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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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Deletion of ffg1, ffac and ffg3 results in fusarubin biosynthesis under unfavorable conditions.The WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2, Δffg3) were grown for 3, 4 (for Northern blot analysis) or 7 days (for chemical analysis) in synthetic ICI medium (6 mM glutamine (Gln)). A) Liquid cultures were directly used for HPLC-DAD analysis (for details see material and methods). Fusarubins (FSR) are highlighted by the red box, bikaverins (BIK) by the purple box. Flasks containing culture broth of the WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2) after 7 days of cultivation are shown on the left, corresponding HPLC-DAD chromatograms at 450 nm on the right. The scale for Δffac was set to 340 mAU due to a higher accumulation of compounds compared to the other strains. B) Photograph of the culture broth and corresponding HPLC-DAD chromatogram at 450 nm of Δffg3. C) Northern blot of WT and cAMP pathway mutants after 3 and 4 days of growth under fusarubin unfavorable conditions. The fusarubin biosynthetic genes, fsr1 and fsr2, and the bikaverin biosynthetic genes, bik1 and bik2, were used for probing.
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pone-0058185-g003: Deletion of ffg1, ffac and ffg3 results in fusarubin biosynthesis under unfavorable conditions.The WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2, Δffg3) were grown for 3, 4 (for Northern blot analysis) or 7 days (for chemical analysis) in synthetic ICI medium (6 mM glutamine (Gln)). A) Liquid cultures were directly used for HPLC-DAD analysis (for details see material and methods). Fusarubins (FSR) are highlighted by the red box, bikaverins (BIK) by the purple box. Flasks containing culture broth of the WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2) after 7 days of cultivation are shown on the left, corresponding HPLC-DAD chromatograms at 450 nm on the right. The scale for Δffac was set to 340 mAU due to a higher accumulation of compounds compared to the other strains. B) Photograph of the culture broth and corresponding HPLC-DAD chromatogram at 450 nm of Δffg3. C) Northern blot of WT and cAMP pathway mutants after 3 and 4 days of growth under fusarubin unfavorable conditions. The fusarubin biosynthetic genes, fsr1 and fsr2, and the bikaverin biosynthetic genes, bik1 and bik2, were used for probing.

Mentions: Similarly to Δffg1, deletion of ffac also caused significant up-regulation of fusarubin biosynthesis under favoring (6 mM sodium nitrate) (data not shown) as well as under non-favoring conditions (6 mM glutamine), while bikaverin biosynthesis is decreased in both conditions (fig. 3A). Surprisingly, neither deletion of ffpka1 nor ffpka2 induced the formation of fusarubin. However, bikaverin production was reduced in the Δffpka1 but not the Δffpka2 mutant (fig. 3A). It is notable that deletion of ffac resulted in an about three times higher accumulation of fusarubins compared to the Δffg1 mutants under both fusarubin-favoring (data not shown) and non-favoring conditions (fig. 3A). Expression of fusarubin-biosynthetic genes in Δffac mutants started earlier, already after 3 days of incubation, while expression in Δffg1 mutants was detectable only after 4 days (fig. 3C). The much stronger effect of FfAC on fusarubin biosynthesis, compared to that of the upstream-acting FfG1, indicates the existence of an additional upstream activator of FfAC besides FfG1 exist. In several filamentous fungi two of the three Gα subunits (class I and III) were shown to stimulate the AC activity [11], [12]. For example, in B. cinerea addition of exogenous cAMP can partially restore the defects of the respective knock-out mutants [17], [53], [26]. To test whether FfG3, a Gα proteins belonging to class III, is also involved in fusarubin regulation we analyzed the ffg3 deletion mutants in more detail. Indeed, we found elevated fusarubin gene expression and a strong accumulation of these pigments under non-favoring conditions indicating the involvement of FfG3 in fusarubin biosynthesis, as it was shown for FfG1 and FfAC (fig. 3). Complementation of Δffac and Δffg3 deletion mutants by homologous reintroduction of the respective gene, driven by its native promoter, restored the wild-type phenotype with regard to fusarubin biosynthesis (fig. S3).


