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Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Elleuche S, Pöggeler S - PLoS ONE (2009)

Bottom Line: No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile.Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant.The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Göttingen, Germany.

ABSTRACT
Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

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Germination rate of S. macrospora wild type and cas mutant strains.(A) Ascospores were isolated from strains grown in ambient air. Only strain Δcas1/2 grew at 5% CO2 (marked by an asterisk); because it develops mature perithecia with asci and ascospores only at elevated CO2 levels (Figure 7). 100 single spores from 10 different perithecia of wild type and mutant strains were isolated and plated on BMM medium with 0.5% sodium acetate. Ascospores were incubated in ambient air or at 5% CO2. Germinated spores were counted after 2–5 days of incubation. The percentage of germination efficiency was determined for each strain. (B) Mycelia from germinated spores of Δcas2, Δcas1/2 and Δcas2/3 are affected in growth velocity and hyphal density. Ten spores were plated from each strain and images were taken after two days of growth. (C) Germlings of Δcas2 mutants are highly vacuolated. Spores were plated on glass slides overlaid with a thin layer of BMM supplied with 0.5% sodium acetate and incubated for one day. Vacuoles in hyphae of Δcas2 are marked by white arrows Scale bar indicates 50 µm.
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pone-0005177-g008: Germination rate of S. macrospora wild type and cas mutant strains.(A) Ascospores were isolated from strains grown in ambient air. Only strain Δcas1/2 grew at 5% CO2 (marked by an asterisk); because it develops mature perithecia with asci and ascospores only at elevated CO2 levels (Figure 7). 100 single spores from 10 different perithecia of wild type and mutant strains were isolated and plated on BMM medium with 0.5% sodium acetate. Ascospores were incubated in ambient air or at 5% CO2. Germinated spores were counted after 2–5 days of incubation. The percentage of germination efficiency was determined for each strain. (B) Mycelia from germinated spores of Δcas2, Δcas1/2 and Δcas2/3 are affected in growth velocity and hyphal density. Ten spores were plated from each strain and images were taken after two days of growth. (C) Germlings of Δcas2 mutants are highly vacuolated. Spores were plated on glass slides overlaid with a thin layer of BMM supplied with 0.5% sodium acetate and incubated for one day. Vacuoles in hyphae of Δcas2 are marked by white arrows Scale bar indicates 50 µm.

Mentions: To assess the role of β-CAs during ascospore germination, we investigated the ascospore-germination rate of single and double mutants (Figure 8a). Under ambient air conditions, the germination efficiency of Δcas2 and Δcas2/3 decreased by about 50% while all other mutants exhibited germination efficiencies similar to wild type. Interestingly, the spore germination defect of Δcas2 and Δcas2/3 was not complemented by 5% CO2 concentration. The Δcas1/2 strain produced ascospores only when grown at 5% CO2, therefore we isolated ascospores from fruiting-bodies developed under this condition. These exhibited a germination defect similar to the Δcas2 single and the Δcas2/3 double knock-out strain. Compared to wild type, the germination efficiency of Δcas1/2 decreased to 58% when spores were germinated under ambient air conditions, and to 53% when spores were germinated under elevated CO2 (Figure 8A). As described for Δcas2 and Δcas2/3, mycelia derived from ascospores of Δcas1/2 showed a decreased growth velocity and hyphal density under ambient air conditions (Figure 8B). In contrast, no defects in mycelial growth were observed when spores were germinated at 5% CO2 (data not shown). The severe growth defect of Δcas2 single and double knockout mutant strains was also seen when ascospore germination was investigated under the microscope (Figure 8c). While wild type, Δcas1 and Δcas3 ascospores form non-vacuolated hyphae, germlings of the Δcas2 mutant exhibited multiple vacuoles.


Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Elleuche S, Pöggeler S - PLoS ONE (2009)

Germination rate of S. macrospora wild type and cas mutant strains.(A) Ascospores were isolated from strains grown in ambient air. Only strain Δcas1/2 grew at 5% CO2 (marked by an asterisk); because it develops mature perithecia with asci and ascospores only at elevated CO2 levels (Figure 7). 100 single spores from 10 different perithecia of wild type and mutant strains were isolated and plated on BMM medium with 0.5% sodium acetate. Ascospores were incubated in ambient air or at 5% CO2. Germinated spores were counted after 2–5 days of incubation. The percentage of germination efficiency was determined for each strain. (B) Mycelia from germinated spores of Δcas2, Δcas1/2 and Δcas2/3 are affected in growth velocity and hyphal density. Ten spores were plated from each strain and images were taken after two days of growth. (C) Germlings of Δcas2 mutants are highly vacuolated. Spores were plated on glass slides overlaid with a thin layer of BMM supplied with 0.5% sodium acetate and incubated for one day. Vacuoles in hyphae of Δcas2 are marked by white arrows Scale bar indicates 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664464&req=5

pone-0005177-g008: Germination rate of S. macrospora wild type and cas mutant strains.(A) Ascospores were isolated from strains grown in ambient air. Only strain Δcas1/2 grew at 5% CO2 (marked by an asterisk); because it develops mature perithecia with asci and ascospores only at elevated CO2 levels (Figure 7). 100 single spores from 10 different perithecia of wild type and mutant strains were isolated and plated on BMM medium with 0.5% sodium acetate. Ascospores were incubated in ambient air or at 5% CO2. Germinated spores were counted after 2–5 days of incubation. The percentage of germination efficiency was determined for each strain. (B) Mycelia from germinated spores of Δcas2, Δcas1/2 and Δcas2/3 are affected in growth velocity and hyphal density. Ten spores were plated from each strain and images were taken after two days of growth. (C) Germlings of Δcas2 mutants are highly vacuolated. Spores were plated on glass slides overlaid with a thin layer of BMM supplied with 0.5% sodium acetate and incubated for one day. Vacuoles in hyphae of Δcas2 are marked by white arrows Scale bar indicates 50 µm.
Mentions: To assess the role of β-CAs during ascospore germination, we investigated the ascospore-germination rate of single and double mutants (Figure 8a). Under ambient air conditions, the germination efficiency of Δcas2 and Δcas2/3 decreased by about 50% while all other mutants exhibited germination efficiencies similar to wild type. Interestingly, the spore germination defect of Δcas2 and Δcas2/3 was not complemented by 5% CO2 concentration. The Δcas1/2 strain produced ascospores only when grown at 5% CO2, therefore we isolated ascospores from fruiting-bodies developed under this condition. These exhibited a germination defect similar to the Δcas2 single and the Δcas2/3 double knock-out strain. Compared to wild type, the germination efficiency of Δcas1/2 decreased to 58% when spores were germinated under ambient air conditions, and to 53% when spores were germinated under elevated CO2 (Figure 8A). As described for Δcas2 and Δcas2/3, mycelia derived from ascospores of Δcas1/2 showed a decreased growth velocity and hyphal density under ambient air conditions (Figure 8B). In contrast, no defects in mycelial growth were observed when spores were germinated at 5% CO2 (data not shown). The severe growth defect of Δcas2 single and double knockout mutant strains was also seen when ascospore germination was investigated under the microscope (Figure 8c). While wild type, Δcas1 and Δcas3 ascospores form non-vacuolated hyphae, germlings of the Δcas2 mutant exhibited multiple vacuoles.

Bottom Line: No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile.Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant.The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Göttingen, Germany.

ABSTRACT
Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Show MeSH
Related in: MedlinePlus