cynT1-77 (NCU04777.3), a 2,271-bp fragment located upstream of the cas1 gene was amplified and sequenced. All together, a 3,558 bp part of the genomic sequence including the 770-bp cas1 coding region with 59 and 39 regions was sequenced. The 586-bp sequence of an internal part of cas2 was amplified with the heterologous primer pair cynT2-a/cynT2-b. Since, heterologous oligonucleotides designed according to N. crassa genes flanking the cas2 open reading frame in N. crassa failed to amplify sequences adjacent to cas2 of S. macrospora, we performed an inverse PCR strategy as described previously [21]. In brief: genomic DNA from S. macrospora was digested with restriction endonuclease Bsp120I. Afterwards, all fragments were self-ligated using T4-Ligase (Fermentas, Germany) and precipitated. The DNA was then used for inverse PCR with primer pair cynT2-c1/cynT2-d1 with Thermozym-Hotstart Taq-Polymerase (Molzyme, Germany). The resulting PCR fragment was cloned into pDrive cloning vector (Qiagen, Germany) and sequenced by means of primer walking, resulting in a 2,418-bp fragment including the complete 855-bp cas2 open reading frame and adjacent 59- and 39-regions. In case of cas3, a 3,242-bp fragment with the entire protein coding sequence and 59 and 39 flanking regions were amplified with the heterologous primer pair T3-1102/T3-1104, based on the sequence of the adjacent gene homologues NCU01104.3 and NCU01102.3 of N. crassa. The 3,242-bp fragment was cloned into pDrive cloning vector (Qiagen) and sequenced. Construction of fluorescence plasmids For construction of cas-egfp fusion constructs under the control of the constitutive A. nidulans gpd promoter, the coding regions of cas2 and cas3 were amplified by PCR with primer pairs cynT2-GFP-f/ cynT2-r and cynT3-GFP-f/cynT3-r (Table S1) from S. macrospora genomic DNA. With this PCR, flanking NcoI restriction sites were generated on each end. The fragments were subcloned into pDrive vector (Qiagen, Germany). Since the cas1 gene contains an internal NcoI restriction site, a site directed point mutation was introduced by overlap extension PCR with primer pairs cynT1- GFP-f/T1-Mut-r and T1-Mut-f/cynT1-r (Table S1). All PCR reactions were accomplished, using HotStarTaq DNA Polymerase (Qiagen, Germany). After sequencing, NcoI restriction resulted in fragments of 773-, 858- and 736-bp, which were cloned into the single NcoI site of egfp containing vector p1783.1 to create plasmids pGFP-CAS1, pGFP-CAS2 and pGFP-CAS3, respectively. Plas- mid p1783.1 [64] contains the egfp gene downstream of the NcoI restriction site, resulting in CAs fused to the N terminus of EGFP. Furthermore, a truncated cas2-DsRed fusion gene was construct- ed by PCR using primers cynT2-56f-NcoI and cynT2-r (Table S1). The amplification resulted in the deletion of the first 165 bp encoding for 55 amino acids. The resulting sequence was verified and the NcoI fragment was cloned into the single NcoI restriction site of DsRed expression vector pRHN1 [65]. This vector is termed pDMito-CAS2-DsRED (Table S2) and allows expression of DsRED fused to CAS2 without its 55 aa N-terminal mitochondrial target sequence but with a newly generated ATG start codon. In a further approach, the first 180 bp encoding the 60 aa of the CAS2 N-terminus including the mitochondrial target sequence were amplified with primer pair cynT2-GFP-f/Mito-r (Table S1) and were also ligated into the NcoI site of pRHN1, resulting in plasmid pMito-DsRED (Table S2). This plasmid was used as a template for an inverse PCR based cloning strategy with oligonucleotides CTG-Mito_f and pEHNnat-r as described previously [66], resulting in the truncation of 105 bp of the mitochondrial target sequence. The final plasmid is termed pCTG-CAS2-DsRED (Table S2) and encodes a CAS2-DsRED fusion protein which starts with a CTG codon instead of an ATG start