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Signaling governed by G proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi.

Studt L, Humpf HU, Tudzynski B - PLoS ONE (2013)

Bottom Line: Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs.In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner.In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media.

View Article: PubMed Central - PubMed

Affiliation: Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität, Münster, Germany.

ABSTRACT
The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs). In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s) exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

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FfG1 has distinct functions as a repressor for fusarubin biosynthesis.The WT, ΔffG1 and ffG1G42R mutants were grown in ICI synthetic media either under fusarubin biosynthesis-repressing (6 mM glutamine (Gln)) or fusarubin biosynthesis-favoring (sodium nitrate (NO3−)) conditions. A) After 7 days of incubation, culture filtrates were used for HPLC-DAD analysis (for details see material and methods). Photographs show the flask with the culture broth after 7 days of growth and the corresponding HPLC chromatogram at 450 nm. Brackets indicate metabolites found in the liquid culture: bikaverins (BIK) and fusarubins (FSR). The scale for ffG1G42R was set to 20 mAU due to a lower accumulation of compounds in the liquid culture compared to the other strains. B) After 3 and 4 days, mycelia were harvested and used for northern blot analysis. The genes fsr1 and fsr2 (as examples for fusarubin biosynthetic genes) and bik2 (as an example for bikaverin biosynthetic genes) were used as probes. C) WT and Δffg1 were grown under fusarubin biosynthesis-favoring conditions. After 4 days of growth either glutamine, sodium nitrate or no nitrogen was added and after another 30 min mycelia were harvested and used for Northern blot analysis, fsr2 was used for probing.
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pone-0058185-g002: FfG1 has distinct functions as a repressor for fusarubin biosynthesis.The WT, ΔffG1 and ffG1G42R mutants were grown in ICI synthetic media either under fusarubin biosynthesis-repressing (6 mM glutamine (Gln)) or fusarubin biosynthesis-favoring (sodium nitrate (NO3−)) conditions. A) After 7 days of incubation, culture filtrates were used for HPLC-DAD analysis (for details see material and methods). Photographs show the flask with the culture broth after 7 days of growth and the corresponding HPLC chromatogram at 450 nm. Brackets indicate metabolites found in the liquid culture: bikaverins (BIK) and fusarubins (FSR). The scale for ffG1G42R was set to 20 mAU due to a lower accumulation of compounds in the liquid culture compared to the other strains. B) After 3 and 4 days, mycelia were harvested and used for northern blot analysis. The genes fsr1 and fsr2 (as examples for fusarubin biosynthetic genes) and bik2 (as an example for bikaverin biosynthetic genes) were used as probes. C) WT and Δffg1 were grown under fusarubin biosynthesis-favoring conditions. After 4 days of growth either glutamine, sodium nitrate or no nitrogen was added and after another 30 min mycelia were harvested and used for Northern blot analysis, fsr2 was used for probing.

Mentions: To study the impact of ffg1 mutations on secondary metabolism we grew the wild type and the Δffg1 and the ffg1G42R mutants in synthetic ICI medium with either 6 mM glutamine or 6 mM sodium nitrate, representing optimal culture conditions for the production of the two red polyketides, bikaverins and fusarubins, respectively [51], [52], [28]. Using sodium nitrate as sole nitrogen source both the wild type and the deletion mutant showed the typical dark red pigmentation characteristic for the accumulation of fusarubins (fig. 2A). Using glutamine, the coloration of the culture broth of Δffg1 differed markedly from that of the wild type. Subsequent analysis using high performance liquid chromatography (HPLC) coupled to a diode array detector (HPLC-DAD) revealed that the Δffg1 mutant produced only trace amounts of bikaverin in contrast to the wild type. However, high concentrations of fusarubins, not present in the wild type under these conditions, were produced in Δffg1 (fig. 2A). Northern blot analysis verified the HPLC data as fusarubin biosynthetic genes (fsr1 encoding the polyketide synthase; fsr2 encoding an O-methyltransferase) were up-regulated in Δffg1 under both fusarubin-favoring (6 mM sodium nitrate) and bikaverin-favoring (6 mM glutamine) conditions (fig. 2B).


Signaling governed by G proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi.

