Figure 2. Expression analyses of cas genes. Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27uC 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 [70]. doi:10.1371/journal.pone.0005177.g002 Mito-Tracker, indicating that CAS2 is a mitochondrial protein (Figure 4A). No co-localization was observed with DsRED-SKL. Co-transformants carrying pDsRED-SKL and pCAS2-GFP displayed a punctuated red fluorescence pattern of the peroxi- somes that did not overlap with the green fluorescent tubular structures of mitochondria (Figure 4B). Some yellow patches in the merge might be because the fluorescent proteins, which were b-Carbonic Anhydrases in Fungi present in the specific organelles, were located in different planes. Overall, the subcellular distribution of CAS2-EGFP was clearly different from that of the DsRED-SKL marker protein. To address the question of whether the predicted mitochondrial target sequence is sufficient for translocation into mitochondria, we constructed plasmids pMito-DsRED and pDMito-CAS2- DsRED. According to MitoProt II-v1.101 prediction program [29], CAS2 should be targeted to mitochondria with a probability of 98%. The N-terminus is predicted to be cleaved between residue His59 and Ser60. Hence, we fused the coding region of the 60 N-terminal residues of CAS2 to DsRed and analyzed the subcellular distribution of the fusion protein after transformation of pMito-DsRED into the S. macrospora wild type. As expected, this protein also localized to tubular or punctate structures, indicating its transport into mitochondria (Figure 4C). Furthermore the expression of cas2 without its mitochondrial target sequence, encoded by plasmid pDMito-CAS2-DsRED, led to fluorescence appearing uniformly distributed throughout the cytoplasm of the hyphae (Figure 4C). This result showed that the N-terminal 60 amino acid residues of CAS2 were essential and sufficient for correct localization of the CA to mitochondria. Generation of cas knock-out strains For a functional characterization of the S. macrospora CAs, we generated Dcas1, Dcas2 and Dcas3 deletion strains by gene replacement. Linear fragments containing flanking regions of the desired S. macrospora cas gene interrupted by the hph-cassette were amplified by PCR from plasmids pCAS1-KO, pCAS2-KO and pCAS3-KO. In the Dcas1 and Dcas2 knock-out constructs, the selectable marker cassette replaced the central part of the gene, including all functional domains, while in the Dcas3 knock-out construct, the complete 59 region of the gene including all regions encoding putative functional domains was deleted (Figure S1A). Linear knock-out fragments were transformed into the S. macrospora 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 mm. doi:10.1371/journal.pone.0005177.g003 PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5177