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)

Deletion of ffg1, ffac and ffg3 results in fusarubin biosynthesis under unfavorable conditions.The WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2, Δffg3) were grown for 3, 4 (for Northern blot analysis) or 7 days (for chemical analysis) in synthetic ICI medium (6 mM glutamine (Gln)). A) Liquid cultures were directly used for HPLC-DAD analysis (for details see material and methods). Fusarubins (FSR) are highlighted by the red box, bikaverins (BIK) by the purple box. Flasks containing culture broth of the WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2) after 7 days of cultivation are shown on the left, corresponding HPLC-DAD chromatograms at 450 nm on the right. The scale for Δffac was set to 340 mAU due to a higher accumulation of compounds compared to the other strains. B) Photograph of the culture broth and corresponding HPLC-DAD chromatogram at 450 nm of Δffg3. C) Northern blot of WT and cAMP pathway mutants after 3 and 4 days of growth under fusarubin unfavorable conditions. The fusarubin biosynthetic genes, fsr1 and fsr2, and the bikaverin biosynthetic genes, bik1 and bik2, were used for probing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585259&req=5

pone-0058185-g003: Deletion of ffg1, ffac and ffg3 results in fusarubin biosynthesis under unfavorable conditions.The WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2, Δffg3) were grown for 3, 4 (for Northern blot analysis) or 7 days (for chemical analysis) in synthetic ICI medium (6 mM glutamine (Gln)). A) Liquid cultures were directly used for HPLC-DAD analysis (for details see material and methods). Fusarubins (FSR) are highlighted by the red box, bikaverins (BIK) by the purple box. Flasks containing culture broth of the WT and cAMP pathway mutants (Δffg1, Δffac, Δffpka1, Δffpka2) after 7 days of cultivation are shown on the left, corresponding HPLC-DAD chromatograms at 450 nm on the right. The scale for Δffac was set to 340 mAU due to a higher accumulation of compounds compared to the other strains. B) Photograph of the culture broth and corresponding HPLC-DAD chromatogram at 450 nm of Δffg3. C) Northern blot of WT and cAMP pathway mutants after 3 and 4 days of growth under fusarubin unfavorable conditions. The fusarubin biosynthetic genes, fsr1 and fsr2, and the bikaverin biosynthetic genes, bik1 and bik2, were used for probing.
Mentions: Similarly to Δffg1, deletion of ffac also caused significant up-regulation of fusarubin biosynthesis under favoring (6 mM sodium nitrate) (data not shown) as well as under non-favoring conditions (6 mM glutamine), while bikaverin biosynthesis is decreased in both conditions (fig. 3A). Surprisingly, neither deletion of ffpka1 nor ffpka2 induced the formation of fusarubin. However, bikaverin production was reduced in the Δffpka1 but not the Δffpka2 mutant (fig. 3A). It is notable that deletion of ffac resulted in an about three times higher accumulation of fusarubins compared to the Δffg1 mutants under both fusarubin-favoring (data not shown) and non-favoring conditions (fig. 3A). Expression of fusarubin-biosynthetic genes in Δffac mutants started earlier, already after 3 days of incubation, while expression in Δffg1 mutants was detectable only after 4 days (fig. 3C). The much stronger effect of FfAC on fusarubin biosynthesis, compared to that of the upstream-acting FfG1, indicates the existence of an additional upstream activator of FfAC besides FfG1 exist. In several filamentous fungi two of the three Gα subunits (class I and III) were shown to stimulate the AC activity [11], [12]. For example, in B. cinerea addition of exogenous cAMP can partially restore the defects of the respective knock-out mutants [17], [53], [26]. To test whether FfG3, a Gα proteins belonging to class III, is also involved in fusarubin regulation we analyzed the ffg3 deletion mutants in more detail. Indeed, we found elevated fusarubin gene expression and a strong accumulation of these pigments under non-favoring conditions indicating the involvement of FfG3 in fusarubin biosynthesis, as it was shown for FfG1 and FfAC (fig. 3). Complementation of Δffac and Δffg3 deletion mutants by homologous reintroduction of the respective gene, driven by its native promoter, restored the wild-type phenotype with regard to fusarubin biosynthesis (fig. S3).

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