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Botrytis cinerea protein O-mannosyltransferases play critical roles in morphogenesis, growth, and virulence.

González M, Brito N, Frías M, González C - PLoS ONE (2013)

Bottom Line: The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs.Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves.Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de La Laguna, La Laguna (Tenerife), Spain.

ABSTRACT
Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea.

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Related in: MedlinePlus

Penetration assays with the Δbcpmt mutants on onion epidermis.Small pieces of mycelia from the three mutants and the wild type (B05.10) were placed on onion epidermis (inner surface up) and incubated for 1–4 days on the surface of water-agar. A) Samples stained with lactophenol trypan-blue. Arrows indicate unstained hyphae that have penetrated the epidermis. B) Fluorescence microscope observation of the same samples stained with lactophenol trypan-blue. Yellow autofluorescence is due to the release of phenolic compounds in the infected onion epidermal cells, and is an indirect indication of penetration. C) Observation under the scanning electron microscope. Arrows indicate the points where the mycelium has entered or is below the surface.
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pone-0065924-g009: Penetration assays with the Δbcpmt mutants on onion epidermis.Small pieces of mycelia from the three mutants and the wild type (B05.10) were placed on onion epidermis (inner surface up) and incubated for 1–4 days on the surface of water-agar. A) Samples stained with lactophenol trypan-blue. Arrows indicate unstained hyphae that have penetrated the epidermis. B) Fluorescence microscope observation of the same samples stained with lactophenol trypan-blue. Yellow autofluorescence is due to the release of phenolic compounds in the infected onion epidermal cells, and is an indirect indication of penetration. C) Observation under the scanning electron microscope. Arrows indicate the points where the mycelium has entered or is below the surface.

Mentions: This hypothesis was further explored by performing penetration assays on onion epidermis inoculated with agar plugs coming from cultures of the three mutants, or the wild type. Figure 9A shows pictures of samples stained with lactophenol trypan-blue, which densely stains only those hyphae that have not penetrated the plant tissue. Weakly stained hyphae inside the onion cells could be observed for the wild type and for Δbcpmt1, but not for Δbcpmt2 or Δbcpmt4. Similarly, Δbcpmt2 and Δbcpmt4 were also unable to stimulate autofluorescence in onion cells (Figure 9B), a phenomenon indicative of a successful infection [46]. Finally, examination of the infected tissues with a Scanning Electron Microscope (Figure 9C) revealed the presence of fungal penetration sites displaying macerated plant tissue in the case of the wild type and the Δbcpmt1 mutant, but these sites could not be found for the two other mutants which displayed, instead, plenty of fungal hyphae growing outside the onion cells.


Botrytis cinerea protein O-mannosyltransferases play critical roles in morphogenesis, growth, and virulence.

González M, Brito N, Frías M, González C - PLoS ONE (2013)

Penetration assays with the Δbcpmt mutants on onion epidermis.Small pieces of mycelia from the three mutants and the wild type (B05.10) were placed on onion epidermis (inner surface up) and incubated for 1–4 days on the surface of water-agar. A) Samples stained with lactophenol trypan-blue. Arrows indicate unstained hyphae that have penetrated the epidermis. B) Fluorescence microscope observation of the same samples stained with lactophenol trypan-blue. Yellow autofluorescence is due to the release of phenolic compounds in the infected onion epidermal cells, and is an indirect indication of penetration. C) Observation under the scanning electron microscope. Arrows indicate the points where the mycelium has entered or is below the surface.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675079&req=5

pone-0065924-g009: Penetration assays with the Δbcpmt mutants on onion epidermis.Small pieces of mycelia from the three mutants and the wild type (B05.10) were placed on onion epidermis (inner surface up) and incubated for 1–4 days on the surface of water-agar. A) Samples stained with lactophenol trypan-blue. Arrows indicate unstained hyphae that have penetrated the epidermis. B) Fluorescence microscope observation of the same samples stained with lactophenol trypan-blue. Yellow autofluorescence is due to the release of phenolic compounds in the infected onion epidermal cells, and is an indirect indication of penetration. C) Observation under the scanning electron microscope. Arrows indicate the points where the mycelium has entered or is below the surface.
Mentions: This hypothesis was further explored by performing penetration assays on onion epidermis inoculated with agar plugs coming from cultures of the three mutants, or the wild type. Figure 9A shows pictures of samples stained with lactophenol trypan-blue, which densely stains only those hyphae that have not penetrated the plant tissue. Weakly stained hyphae inside the onion cells could be observed for the wild type and for Δbcpmt1, but not for Δbcpmt2 or Δbcpmt4. Similarly, Δbcpmt2 and Δbcpmt4 were also unable to stimulate autofluorescence in onion cells (Figure 9B), a phenomenon indicative of a successful infection [46]. Finally, examination of the infected tissues with a Scanning Electron Microscope (Figure 9C) revealed the presence of fungal penetration sites displaying macerated plant tissue in the case of the wild type and the Δbcpmt1 mutant, but these sites could not be found for the two other mutants which displayed, instead, plenty of fungal hyphae growing outside the onion cells.

Bottom Line: The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs.Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves.Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de La Laguna, La Laguna (Tenerife), Spain.

ABSTRACT
Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea.

Show MeSH
Related in: MedlinePlus