Studt L, Humpf HU, Tudzynski B - PLoS ONE (2013)

FfG1 has distinct functions as a repressor for fusarubin biosynthesis.The WT, ΔffG1 and ffG1G42R mutants were grown in ICI synthetic media either under fusarubin biosynthesis-repressing (6 mM glutamine (Gln)) or fusarubin biosynthesis-favoring (sodium nitrate (NO3−)) conditions. A) After 7 days of incubation, culture filtrates were used for HPLC-DAD analysis (for details see material and methods). Photographs show the flask with the culture broth after 7 days of growth and the corresponding HPLC chromatogram at 450 nm. Brackets indicate metabolites found in the liquid culture: bikaverins (BIK) and fusarubins (FSR). The scale for ffG1G42R was set to 20 mAU due to a lower accumulation of compounds in the liquid culture compared to the other strains. B) After 3 and 4 days, mycelia were harvested and used for northern blot analysis. The genes fsr1 and fsr2 (as examples for fusarubin biosynthetic genes) and bik2 (as an example for bikaverin biosynthetic genes) were used as probes. C) WT and Δffg1 were grown under fusarubin biosynthesis-favoring conditions. After 4 days of growth either glutamine, sodium nitrate or no nitrogen was added and after another 30 min mycelia were harvested and used for Northern blot analysis, fsr2 was used for probing.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585259&req=5

pone-0058185-g002: FfG1 has distinct functions as a repressor for fusarubin biosynthesis.The WT, ΔffG1 and ffG1G42R mutants were grown in ICI synthetic media either under fusarubin biosynthesis-repressing (6 mM glutamine (Gln)) or fusarubin biosynthesis-favoring (sodium nitrate (NO3−)) conditions. A) After 7 days of incubation, culture filtrates were used for HPLC-DAD analysis (for details see material and methods). Photographs show the flask with the culture broth after 7 days of growth and the corresponding HPLC chromatogram at 450 nm. Brackets indicate metabolites found in the liquid culture: bikaverins (BIK) and fusarubins (FSR). The scale for ffG1G42R was set to 20 mAU due to a lower accumulation of compounds in the liquid culture compared to the other strains. B) After 3 and 4 days, mycelia were harvested and used for northern blot analysis. The genes fsr1 and fsr2 (as examples for fusarubin biosynthetic genes) and bik2 (as an example for bikaverin biosynthetic genes) were used as probes. C) WT and Δffg1 were grown under fusarubin biosynthesis-favoring conditions. After 4 days of growth either glutamine, sodium nitrate or no nitrogen was added and after another 30 min mycelia were harvested and used for Northern blot analysis, fsr2 was used for probing.
Mentions: To study the impact of ffg1 mutations on secondary metabolism we grew the wild type and the Δffg1 and the ffg1G42R mutants in synthetic ICI medium with either 6 mM glutamine or 6 mM sodium nitrate, representing optimal culture conditions for the production of the two red polyketides, bikaverins and fusarubins, respectively [51], [52], [28]. Using sodium nitrate as sole nitrogen source both the wild type and the deletion mutant showed the typical dark red pigmentation characteristic for the accumulation of fusarubins (fig. 2A). Using glutamine, the coloration of the culture broth of Δffg1 differed markedly from that of the wild type. Subsequent analysis using high performance liquid chromatography (HPLC) coupled to a diode array detector (HPLC-DAD) revealed that the Δffg1 mutant produced only trace amounts of bikaverin in contrast to the wild type. However, high concentrations of fusarubins, not present in the wild type under these conditions, were produced in Δffg1 (fig. 2A). Northern blot analysis verified the HPLC data as fusarubin biosynthetic genes (fsr1 encoding the polyketide synthase; fsr2 encoding an O-methyltransferase) were up-regulated in Δffg1 under both fusarubin-favoring (6 mM sodium nitrate) and bikaverin-favoring (6 mM glutamine) conditions (fig. 2B).

Bottom Line: Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs.In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner.In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media.

View Article: PubMed Central - PubMed

Affiliation: Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität, Münster, Germany.

ABSTRACT
The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs). In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA), to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s) exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

Show MeSH
Related in: MedlinePlus