b-Carbonic Anhydrases in Fungi 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. doi:10.1371/journal.pone.0005177.g005 Dku70 strain, which is impaired in the repair of DNA double- strand breaks and has been shown to be an ideal recipient strain for gene targeting experiments [30]. Since S. macrospora primary transformants are often heterokaryotic, we isolated single spore isolates to segregate the wild-type and mutant alleles of cas genes in a Dku70 background. Strains that were homokaryotic for the desired cas deletion and carried the ku70-deletion were further crossed against color spore mutant fus1-1. By conventional genetic analyses, we succeeded in isolating hygromycin resistant spores without the Dku70 (nourseothricin-resistant) background, indicat- ing that cas genes are not coupled to ku70 and that none of the cas genes is an essential gene. PCR amplification with primer pairs specific for external flanking regions in combination with primers specific for the integrated hph-cassette confirmed the integration of the hygromycin-resistant cassette at the desired cas-locus (Figure S1). By conventional crossing of the single mutant strains, we generated all three possible double knock-out mutants. The genotype of double knock-outs was confirmed by PCR (Figure S1B). In addition, using primer pairs cynT1-GFP-f/cynT1-r, cynT2-GFP-f/cynT2-r and cynT3-GFP-f/cynT3-r, we verified by RT-PCR that transcripts of the deleted genes were absent in all single and double knock-out mutant strains (Figure 5). Phenotypic characterization of carbonic anhydrase mutants The characterization of Dcas1 and Dcas3 single knock-out mutants revealed no obvious phenotypic changes compared to the wild type. Almost no alterations in vegetative growth behavior were detectable, when measuring the growth rate under ambient air conditions (Figure 6). Only Dcas1 showed a 14% elevated growth velocity at 5% CO2. Furthermore, neither Dcas1 nor Dcas3 displayed irregularities in mycelial morphology or the formation of sexual reproductive structures (Figure 7). When compared to the wild type or single knock-out strains, the Dcas1/3 double deletion strain also showed no changes in vegetative morphology or reproductive structures. Interestingly, similar to Dcas1, Dcas3 and Dcas1/3 sexual reproduction of the S. macrospora wild type was not affected by high CO2 concentrations, as recently reported for the basidiomycete C. neoformans [16]. 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. doi:10.1371/journal.pone.0005177.g006 PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5177