b-Carbonic Anhydrases in Fungi Table 1. Sordaria macropsora strains used in this study Strain S17736 S23442 S66001 Dcas1 Dcas1-fus Dcas2 Dcas2-fus Dcas3 Dcas3-fus Dcas1/2 Dcas1/2-fus Dcas2/3 Dcas1/3 Dcas1/2-fus:gpd-cas1ect Dcas1/2-fus:gpd-cas2ect Dcas1/2-fus:gpd-cas3ect Dcas1/2-fus:gpd-CTG-cas2ect wt_CAS1-GFP wt_CAS2-GFP wt_CAS3-GFP wt_CAS2-GFP:DsRED-SKL wt_DMito-CAS2-DsRED wt_Mito-DsRED wt_CTG-Mito-DsRED Genotype and Phenotype1 Wild type fus1-1, spore color mutant Dku70::natRhphS Dcas1::hphR, single spore isolate, fertile Dcas1:: hphR/fus1-1, single spore isolate, fertile Dcas2::hphR, single spore isolate, fertile Dcas2:: hphR/fus1-1, single spore isolate, fertile Dcas3::hphR, single spore isolate, fertile Dcas3:: hphR/fus1-1, single spore isolate, fertile Dcas1::hphR/Dcas2::hphR , single spore isolate, sterile Dcas1::hphR/Dcas2::hphR/fus1-1, single spore isolate, sterile Dcas2::hphR/Dcas3::hphR, single spore isolate, fertile Dcas1::hphR/Dcas3::hphR, single spore isolate, fertile Dcas1::hphR/Dcas2::hphR/fus1-1, ectopic copy of cas1 under control of A. nidulans gpd promoter, natR , single spore isolate, fertile Dcas1::hphR/Dcas2::hphR/fus1-1, ectopic copy of cas2 under control of A. nidulans gpd promoter, natR , single spore isolate, fertile Dcas1::hphR/Dcas2::hphR/fus1-1, ectopic copy of cas3 under control of A. nidulans gpd promoter, natR , single spore isolate, affected in ascosporogenesis Dcas1::hphR/Dcas2::hphR/fus1-1, ectopic copy of cas2 (deletion of nucleotides 1–108) under control of A. nidulans gpd promoter, natR , primary transformant, blocked in ascosporogenesis Primary transformant of S17736 with pGFP-CAS1 Primary transformant of S17736 with pGFP-CAS2 Primary transformant of S17736 with pGFP-CAS3 Primary transformant of S17736 with pGFP-CAS2 and pDsRED-SKL Primary transformant of S17736 with pDMito-CAS2-DsRED S17736 transformed with pMito-DsRED, single spore isolate S17736 transformed with pCTG-Mito-DsRED, single spore isolate Source Lab Collection2 Lab Collection2 [30] This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study 1hygR – hygromcin resistant, natR – nourseothricin resistant 2Department of General and Molecular Botany, Ruhr-University Bochum doi:10.1371/journal.pone.0005177.t001 (10 min, 20uC, 12,000 rpm). Then, 1 ml from the supernatant was mixed with 0.2 ml chloroform and centrifuged again. One volume of isopropanol was then incubated at 280uC supplemented with 500 ml from the supernatant. After precipitation (10 min, 20uC, 12,000 rpm), the pellet was washed with 500 ml 75% ethanol (10 min, 20uC, 12,000 rpm) and dried at room temperature. The precipitated RNA was resolved in 100 ml of sterile DNA free water and tested on a gel, before it was used for cDNA synthesis and qPCR analysis. Quantitative real-time PCR Reverse transcription was done using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany) as described by the manufacturers. Quantitative RT-PCR was performed as described previously [63]. The qPCR MasterMix Plus for SYBR green Kit (Eurogentec, Belgium) was used for real-time PCR performance carried out with a MastercyclerH ep realplex (Eppendorf, Germany). Primer pair CynT1-RT_f/CynT1-RT_r was used to amplify a 133 bp part of the cas1 gene. Combinations of primers CynT2-RT_f/ CynT2-RT_r and CynT3-RT_f/CynT3-RT_r resulted in ampli- cons of 134 bp in case of cas2 and 146 bp in case of cas3. Primers used are given in Table S1. All real-time experiments were performed at least two times in triplicates with independent biological samples. An amplicon derived from the small-subunit rRNA was used as a reference for normalization (primer pair SSU- f/SSU-r) according to Po¨ggeler et al. [63]. Identification and isolation of three genes encoding for carbonic anhydrases Two different strategies were used to isolate cas genes encoding for putative carbonic anhydrases in S. macrospora. The genes cas1 and cas3 and their flanking sequences were in parts or directly amplified from S. macrospora genomic DNA using heterologous oligonucleotides derived from sequences of homologous N. crassa genes, while an inverse PCR strategy was used in case of the cas2 gene according to Nowrousian et al. [21]. Oligonucleotides used for cloning are given in Table S1. Firstly, primer pair cynT1-a/ cynT1-b, whose nucleotide sequences are based on the sequence of the appropriate N. crassa homologue, was used to amplify an internal 593-bp cas1 fragment, followed by the design of S. macrospora specific primers cynT1-c1 and cynT1-d. Primer cynT1- c1 was used in combination with the heterologous primer cynT1- 79 (based on the sequence of NCU04779.3 from N. crassa) to amplify a fragment of approximately 2800 bp. Only 689 bp downstream of the cas1 gene were sequenced and used for further analysis. With primer cynT1-d and the heterologous primer PLoS ONE | www.plosone.org 13 April 2009 | Volume 4 | Issue 4 | e5177