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<Supporting information>
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Table S1. List of oligonucleotides used during this study
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Table S2. MIC (48 h) and MEC (24 h) values for Aspergillus conidia according to the
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EUCAST methodology. MEC, (microscopic) minimal effective concentration (µg/ml) at 24 h;
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MIC, minimal inhibitory concentration (µg/ml) at 48 h
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Table S3. E-test values for Aspergillus parental strain and och1-4Δ mutant strains. MIC,
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minimal inhibitory concentration (µg/ml) at 24 h
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Figure S1. Alignment of protein sequences of Och1p orthologues in filamentous and yeast
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ascomycetous species. This alignment was used for the construction of the tree in Figure 1.
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This aligment was carried out using Clustal X 1.8. Conserved amino acids are indicated in
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black (> 80%), dark grey (> 60%) and light grey (> 20%)
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Figure S2. Deletion of the AfOCH1 gene by targeted gene replacement using the HPH gene.
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Restriction maps of genomic fragments containing the AfOCH1 wild-type (A), the
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Afoch1::HPH deletion allele (B). The black boxes indicate the AfOCH1 5- and 3-flanking
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sequences used for homologous recombination. (D) Southern hybridization of AfOCH1 wild-
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type strain (akuB) and deletion mutant strain (Afoch1::HPH). Genomic DNA of each strain
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was digested with BamHI or EcoRI and hybridized and probed with LB or RB DNA
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fragments. LB, left border flanking 5 sequences of the target gene; RB, right border flanking
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3 sequences of the target gene
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Figure S3. Deletion of AfOCH1 gene by targeted gene replacement using the BLE gene.
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Genomic fragments containing the AfOCH1 wild-type (A) and the Afoch1::lox BLE deletion
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allele (B). The black boxes indicate the AfOCH1 5- and 3-flanking sequences used for
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homologous recombination. (C) PCR products obtained with the primer pairs: LB-Och1-
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loxble-F and LB-Och1-loxble-R (C1); RB-Och1-loxble-F and RB-Och1-loxble-R (C2); and
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Och1-F and Och1-R (C3), and using genomic DNA from transformed (1–3) and wild-type
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(akuB) strains as template. LB, left border 5-flanking sequences of the target gene; RB, right
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border 3-flanking sequences of the target gene
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Figure S4. Deletion of AfOCH2 gene by targeted gene replacement. Restriction maps of
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genomics fragments containing the AfOCH2 wild-type (A), the Afoch2::BLE deletion allele
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(B). The black boxes indicate the AfOCH2 5- and 3-flanking sequences used for homologous
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recombination. (D) Southern hybridization of AfOCH2 wild-type strain (akuB) and deletion
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mutant strain (Afoch2::BLE). Genomic DNA of each strain was digested with EcoRI or
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HindIII and hybridized and probed with LB or RB DNA fragments. LB, left border 5-
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flanking sequences of the target gene; RB, right border 3-flanking sequences of the target
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gene
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Figure S5. Deletion of AfOCH3 gene by targeted gene replacement. Restriction maps of
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genomics fragments containing the AfOCH3 wild-type (A) and the Afoch3::PYRG deletion
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allele (B). The black boxes indicate the AfOCH3 5- and 3-flanking sequences used for
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homologous recombination. (D) Southern hybridization of AfOCH3 wild-type strain (akuB)
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and deletion mutant strain (Afoch3::PYRG). Genomic DNA of each strain was digested with
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EcoRI or EcoRV and hybridized and probed with LB or RB DNA fragments. LB, left border
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5-flanking sequences of the target gene; RB, right border 3-flanking sequences of the target
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gene
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Figure S6. Deletion of AfOCH4 gene by targeted gene replacement. Restriction maps of
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genomics fragments containing the AfOCH4 wild-type (A) and the Afoch4::PTRA deletion
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allele (B). The black boxes indicate the AfOCH4 5- and 3-flanking sequences used for
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homologous recombination. (D) Southern hybridization of AfOCH4 wild-type strain (akuB)
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and deletion mutant strain (Afoch4::PTRA). Genomic DNA of each strain was digested with
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BamHI or XhoI and hybridized and probed with LB or RB DNA fragments. LB, left border 5-
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flanking sequences of the target gene; RB, right border 3-flanking sequences of the target
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gene
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Figure S7. PCR analysis of three A. fumigatus quadruple mutants (1, 2, 3)
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(Afoch1::HPH/Afoch2::BLE/Afoch3::PYRG/Afoch4::PTRA) and the parental strain
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akuB (WT). PCR products were obtained using genomic DNA from mutant and wild-type
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strains as template and the primers shown in Table S1. (A) The presence of a band at 1514 bp
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with the primer pairs RB-Och1-F/RB-Och1-R in the mutant strains and a band at 487 bp using
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Och1-F/Och1-R primers in the WT strain indicated the right deletion of AfOCH1. (B) PCR
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products obtained with the primer pairs RB-Och2-F/RB-Och2-R (2217 bp) and Och2-F/Och2-
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R (855 bp; AfOCH2 ORF amplification) was in agreement with AfOCH2 deletion. (C)
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Products obtained with the primer pairs RB-Och3-F/RB-Och3-R (2058 bp) and Och3-
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F/Och3-R (1159 bp; AfOCH3 ORF amplification) showed the deletion of AfOCH3. (D)
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Products obtained with the primer pairs RB-Och4F/RB-Och4R (2097 bp) and Och4-F/Och4-
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R (881 bp; AfOCH4 ORF amplification) indicated the right deletion of AfOCH4
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Figure S8. Expression of A. fumigatus OCH1-4 genes in S. cerevisiae. Gene expression of the
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AfOCH1, AfOCH2, AfOCH3 and AfOCH4 genes was determined by RT–PCR in
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Scoch1/pRep3-ADH, Scoch1/pRep3-AfOCH1, Scoch1/pRep3-AfOCH2, Scoch1/pRep3-
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AfOCH3 and Scoch1/pRep3-AfOCH4, respectively. Specific primer pairs (see Supporting
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information, Table S1) were used for cDNA amplification (see Supporting information, Table
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S1). The sizes of amplified products are indicated. The gene encoding for actin was used as
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the positive control
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