number of mature ascospores. While perithecia produced by S. macrospora wild type contain 105.2619.9 asci, only 21.1612.8 asci per perithecium were produced in the Dcas1/2 mutant strain. RT-PCR analyses of cas gene expression in single and double knock-out strains revealed that cas1 was significantly down- regulated in Dcas3 after five days of growth under ambient air conditions, but not in Dcas2. A slight down-regulation of cas1 was also obvious in Dcas2/3. The cas2 transcript was also significantly reduced in the Dcas3 knockout strain, whereas a de-regulation of the transcriptional cas2 expression was measured in the Dcas1 and Dcas1/3 strains. In contrast, the cas3 transcript showed no up- or down-regulation in Dcas1, Dcas2 or Dcas1/2 after five days of growth (data not shown). Since CA mutants of S. cerevisiae are more sensitive to several stress-inducing conditions [14], we tested growth and sexual development of wild type and mutant strains on stress inducing media. Neither hyperosmotic media, nor H2O2, which induces oxidative stress, nor sodium dodecylsulphate, which induces cell- wall damage, led to marked phenotypic differences between wild type and mutant strains (data not shown). CAS2 is needed for proper ascospore germination To assess the role of b-CAs during ascospore germination, we investigated the ascospore-germination rate of single and double mutants (Figure 8a). Under ambient air conditions, the germina- tion efficiency of Dcas2 and Dcas2/3 decreased by about 50% while all other mutants exhibited germination efficiencies similar to wild type. Interestingly, the spore germination defect of Dcas2 and Dcas2/3 was not complemented by 5% CO2 concentration. The Dcas1/2 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 Dcas2 single and the Dcas2/3 double knock- out strain. Compared to wild type, the germination efficiency of Dcas1/2 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 Dcas2 and Dcas2/3, mycelia derived from ascospores of Dcas1/2 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 Dcas2 single and double knockout mutant strains was also seen when ascospore germina- tion was investigated under the microscope (Figure 8c). While wild type, Dcas1 and Dcas3 ascospores form non-vacuolated hyphae, germlings of the Dcas2 mutant exhibited multiple vacuoles. Complementation of Dcas1/2-fus strain by overexpression of cas1, cas2 and cas3 To address the question whether the overexpression of one of the b-CAs of S. macrospora can complement the severe develop- mental defects in the Dcas1/2 double mutant, we transformed this strain with overexpression constructs of cas1, cas2, cas3 and cas2- CTG. The latter construct contains a variant of cas2 that lacks the mitochondrial target sequence and starts with a CTG codon instead of an ATG. This construct was made because the N. crassa homologue of cas2, ORF NCU08133.3, is annotated with a CTG start codon. The CTG is conserved in the predicted S. macrospora cas2 ORF, but if the homologous CTG was used as a start codon, the CAS2 protein would lack 35 N-terminal residues and have a truncated mitochondrial target sequence. In all transformants, the cas gene was ectopically integrated and under control of the strong constitutive gpd promoter of A. nidulans. Overexpression of an ectopic cas2 did almost fully complement the b-Carbonic Anhydrases in Fungi mutant phenotype of Dcas1/2 (strain Dcas1/2-gpd-cas2ect). Mature perithecia with a wild type-like number of asci developed, and mycelial colonies exhibited a wild type-like hyphal morphol- ogy as well as a wild type-like colony density. However, the number of germinated ascospores (63%) was only slightly increased in comparison to the mutant strain (53%) (Figure 9). Thus, it seems that only transcription from the endogenous cas2 promoter led to a complementation of all developmental defects. Overexpression of cas2-CTG complemented the defects of Dcas1/ 2 even worse. Transformants carrying pGPD-CAS2-CTG develop fruiting-bodies, but discharge no mature ascospores. Cracking of the fruiting-bodies revealed the presence of only undifferentiated asci (Figure 9B). This result indicated that S. macrospora is able to translate a cas2-CTG ORF, but that the truncated CAS2 is not able to fully complement sterility of Dcas1/2. To investigate whether full complementation depends on translocation of CAS2 into the mitochondria, we tested whether the truncated mito- chondrial target sequence encoded by pCTG-CAS2-DsRED can target DsRED into mitochondria. For this experiment, plasmid pCTG-CAS2-DsRED was transformed into a S. macrospora wild type strain. As shown in Figure S2, the CTG-CAS2-DsRED fusion construct was mainly distributed throughout the cytoplasm of the hyphae, and in only a few hyphal tips were fluorescent tubular structures detectable, indicating that the construct was inefficiently targeted to mitochondria. This result shows that both CAS2 variants may be produced in vivo, but that only the full length version of the cas2 is able to fulfill its complete function. In contrast to the truncated version of cas2, overexpression of cas1 lead to the formation of fertile fruiting-bodies with asci and mature ascospores under ambient air, whereas overexpression of cas3 resulted in a reduced number of asci and ascospores. Moreover, these generated only after a prolonged incubation of 18 days (Figure 9B). Determination of the ascospore germination rate of Dcas1/2 strains overexpressing cas1 or cas3 resulted in a decreased germination efficiency, which showed that the germi- nation defect was neither complemented by cas1 nor by cas3 (Figure 9C). Complementation of Dcas1/2-fus strain by the addition of fatty acids Since HCO32 is an important key substrate for decarboxylating enzymes involved in various biosynthetic pathways, it has been hypothesized that the lack of CA activity might affect the acetyl- CoA carboxylase dependent biosynthesis of fatty acids [16,31]. Therefore, we tried to complement the sterile mutant strain Dcas1/2-fus by the addition of a mixture of fatty acids (palmitic, myristic and stearic acid and 1% Tween 80 as surfactant) in different concentrations. Corn meal medium supplemented with 15 or 30 mg/l of fatty acids completely restored the vegetative growth defect of Dcas1/2-fus, whereas 60 mg/l leads to an increased production of aerial hyphae. Sexual development was slightly revised, because perithecia were formed earlier in contrast to ambient air conditions without fatty acids and they were equally distributed all over the plate (data not shown). Nevertheless, no mature ascospores were produced by the Dcas1/2-fus deletion strain after 14 days of growth under the conditions tested. The Dcas1/2-fus:gpd-cas2ect and wt were not influenced by the addition of fatty acids. Discussion Although the main function of CAs in all organisms is to accomplish the regulation of CO2/HCO32 homeostasis little is known about how CO2 is metabolized and transported in mycelial PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5177