Genelute plasmid miniprep kit

PCR fragments were amplified using the PfuX7 polymerase [21 (link)] with primers purchased from Integrated DNA Technology, Belgium (S1 Table). Construction of vectors was carried out by Uracil-Specific Excision Reagent (USER) fusion of PCR fragment into compatible plasmids [22 (link)]. The deletion plasmids pD-hyg-talA and pD-hyg-albA were constructed by amplification of approximately 2-kb up- and downstream fragments followed by cloning into two distinct PacI/Nt.BbvCI USER cassettes located on each side of the hygromycin resistance gene. The sgRNA was introduced into the CRISPR-Cas9 vector pFC330 via the tails of two primers as described by Nødvig et al. [4 (link)]. Plasmids were purified using the GenElute^TM Plasmid Miniprep Kit (Sigma-Aldrich), and verified by restriction analysis. A list of all plasmids from this study is presented in S2 Table. Deletions were achieved using the CRISPR-Cas9 system described by Nødvig et al. [4 (link)]. A circular deletion plasmid (gene-targeting substrate) was co-transformed with an AMA1-based CRISPR-Cas9 vector containing the guide RNA and the Streptococcus pyogenes cas9 gene codon optimized for A. niger. The CRISPR-Cas9 vector also contained the pyrG auxotrophic marker; however, only the deletion plasmid, containing the hygromycin resistance gene, was selected for during transformation.

The cDNA sequences of the 5′ and 3′ends of RiCTR1-3 were confirmed and completed, when necessary, by RACE using the SMARTer^® RACE 5′/3′ kit (Clontech) according to the manufacturer’s protocol. The primers used for RACE reactions are listed in Supplementary Table S2. Genomic clones and full length cDNAs were obtained by PCR amplification of R. irregularis genomic DNA and cDNA, respectively, from ERM grown under control conditions in monoxenic cultures, using a set of primers flanking the complete open reading frames (Supplementary Table S2). PCR products were cloned into the pGEM-T Easy vector (Promega), following manufacturer’s instructions. Plasmids were amplified by transformation of chemically Escherichia coli DH5α competent cells according to standard procedures and purified using the GenElute^TM Plasmid Miniprep Kit (Sigma-Aldrich). All plasmids were checked by sequencing (ABI PRISM 3130xl Genetic Analyzer, Applied Biosystems, Carlsbad, CA, United States).

Human aquaporin (AQP3, AQP7, and AQP9) complementary DNA (cDNA) was PCR-amplified from the pWPi-DEST plasmid [49 (link)] and C-terminally fused to a green fluorescent protein (GFP) of the centromeric plasmid, pUG35 [60 (link)]. Escherichia coli DH5α was used as a host for routine propagation, and plasmids were purified with a GenElute^TM Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). E. coli transformants were maintained and grown in Luria–Bertani broth (LB) with ampicillin (100 µg∙mL⁻¹) at 37 °C [36 (link)]. For functional studies, Saccharomyces cerevisiae, 10560-6B MATα leu2::hisG trp1::hisG his3::hisG ura352 aqy1D::KanMX aqy2D::KanMX (YSH1770) was used as a host strain for heterologous expression of AQP3, AQP7, and AQP9. Yeast cultures were grown at 28 °C with orbital shaking in YNB (yeast nitrogen base, DIFCO) without amino acids, 2% (w/v) glucose supplemented with the adequate requirements for prototrophic growth [61 (link)]. For subcellular localization of GFP-tagged AQP3, AQP7, and AQP9 in S. cerevisiae, yeast transformants in the mid-exponential phase were observed with a Zeiss Axiovert 200 fluorescence microscope, at 495 nm excitation and 535 nm emission wavelengths, confirming more than 80% localization at the yeast plasma membrane.

PCR was performed with PrimeSTAR® Max DNA Polymerase (Takara Bio) unless otherwise noted. PCR products and linearized plasmids were purified with QIAEX® II Gel Extraction Kit (QIAGEN). DNA cloning experiments were performed using E. coli DH5α (Competent Quick, TOYOBO) following standard techniques and recombinant plasmids were extracted with GenElute^TM Plasmid Miniprep Kit (Sigma-Aldrich) from E. coli cells.

Complete ldpA and ldpB CDSs were amplified from pCR2.1-LdpA and pCR2.1-LdpB. The PCR products were inserted into pHAN02-GFP (Fig. S3) between the HindIII and SmaI sites by using the In-Fusion HD Cloning Kit (Takara Bio). The resulting plasmids were transformed into chemically competent E. coli strain HST08. The plasmids were extracted using the GenElute^TM Plasmid Miniprep kit (Sigma-Aldrich) and linearised using the restriction enzymes BamHI or EcoRI. The A. fumigatus niaD⁻ mutant AfS35-niaD⁻ was isolated by positive selection using chlorate according to the methods described by Unkles et al.^{35 (link)} and Ishi et al.^{36 (link)}. The resulting plasmids were used for the transformation of AfS35-niaD⁻ by PEG-mediated protoplast transformation methods. The niaD⁺ revertants were selected for growth on Czapek–Dox agar (Oxoid), which contains 1.2 M sorbitol and sodium nitrate as the sole source of nitrogen. Gene integration was confirmed by PCR.

Plasmid (pcDNA3) with Rattus norvegicus AQP5 cDNA (pcDNA3-AQP5), kindly provided by Prof. Miriam Eschevarria, Virgen del Rocio University Hospital, Seville, Spain, was used for AQP5 cDNA amplification. The centromeric plasmid pUG35 was used for cloning AQP5, conferring C-terminal GFP tagging, MET25 promoter, and CYC1-T terminator [68 (link)].
For plasmids propagation, Escherichia coli DH5α was used as host [69 ]. E. coli transformants were maintained and grown in Luria-Bertani broth (LB) supplemented with ampicillin (100 µg·mL⁻¹), at 37 °C [70 ]. Plasmid DNA was extracted from E. coli using a GenElute^TM Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA).
Saccharomyces cerevisiae (10560-6B MATa leu2::hisG tpr1::hisG his3::hisG ura3-52 aqy1D::KanMX aqy2D::KanMX) from now on designated as aqy-null, was used as host strain for heterologous expression of AQP5. The aqy-null strain was grown and maintained in YPD medium (2% w/v peptone, 1% w/v yeast extract, 2% w/v glucose). Transformed yeast strain was grown in YNB medium (2% w/v glucose, 0.67% (DIFCO) Yeast Nitrogen Base) supplemented with the adequate requirements for prototrophic growth [71 (link)] and maintained in the same medium with 2% (w/v) agar. For stopped-flow assays, the same medium was used for yeast cell growth. For all experiments, cells were grown to mid exponential phase (OD₆₀₀ 1.0).