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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

2D Electrophoresis of the glyco-secretome. 10 μg of purified protein sample were fractionated by 2D electrophoresis and stained with silver. Numbers indicate proteins identified in each spot by MALDI-TOF/TOF (Additional file 1). Boxes identify charge trains for which the same protein was identified in several spots.
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Fig2: 2D Electrophoresis of the glyco-secretome. 10 μg of purified protein sample were fractionated by 2D electrophoresis and stained with silver. Numbers indicate proteins identified in each spot by MALDI-TOF/TOF (Additional file 1). Boxes identify charge trains for which the same protein was identified in several spots.

Mentions: 2D electrophoresis of purified glycoproteins was carried out for B05.10 and the Δbcpmt1 mutant, and resulted in a relatively simple spot pattern (Figure 2), with not too many differences for the two samples. Some spots, however, showed clear differences in intensity, such as spot 18, which is over-expressed in the Δbcpmt1 sample. A total of 29 spots obtained from the wild-type or the Δbcpmt1 2D gels were excised and analyzed by MALDI-TOF/TOF, resulting in the identification of 18 proteins (Additional file 1). In some cases (spots 4, 10, 12 and 13), the same protein was identified in several spots forming charge trains (different pIs), or even in spots (spots 4 and 12) with different mobility in the second dimension (different apparent molecular weight).Figure 2


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

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

2D Electrophoresis of the glyco-secretome. 10 μg of purified protein sample were fractionated by 2D electrophoresis and stained with silver. Numbers indicate proteins identified in each spot by MALDI-TOF/TOF (Additional file 1). Boxes identify charge trains for which the same protein was identified in several spots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: 2D Electrophoresis of the glyco-secretome. 10 μg of purified protein sample were fractionated by 2D electrophoresis and stained with silver. Numbers indicate proteins identified in each spot by MALDI-TOF/TOF (Additional file 1). Boxes identify charge trains for which the same protein was identified in several spots.
Mentions: 2D electrophoresis of purified glycoproteins was carried out for B05.10 and the Δbcpmt1 mutant, and resulted in a relatively simple spot pattern (Figure 2), with not too many differences for the two samples. Some spots, however, showed clear differences in intensity, such as spot 18, which is over-expressed in the Δbcpmt1 sample. A total of 29 spots obtained from the wild-type or the Δbcpmt1 2D gels were excised and analyzed by MALDI-TOF/TOF, resulting in the identification of 18 proteins (Additional file 1). In some cases (spots 4, 10, 12 and 13), the same protein was identified in several spots forming charge trains (different pIs), or even in spots (spots 4 and 12) with different mobility in the second dimension (different apparent molecular weight).Figure 2

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