codon and has a b-Carbonic Anhydrases in Fungi truncation of the first 35 aa of the mitochondrial target sequence of CAS2. All fluorescence plasmids were transformed into S. macrospora wild type strain to investigate fluorescence patterns. Generation and complementation of S. macrospora knock-out strains Knock-out constructs for homologous recombination were produced utilizing homologous recombination in yeast as described previously [67]. The 59- and 39-regions of the genes cas1, cas2 and cas3 were amplified from genomic DNA of S. macrospora wild type strain using primer pairs cynT1-5f/cynT1-5r, cynT1-3f/cynT1-3r, cynT2-5f/cynT2-5r, cynT2-3f/cynT2-3r, cynT3-5f/cynT3-5r and cynT3-3f/cynT3-3r (Table S1). The PCR reactions were conduced to add specific overhangs to the 59- and 39-regions of the respective cas gene, homologous to the hph- cassette and the yeast plasmid pRS426, respectively. The hph- cassette was generated with primers hph-f and hph-r using pCYN1-KO as a template [50]. In each case, three PCR fragments together with the EcoRI/XhoI linearized vector pRS426 [68] were co-transformed into yeast strain PJ69-4A [69], where homologous recombination took place. Transformants were selected on SD plates lacking uracil, afterwards plasmid DNA was isolated with the NucleoSpinH Plasmid Kit (Macherey-Nagel, according to Nolting and Po¨ggeler [20]). To isolate the resulting plasmids pCAS1-KO, pCAS2-KO and pCAS3-KO (Table S2), total DNA was subsequently transformed into E. coli strain SURE and plasmid DNA isolation was carried out. The KO-plasmids were used as templates to amplify the CA-hph cassettes with oligonucleotide pairs cynT1-5f/cynT1-3r, cynT2-5f/cynT2-3r or cynT3-5f/cynT3-3r (Table S2 and Figure S1). To generate the Dcas1, Dcas2 and Dcas3 mutant strains, the resulting PCR amplicons (cas1: 3,619 bp; cas2: 3,071 bp; cas3: 3,258 bp) were transformed into S. macrospora Dku70 which is impaired in heterologous recombination [30]. In each strain the major part of the CA coding region including its putative catalytic domain is substituted against the hph gene (Figure S1). Transformants were screened by PCR with primer pairs which are specific for external flanking regions and the integrated hph-cassette (Table S1, Figure S1), and with a primer pair specific to the external flanking 59 region (Table S1, Figure S1). Additionally, to prove that mutant strains do not contain the respective wild type cas gene, primer pairs matching the substituted coding region of the cas gene were used, and resulted in amplicons only in the wild type and not in the deletion mutants (data not shown). Furthermore RT-PCR experiments were performed to confirm the knock-out mutants on their transcriptional levels using primer pairs cynT1-GFP-f/ cynT1-r, cynT2-GFP-f/cynT2-r and cynT3-GFP-f/cynT3-r. To complement the phenotypes, rescue plasmids pGPD-CAS1, pGPD-CAS2 and pGPD-CAS3 were constructed by amplifying the entire coding regions with primer pairs cynT1-GFP-f/cynT1- BamHI-r, cynT2GFP-f/cynT2-BamHI-r and cynT3GFP-f/cy- nT3BamHI-r, respectively. Oligonucleotides used generated NcoI and BamHI restriction sites. Genomic DNA was used as template and in case of cas1 (because of its internal NcoI restriction site) with plasmid pGFP-CAS1 as template. After subcloning into pDrive (Qiagen, Germany) and sequencing, specific NcoI/BamHI frag- ments were cloned into pEHN-nat1 (Table S2), containing the nourseothricin resistance cassette. In all constructs, the CA gene is under control the constitutive A. nidulans gpd promoter and trpC terminator. Plasmid pGPD-CAS2-CTG was constructed, by using primers cynT2-CTG-f and cynT2-BamHI-r to generate an amplicon with NotI and BamHI restriction sites. After sequencing, a specific NotI/BamHI fragment was cloned into pEHN-nat1. In PLoS ONE | www.plosone.org 14 April 2009 | Volume 4 | Issue 4 | e5177