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

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.

Figure 1. Multiple sequence alignment of the zinc coordinating region from fungal β-class CAs.

(A) ClustalX alignment was created using the following sequences: Sma1 [S. macrospora, Accession No. FM878639], Sma2 [FM878640], Sma3 [FM878641], Ncr1 [Neurospora crassa, Q7S631], Ncr2 [Q7S4J8], Ncr3 [Q8X0H0], Afu1 [Aspergillus fumigatus, Q4WQ18], Afu2 [A4DA32], Afu3 [Q4WPJ0], Cne1 [Cryptococcus neoformans, Q3I4V7], Cne2 [Q30E79], Sce [Saccharomyces cerevisiae, P53615], Spo [Schizosaccharomyces pombe, O94255] and Ror [Rhizopus oryzae, RO3G_10751.1]. Conserved amino acids important for Zn²⁺-coordination are marked by an asterisk. Identical amino acids, which are conserved in all proteins, are shaded in black; residues conserved in at least 13 of 15 sequences are shaded in dark grey and residues conserved in at least ten sequences are shaded in light grey. Arrows indicate S. macrospora cas genes with introns are given as grey boxes. The dashed box marks the region encoding the part of the protein which was used for the alignment at the top. The coding region for the mitochondrial target sequence of CAS2 is indicated as black box. (B) Amino acid identity in % is given for all sequences in pair-wise comparisons. Percentages given are based on amino acid comparison of the conserved region shown in (A).

Figure 2. Expression analyses of cas genes.

Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27°C in liquid BMM for three, five and seven days under ambient air conditions or 5% CO₂. Comparisons are given as logarithmic values of the ambient air/5% CO₂ ratios and are mean expression ratios from two independent biological replicates, each done in triplicate. For normalization, transcript levels of the SSUrRNA were calculated as described in Material and Methods. Asterisk indicate significance according to REST .

Figure 3. Fluorescence microscopic analyses of S. macrospora wild type strains expressing cas1-egfp or cas3-egfp.

The images illustrate the fluorescence of CAS1-EGFP and CAS3-EGFP caused by the transformation of plasmids pGFP-CAS1 or pGFP-CAS3, respectively. Transformants were analyzed after growth for two days on solid SWG medium supplemented with hygromycin. DIC: differential interference contrast. Scale bar indicates 20 µm.

Figure 4. Localization of the carbonic anhydrase CAS2.

(A) Fluorescence microscopic analysis of a S. macrospora wild type strain carrying plasmid pGFP-CAS2. Expression of full-length cas2-egfp leads to EGFP import into mitochondria, visible as tubular structures. CAS2-EGFP co-localizes to mitochondria stained with Mito-Tracker (Invitrogen, Germany) (B) Co-transformation of pGFP-CAS2 and pDsRED-SKL results in clearly distinguishable signals from green fluorescent mitochondria and red peroxisomes. (C) Fluorescence microscopic analysis of a S. macrospora wild type strain carrying either pMito-DsRED (N-terminal mitochondrial signal sequence of cas2 fused to DsRed) or pΔMito-CAS-DsRED (cas2 gene without the signal sequence fused to DsRed). DIC: differential interference contrast. Scale bar indicates 20 µm.

Figure 5. RT-PCR analyses of wild type, single and double knock-out strains to confirm gene deletions.

The coding sequence of the cas genes were amplified with primer combinations cynT1-GFP-f/cynT1-r, cynT2-GFP-f/cynT2-r and cynT3-GFP-f/cynT3-r, respectively. Equal concentrations of DNA were loaded in each lane. Sizes of amplicons from genomic DNA control (gDNA) and cDNA is indicated aside. Control: negative control, without DNA template.

Figure 6. Vegetative growth rate of S. macrospora wild type and cas mutant strains in ambient air and at elevated CO₂.

Strains were grown for 3 days on solid BMM either in ambient air (black bars) or at 5% CO₂ (white bars). Growth rate of wild type in ambient air was defined as 100%. Growth rates shown are averages from nine measurements of three independent experiments. Error bars are given as indicated.

Figure 7. Fruiting-body developmental of wild type, single and double knock-out strains.

(A) Strains were grown on BMM medium for 7 days under natural environmental conditions (low CO₂) or at high CO₂ concentrations (5%). No morphological changes were observed in single and double Δcas1/3 and Δcas2/3 knock-out strains, respectively. Mature perithecia of Δcas1/2 strain in ambient air were not detectable after 7 days. Elevated CO₂ concentrations complemented this phenotype. Scale bar indicates 500 µm (B) CO₂/HCO₃ ⁻ accumulates in the fungal cell. Growth defect of Δcas2 is severely increased when mycelium is grown from germinated single spores (Δcas2_ssi: single spore isolate). BMM plate supplied with 0.5% sodium acetate was either inoculated with Δcas2 mutant strain or with an isolated single ascospore of Δcas2. Plates were incubated with or without CO₂ for three days and images were taken from young mycelia. After prolonged incubation, Δcas2_ssi develops less fruiting-bodies under ambient air than a consecutively transferred mutant strain after nine days of growth. The defect is complemented at 5% CO₂. Scale bar indicates 1 mm (C) Double knock-out mutant strain Δcas1/2 produced only few perithecia after 21 days of growth. Fruiting-bodies exhibit a wild-type phenotype, but are accumulated at several places on the plate. No mature ascospores were discharged from perithecia developed from Δcas1/2 strain.

Figure 8. 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.

Figure 9. Complementation of the mutant phenotype of Δcas1/2 by ectopically integrated cas1, cas2 and cas3, respectively.

(A) Morphological characterization of Δcas1/2 strain complemented with different cas genes. The images show vegetative hyphae in case of Δcas1/2 strain grown in ambient air and fruiting-body development when grown at 5% CO₂ or complemented with cas1, cas2, or cas3. The gene cas2 starts either with an ATG or CTG start codon; all constructs are driven by the constitutive gpd promoter of A. nidulans. Scale bar indicates 1 mm (B) At ambient air condition, mature ascospores were produced and discharged, only when Δcas1/2 is grown at elevated CO₂-levels or complemented either with cas1 or ATG-cas2. After a prolonged incubation time of 18 days, complementation with cas3 also results in discharge of few mature ascospores. Scale bar indicates 50 µm (C) Germination rate and restoration of vegetative growth defects is partially complemented by constitutively expressed cas2 starting with ATG (+), but not by cas1, cas2-CTG and cas3 (—). Asterisk indicates that spores were isolated from a strain grown at 5% CO₂, because no ascospores were developed in ambient air.