James JE , Lamping E , Santhanam J , Cannon RD .

	FIGURE 1. A one-step cloning strategy optimised for the overexpression of fungal PDR transporters in S. cerevisiae ADΔΔ. (A) The multifunctional pABC3-GFP (Lamping et al., 2007) derivative plasmid pABC3-XLmGFPHis was designed for optimum expression, detection and purification of C-terminally tagged ORFs. Improvements are highlighted in the grey box. pABC3-XLmGFPHis can be used for conventional cloning of any ORF into the PacI/NotI restriction sites, as described in Lamping et al., 2007, or it can be used as a template for a much more efficient and faster one-step cloning strategy of any ORF of interest. The grey coloured region represents the pBluescript IISK(+) backbone while the colour-coded region highlights the transformation cassette which comprises the S. cerevisiae PDR5 promoter followed by the XLmGFPHis double tag, the PGK1 terminator, the URA3 selection marker and part of the PDR5 downstream region. (B) FkABC1 (red) and FkABC2 (blue) ORFs were PCR amplified from first-strand cDNA using DNA oligomer primers (half arrows) designed to overlap by 25 bp with the left arm (blue) and right arm (green) DNA fragments. The left and right arm fragments were prepared as gel-purified DNA stocks by PCR amplification from plasmid pABC3-XLmGFPHis using the indicated primers. Due to technical difficulties the FkABC2 ORF was amplified as two separate DNA fragments that overlapped by 25 bp. Equimolar amounts (∼1 μg total) of three (FkABC1) or four (FkABC2) PCR amplified DNA fragments were used to transform S. cerevisiae ADΔΔ and positive Ura+ transformants were tested for correct integration of the entire transformation cassette with primers that bound to regions ∼40 bp upstream and downstream of the integration site. Correct integration occurred with 100% accuracy via four (FkABC1) or five (FkABC2) homologous recombination events (dashed crossed lines).
	FIGURE 2. Maximum likelihood phylogram of the entire repertoire of full-size PDR transporters of four FSSC species (i.e., FSSC5, FSSC10, FSSC11, N. haematococca). The PDR transporters belong to four (B,C,F,H1,H2) of the eight major clusters (A-H) of fungal PDR transporters (Lamping et al., 2010). The 21 PDR transporters of Nectria haematococca (Nh) are highlighted in red. Numbers in brackets on alternating grey or blue background denote nine distinct cluster B, three cluster C, three cluster H1, four cluster H2 and two cluster F lineages, each showing 100% bootstrap support. NhAbc1 and NhAbc2, cluster B lineages 1 and 2, respectively, are shown in brackets. The percentage bootstrap support of 1,000 replicates is shown for all major branches. Inset: The phylogenetic relationship of NhAbc1 and NhAbc2 orthologues of the FSSC with those of related Fusarium species that have been shown to be involved in azole antifungal and/or phytoalexin resistance (red). F. keratoplasticum (i.e., FSSC2) Abc1 (red) and Abc2 (blue) were also included. Lineage 3 cluster B PDR transporters of the FSSC were used as the outgroup. The percentage bootstrap support of 1,000 replicates is shown for all branches. The scale bars indicate the number of amino acid substitutions per position.
	FIGURE 3. Graphical illustration of F. keratoplasticum ABC1 (red) and ABC2 (blue) ORFs. Boxes indicate ORF sequences, black lines indicate introns (intron sizes are listed) and bidirectional arrows indicate the size of the entire ORF including introns.
	FIGURE 4. F. keratoplasticum ABC1 and ABC2 mRNA expression levels in response to VRC exposure. Logarithmic phase F. keratoplasticum 2781 cells were incubated for 4 h at 30°C in PDB medium in the presence of sub-growth inhibitory concentrations of VRC (16 mg/L). (A) GAPDH normalised ABC1 and ABC2 mRNA expression levels (2–ΔCq) of F. keratoplasticum 2781 cells harvested at the indicated times of VRC induction (16 mg/L). (B) Graph of the fold up- or down-regulation of GAPDH normalised ABC1 and ABC2 mRNA expression levels after 20, 40, 80, and 240 min VRC exposure relative to their mRNA expression levels at time zero. Numbers on the top of each bar indicate the fold changes.
	FIGURE 5. Expression and localisation of FkAbc1 and FkAbc2 in S. cerevisiae ADΔΔ. (A) SDS-PAGE of plasma membranes (10 μg protein) isolated from S. cerevisiae ADΔΔ cells (lane 1; negative control) or from ADΔΔ cells overexpressing C. albicans Cdr1-GFP (lane 2; positive control), F. keratoplasticum 2781 Abc1 (lane 3), Abc1-XLmGFPHis (lane 4), Abc2 (lane 5), and Abc2-XLmGFPHis (lane 6); lane M is the molecular weight markers [molecular weights (kDa) are indicated]. The image underneath shows the green fluorescence signals of the C-terminally GFP tagged proteins that were used to quantify the expression levels, expressed as % of Cdr1-GFP. The image above was obtained from the same SDS-PAGE gel after Coomassie Blue R-250 staining. Arrowheads point to GFP-tagged (green) or untagged (red) protein bands, respectively. (B) Confocal microscopy of ADΔΔ cells overexpressing CaCdr1-GFP, FkAbc1-XLmGFPHis, and FkAbc2-XLmGFPHis. GFP signals were detected with a LSM800 confocal microscope (Zeiss, Germany) at 630 × magnification. For optimum visualisation the argon laser intensities were adjusted to 4% for CaCdr1-GFP and 7% for FkAbc1-XLmGFPHis and FkAbc2-XLmGFPHis, respectively. Images on the right are light microscopy images of the same cells.
	FIGURE 6. Quantification of drug resistance levels of S. cerevisiae ADΔΔ cells overexpressing CaCDR1 (green), FkABC1 (red), or FkABC2 (blue). The increased drug resistance levels (x-axis) of 10 test compounds (y-axis) were expressed as fold increased MICs relative to the sensitive control strain, S. cerevisiae ADΔΔ. The 12 test compounds are arranged, from bottom to top, according to their molecular weight (MW): ANI (MW = 265), AOR (265), CHX (281), TRB (291), TRD (292), FLC (306), CLT (345), VRC (349), DFC (406), R6G (479), KTC (531), and NIG (725). One (*) or two (**) asterisks indicate no transport by FkAbc1 or FkAbc2, respectively. MIC measurements were performed twice and gave identical values.

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