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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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Growth of Δbcpmt strains in axenic culture.Growth rates (mean values and standard deviation) of the three mutant strains and the wild type (B05.10) in different media. Media compositions are given in Experimental Procedures. Asterisks indicate a statistically significant difference with the wild type in the same medium (n≥3, p<0.05).
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pone-0065924-g002: Growth of Δbcpmt strains in axenic culture.Growth rates (mean values and standard deviation) of the three mutant strains and the wild type (B05.10) in different media. Media compositions are given in Experimental Procedures. Asterisks indicate a statistically significant difference with the wild type in the same medium (n≥3, p<0.05).

Mentions: All three Δbcpmt mutants showed a lower growth rate than the wild-type strain in all media tested (Figure 2). Δbcpmt2 was not able to grow at all in 3 of the most standard B. cinerea growth media, Gamborg’s B5, MEA and PDA, while Δbcpmt1 and Δbcpmt4 grew in these three media with a 23–62% reduction in the growth rate, as compared with the wild-type strain. These differences were smaller in the richer medium YGG and in SH, and these media even allowed the growth of Δbcpmt2. Other studies in yeast and filamentous fungi have shown that pmt mutants partially recover the normal growth rate levels in media supplemented with osmotic stabilizers [8], [33], [34]. The addition of 1 M sorbitol, 1 M sucrose, or 0.5 M KCl to the cultures in MEA (Figure 3) increased the growth rate of the wild-type strain B05.10, surprisingly, by 18%. This increase was only slightly higher for Δbcpmt1 (17–40%), but a clearer recovery of the growth rate was observed in the case of Δbcpmt4 (28–62%), and especially in the case of Δbcpmt2 (Figure 3), which did not growth at all in MEA but recovered the growth rate to 56–74% of that of the wild type with the osmotic stabilizers. The stabilizing effect of the increased osmolarity is usually interpreted by a weakened cell wall structure. A cell wall not fully able to cope with the osmotic pressure would be a growth-limiting factor at low osmolarities, but not so when osmolarities of the cell interior and the surrounding medium are similar. The fact that it is especially Δbcpmt2 the mutant that most clearly recovers the growth rate in the presence of osmotic stabilizers may represent that BcPMT2 is the main contributor to the O-glycosylation of key structural cell-wall proteins.


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

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

Growth of Δbcpmt strains in axenic culture.Growth rates (mean values and standard deviation) of the three mutant strains and the wild type (B05.10) in different media. Media compositions are given in Experimental Procedures. Asterisks indicate a statistically significant difference with the wild type in the same medium (n≥3, p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065924-g002: Growth of Δbcpmt strains in axenic culture.Growth rates (mean values and standard deviation) of the three mutant strains and the wild type (B05.10) in different media. Media compositions are given in Experimental Procedures. Asterisks indicate a statistically significant difference with the wild type in the same medium (n≥3, p<0.05).
Mentions: All three Δbcpmt mutants showed a lower growth rate than the wild-type strain in all media tested (Figure 2). Δbcpmt2 was not able to grow at all in 3 of the most standard B. cinerea growth media, Gamborg’s B5, MEA and PDA, while Δbcpmt1 and Δbcpmt4 grew in these three media with a 23–62% reduction in the growth rate, as compared with the wild-type strain. These differences were smaller in the richer medium YGG and in SH, and these media even allowed the growth of Δbcpmt2. Other studies in yeast and filamentous fungi have shown that pmt mutants partially recover the normal growth rate levels in media supplemented with osmotic stabilizers [8], [33], [34]. The addition of 1 M sorbitol, 1 M sucrose, or 0.5 M KCl to the cultures in MEA (Figure 3) increased the growth rate of the wild-type strain B05.10, surprisingly, by 18%. This increase was only slightly higher for Δbcpmt1 (17–40%), but a clearer recovery of the growth rate was observed in the case of Δbcpmt4 (28–62%), and especially in the case of Δbcpmt2 (Figure 3), which did not growth at all in MEA but recovered the growth rate to 56–74% of that of the wild type with the osmotic stabilizers. The stabilizing effect of the increased osmolarity is usually interpreted by a weakened cell wall structure. A cell wall not fully able to cope with the osmotic pressure would be a growth-limiting factor at low osmolarities, but not so when osmolarities of the cell interior and the surrounding medium are similar. The fact that it is especially Δbcpmt2 the mutant that most clearly recovers the growth rate in the presence of osmotic stabilizers may represent that BcPMT2 is the main contributor to the O-glycosylation of key structural cell-wall proteins.

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