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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

Growth inhibition and necrosis of seedlings caused byO-glycosylated proteins. Tobacco seedlings were treated for 9 days with culture media from the strains overexpressing the indicated O-glycosylated proteins and then assessed for necrosis and growth inhibition. A: Western-blot (anti-c-myc) showing the relative amounts of recombinant proteins in the culture media from the overexpressing strains. Medium from the wild-type strain (B05.10) and uninoculated medium (YGG-low) were used as controls. Each lane contained proteins precipitated from 1 ml of medium. Molecular weight makers are show to the left of each lane (kDa). B: Example seedlings treated with the culture media. C: Average weight (n=6) of treated seedling after the 9-day incubation. Different letters on bars indicate statistically significant differences with 0.99 confidence.
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Fig7: Growth inhibition and necrosis of seedlings caused byO-glycosylated proteins. Tobacco seedlings were treated for 9 days with culture media from the strains overexpressing the indicated O-glycosylated proteins and then assessed for necrosis and growth inhibition. A: Western-blot (anti-c-myc) showing the relative amounts of recombinant proteins in the culture media from the overexpressing strains. Medium from the wild-type strain (B05.10) and uninoculated medium (YGG-low) were used as controls. Each lane contained proteins precipitated from 1 ml of medium. Molecular weight makers are show to the left of each lane (kDa). B: Example seedlings treated with the culture media. C: Average weight (n=6) of treated seedling after the 9-day incubation. Different letters on bars indicate statistically significant differences with 0.99 confidence.

Mentions: Culture media from the three strains overexpressing the O-glycosylated proteins were also tested for the ability to elicit defense responses in plants by seedling growth inhibition assay [44], a sensitive and quantitative test which correlates with typical plant defence responses such as callose deposition, production of reactive oxygen species, or pathogenesis-related gene expression [45,46]. In this assay, culture media from the three overexpressing strains revealed considerably more efficient in inhibiting growth of tobacco seedling, as compared with the media obtained with the wild-type strain (Figure 7A-C). Moreover, the seedlings treated with media from the overexpressing strains showed necrotic symptoms that were clearly more intense than with the wild-type strain.Figure 7


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

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

Growth inhibition and necrosis of seedlings caused byO-glycosylated proteins. Tobacco seedlings were treated for 9 days with culture media from the strains overexpressing the indicated O-glycosylated proteins and then assessed for necrosis and growth inhibition. A: Western-blot (anti-c-myc) showing the relative amounts of recombinant proteins in the culture media from the overexpressing strains. Medium from the wild-type strain (B05.10) and uninoculated medium (YGG-low) were used as controls. Each lane contained proteins precipitated from 1 ml of medium. Molecular weight makers are show to the left of each lane (kDa). B: Example seedlings treated with the culture media. C: Average weight (n=6) of treated seedling after the 9-day incubation. Different letters on bars indicate statistically significant differences with 0.99 confidence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Growth inhibition and necrosis of seedlings caused byO-glycosylated proteins. Tobacco seedlings were treated for 9 days with culture media from the strains overexpressing the indicated O-glycosylated proteins and then assessed for necrosis and growth inhibition. A: Western-blot (anti-c-myc) showing the relative amounts of recombinant proteins in the culture media from the overexpressing strains. Medium from the wild-type strain (B05.10) and uninoculated medium (YGG-low) were used as controls. Each lane contained proteins precipitated from 1 ml of medium. Molecular weight makers are show to the left of each lane (kDa). B: Example seedlings treated with the culture media. C: Average weight (n=6) of treated seedling after the 9-day incubation. Different letters on bars indicate statistically significant differences with 0.99 confidence.
Mentions: Culture media from the three strains overexpressing the O-glycosylated proteins were also tested for the ability to elicit defense responses in plants by seedling growth inhibition assay [44], a sensitive and quantitative test which correlates with typical plant defence responses such as callose deposition, production of reactive oxygen species, or pathogenesis-related gene expression [45,46]. In this assay, culture media from the three overexpressing strains revealed considerably more efficient in inhibiting growth of tobacco seedling, as compared with the media obtained with the wild-type strain (Figure 7A-C). Moreover, the seedlings treated with media from the overexpressing strains showed necrotic symptoms that were clearly more intense than with the wild-type strain.Figure 7

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