl@hotmail.com.

ABSTRACT

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.

Results: We 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.

Conclusions: Glycosylation 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.

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Related in: MedlinePlus

Glyco-secretome purification. Electrophoretic analysis of glycoproteins purified from the extracellular fraction in cultures of wild-type B. cinerea (B05.10) and the indicated Δbcpmt mutants. A: SDS-PAGE showing all proteins precipitated from 150 μl of culture medium. B: SDS-PAGE of the purified glycoprotein samples (150 μl). Black arrow: BcAp8, a protein with no predicted glycosylation sites. White arrows: example bands appearing/disappearing in the mutant samples as compared with the corresponding wild-type sample.
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Fig1: Glyco-secretome purification. Electrophoretic analysis of glycoproteins purified from the extracellular fraction in cultures of wild-type B. cinerea (B05.10) and the indicated Δbcpmt mutants. A: SDS-PAGE showing all proteins precipitated from 150 μl of culture medium. B: SDS-PAGE of the purified glycoprotein samples (150 μl). Black arrow: BcAp8, a protein with no predicted glycosylation sites. White arrows: example bands appearing/disappearing in the mutant samples as compared with the corresponding wild-type sample.

Mentions: We have previously identified more than one hundred proteins in the B. cinerea secretome [7], and we have also observed that the appearance of the secretome in 1D- and 2D-PAGE changes radically for mutants affected in PMTs, which catalyse the first step of O-glycosylation [28]. This prompted us to better characterize the set of glycoproteins secreted by wild-type B. cinerea strain B05.10, as well as by the mutants lacking each one of the three B. cinerea PMTs [28]. Preliminary observations allowed us to establish static liquid cultures in Petri dishes with YGG-low medium, inoculated with mycelial plugs and incubated for 4 days, as the optimum conditions to maximize isolation of secretory proteins in the case of Δbcpmt mutants. Culture media obtained from these plates contained plenty of proteins (Figure 1A) and the band pattern in SDS-PAGE was different for the wild type and for the Δbcpmt mutants. Purification of glycoproteins from the culture medium by affinity chromatography with Concanavalin-A resulted in quite different band patterns (Figure 1B), with some proteins clearly disappearing and others being enriched. Notably, the band corresponding to the most abundant protein in the secretome of B. cinerea, the 35-kDa aspartic protease BcAp8 [30] (black arrow in Figure 1A), is completely absent after purification of glycoproteins, in accordance with the fact that no N- or O-glycosylated sites are predicted in silico for this protein by the NetNGlyc 1.0 [31] and NetOGlyc 4.0 [32,33] servers. Moreover, an enrichment in proteins with high molecular weight (>50 kDa) was observed for all samples after glycoprotein purification, in agreement with the fact that lectin-blot experiments also show that glycosylation is more prominent for these proteins [28]. As expected, the SDS-PAGE band pattern of purified glycoprotein samples is different for the wild type and the three Δbcpmt mutants, especially in the case of Δbcpmt2 and Δbcpmt4.Figure 1


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

González M, Brito N, González C - BMC Microbiol. (2014)

Glyco-secretome purification. Electrophoretic analysis of glycoproteins purified from the extracellular fraction in cultures of wild-type B. cinerea (B05.10) and the indicated Δbcpmt mutants. A: SDS-PAGE showing all proteins precipitated from 150 μl of culture medium. B: SDS-PAGE of the purified glycoprotein samples (150 μl). Black arrow: BcAp8, a protein with no predicted glycosylation sites. White arrows: example bands appearing/disappearing in the mutant samples as compared with the corresponding wild-type sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197228&req=5

Fig1: Glyco-secretome purification. Electrophoretic analysis of glycoproteins purified from the extracellular fraction in cultures of wild-type B. cinerea (B05.10) and the indicated Δbcpmt mutants. A: SDS-PAGE showing all proteins precipitated from 150 μl of culture medium. B: SDS-PAGE of the purified glycoprotein samples (150 μl). Black arrow: BcAp8, a protein with no predicted glycosylation sites. White arrows: example bands appearing/disappearing in the mutant samples as compared with the corresponding wild-type sample.
Mentions: We have previously identified more than one hundred proteins in the B. cinerea secretome [7], and we have also observed that the appearance of the secretome in 1D- and 2D-PAGE changes radically for mutants affected in PMTs, which catalyse the first step of O-glycosylation [28]. This prompted us to better characterize the set of glycoproteins secreted by wild-type B. cinerea strain B05.10, as well as by the mutants lacking each one of the three B. cinerea PMTs [28]. Preliminary observations allowed us to establish static liquid cultures in Petri dishes with YGG-low medium, inoculated with mycelial plugs and incubated for 4 days, as the optimum conditions to maximize isolation of secretory proteins in the case of Δbcpmt mutants. Culture media obtained from these plates contained plenty of proteins (Figure 1A) and the band pattern in SDS-PAGE was different for the wild type and for the Δbcpmt mutants. Purification of glycoproteins from the culture medium by affinity chromatography with Concanavalin-A resulted in quite different band patterns (Figure 1B), with some proteins clearly disappearing and others being enriched. Notably, the band corresponding to the most abundant protein in the secretome of B. cinerea, the 35-kDa aspartic protease BcAp8 [30] (black arrow in Figure 1A), is completely absent after purification of glycoproteins, in accordance with the fact that no N- or O-glycosylated sites are predicted in silico for this protein by the NetNGlyc 1.0 [31] and NetOGlyc 4.0 [32,33] servers. Moreover, an enrichment in proteins with high molecular weight (>50 kDa) was observed for all samples after glycoprotein purification, in agreement with the fact that lectin-blot experiments also show that glycosylation is more prominent for these proteins [28]. As expected, the SDS-PAGE band pattern of purified glycoprotein samples is different for the wild type and the three Δbcpmt mutants, especially in the case of Δbcpmt2 and Δbcpmt4.Figure 1

Bottom Line: 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.Glycosylation 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.

View Article: PubMed Central - PubMed

Affiliation: U.D. Bioquímica y Biología Molecular, Universidad de La Laguna, 38206, La Laguna (Tenerife), Spain. mario_hztl@hotmail.com.

ABSTRACT

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.

Results: We 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.

Conclusions: Glycosylation 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.

Show MeSH
Related in: MedlinePlus