b-Carbonic Anhydrases in Fungi 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 Dcas1/3 and Dcas2/3 knock-out strains, respectively. Mature perithecia of Dcas1/2 strain in ambient air were not detectable after 7 days. Elevated CO2 concentrations complemented this phenotype. Scale bar indicates 500 mm (B) CO2/HCO32 accumulates in the fungal cell. Growth defect of Dcas2 is severely increased when mycelium is grown from germinated single spores (Dcas2_ssi: single spore isolate). BMM plate supplied with 0.5% sodium acetate was either inoculated with Dcas2 mutant strain or with an isolated single ascospore of Dcas2. Plates were incubated with or without CO2 for three days and images were taken from young mycelia. After prolonged incubation, Dcas2_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 Dcas1/2 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 Dcas1/2 strain. doi:10.1371/journal.pone.0005177.g007 In contrast to Dcas1 and Dcas3, the mycelial growth rate of Dcas2 was reduced by 37% under ambient air conditions. This growth defect was fully restored by incubating the mutant strain under elevated CO2 conditions (Figure 6). The vegetative growth defect was not rescued by the addition of NaHCO3 under ambient air conditions (data not shown). In addition to the reduced growth rate, Dcas2 exhibited a delay in fruiting-body production. While the wild type develops mature perithecia with asci and ascospores after seven days grown on solid corn meal medium under ambient air conditions, the number of fruiting-bodies was slightly reduced in the Dcas2 strain. This defect was also rescued by incubation in high CO2 (Figure 7A). Interestingly, both defects of Dcas2, the vegetative growth defect and the reduced number of perithecia, were more evident when the mycelium analyzed was derived from a single germinated Dcas2 ascospore (Figure 7B). Mycelia derived from single spores showed only faint hyphae after three days of growth under ambient air conditions, but this defect was complemented in 5% CO2. Furthermore, an apparent decline in the number of mature perithecia was clearly visible when mycelia derived from a single germinated Dcas2 ascospore were grown for nine days in ambient air (Figure 7B). After consecutive transfers of the mycelium from the single spore isolate, this marked effect was reduced and only a slight growth reduction was detectable when compared to the wild type (Figure 6). Thus, CO2 and/or HCO32 seem to accumulate in the hyphae but not in the ascospore. The phenotypic characteristics of the Dcas2 were also present in the Dcas2/3 double knock-out strain (Figure 7, data not shown). However, in the Dcas1/2 double knock-out strain, fruiting-body development was drastically inhibited under ambient air condi- tions (Figure 7A). The double mutant produced very few fruiting- bodies after 21 days of growth, and they accumulated only on a few distinct positions of the plate (Figure 7C). Moreover, fruiting- bodies made by Dcas1/2 under ambient air conditions never produced mature ascospores. Only under elevated CO2 condi- tions, after 7–10 days, was the Dcas1/2 mutant able to form a few fruiting-bodies, and then only 75% of these generated a small PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5177