Identification of glycerol utilization mutants

To determine whether U. maydis is suitable for the study of glycerol bioconversion, glycolipid-producing strain L8 was tested for the production of glycolipids in various media with glycerol as the sole carbon source. Synthetic minimal medium with glycerol (MinG) was found competent in producing both UA and MEL. We assumed that mutants with enhanced glycerol utilization capability would grow faster than most other colonies in the background. We created a random insertional mutants library of U. maydis L8 strain by the Agrobacterium tumefaciens-mediated transformation (ATMT) technique, and screened approximately 20,000 T-DNA insertional mutants on plates with glycerol as the sole carbon source. Twenty-four colonies that appeared significantly larger in size were selected and the screening was repeated in MinG agar medium by comparing the growth rates of the cells under various dilutions. As shown in Figure 2A, two best mutants showing clearly faster growth were selected and named as Glycerol Utilization Mutants No. 1 and 2 (GUM1 and GUM2). When cultured in glycerol-containing medium, GUM2 was severely defective in secretion of glycolipids while GUM1 largely retained the glycolipids profile found in the wild-type strain (WT) using either glucose or glycerol as carbon source (Figure 2B). GUM1 showed no obvious change in cell morphology and growth rate when cultured in glucose-containing medium (data not shown). Hence, we focused on the characterization of GUM1 in this report.

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Figure 2. Phenotypes of GUM1 and GUM2.

A. Screening for glycerol utilization mutants. Cultures of selected mutants were normalized to 1.0 OD₆₀₀ and spotted on MinG agar plate in 10-fold serial dilutions. The photo was taken after incubation at 28°C for 2 days. B. Glycolipid profiles of WT, GUM1 and GUM2 in glucose or glycerol-containing medium. The MEL and UA species are indicated on the left. WT: wild-type strain (U. maydis L8 strain); D: dextrose as carbon source; G: glycerol as carbon source.

https://doi.org/10.1371/journal.pone.0016438.g002

Characterization of GUM1 and GUM2

Southern blot analysis of GUM1 and GUM2 showed that both mutants contained a single-copy T-DNA integrated into the genome (Figure 3A). The T-DNA-tagged sequences were obtained by inverse PCR. BLASTn search showed that GUM1 contained a T-DNA inserted between the genomic locus of UM02592 and UM10280 (MIPS Ustilago maydis DataBase, MUMDB, with GenBank accession number XM_753646 and XM_753647 respectively), 236 bp upstream of the UM02592 locus and 274 bp upstream of UM10280 (Figure 3B). Northern blot hybridization showed that um02592 mRNA was expressed at higher level in GUM1 compared with WT even when cultured in glucose-containing medium, whereas um10280 mRNA expression was not significantly different from WT (Figure 3C). Noticeably, um02592 appeared to be inducible by glycerol in GUM1 (Figure 3C). These results suggest that um02592 is likely to be involved in the observed phenotype of GUM1.

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Figure 3. Characterization of GUM1.

A. Southern blot analysis of GUM1 and GUM2. Genomic DNA (10 µg) of WT, GUM1 and GUM2 were digested with BamHI and probed with a digoxigenin-labeled hpt DNA fragment. B. Schematic illustration of T-DNA insertion site in GUM1. LB: left border of T-DNA; Pgpd: gpd promoter; hpt: coding sequence of Hygromycin resistance gene; tsr1L and tsr1R: homologous regions used for knockout of tsr1. C. Expression of um10280 and um02592. WT and GUM1 cells were shifted to YPG medium and harvested after the time indicated (hours), and mRNAs were detected using the probes as illustrated in Figure 2B. Hybridization with ppi probe was used as the control for constitutive expression.

https://doi.org/10.1371/journal.pone.0016438.g003

Molecular analysis of um02592 gene

Based on the sequence information on U. maydis 521 strain (MUMDB), the ORF of Um02592 was amplified by PCR from the genomic DNA of L8 strain. It contains two silent nucleotide substitutions (G93A and G543A) and the third (A234T) substitution converts the 78^th residue from glutamic acid to aspartic acid. A BLASTp search of Um02592 (acc. no. EAK83762) revealed that it shares 44% identity to a putative β-hydroxyacid dehydrogenase from Cryptococcus neoformans (acc. no. EAL21888) and a long list of other related β-hydroxyacid dehydrogenases from bacteria, fungi, plants and animals, including 6-phosphogluconate dehydrogenases (PGD), 3-hydroxyisobutyrate dehydrogenases (HIBD) and tartronate semialdehyde reductases (TSR). We selected a few representative proteins from each class, which included at least one biochemically characterized enzyme, to analyze the phylogenic relationship (Figure 4A; Support information Figure S1). It was obvious that Um02592 and two other related sequences of fungal origin (CnTsr and PpTsr) belonged to a distinct clad and were most divergent from the PGD clad. However, it was not possible to clearly distinguish Um02592 from the HIBD and bacterial TSR clads. Since the U. maydis genome contains another protein (Um02577) more related to the PGDs and HIBDs (Um02189), we predicted that Um02592 is more likely to be a homolog of TSR. A detailed examination of Um02592 revealed that it contains the majority of residues found in the signature sequences of β-hydroxyacid dehydrogenases, including the cofactor binding, substrate binding and catalytic domains [4] (Figure 4B). However, we noticed that Um02592 contains a high degree of divergence in the substrate-binding and catalytic domains, which are highly conserved between TSRs and HIBDs.

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Figure 4. Sequence analysis of β-hydroxyacid dehydrogenases.

A. Phylogenic tree of selected β-hydroxyacid dehydrogenases. Sequence alignment and phylogenic tree was created with Jalview 2.5 program [36] using the average distance method with BLOSUM62 matrix. The sequences and origins can be found in the following Protein Identifiers from GenBank: 145698315 (EcTSR, Escherichia coli str. K-12 substr. MG1655); 16766546 (SeTSR, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2); 152972053 (KpTSR, Klebsiella pneumoniae subsp. pneumoniae MGH 78578); 57226089 (CnTsr, putative TSR of Cryptococcus neoformans var. neoformans JEC21); 46098529 (Um02592, Ustilago maydis 521); 242207395 (PpTsr, putative Tsr of Postia placenta Mad-698-R); 16272945 (HiHIBD, Haemophilus influenzae Rd KW20); 23308751 (HsHIBD, Homo sapiens); 83977457 (RnHIBD, Rattus norvegicus); 39952089 (MgHIBD, Magnaporthe grisea 70-15); 32411867 (NcHIBD, Neurospora crassa OR74A); 18415593 (AtHIBD, Arabidopsis thaliana); 19111887 (SpPGD, Schizosaccharomyces pombe); 40068518 (HsPGD, Homo sapiens); 145611724 (MgPGD, Magnaporthe grisea 70-15); 458910 (ScPGD, Saccharomyces cerevisiae); 15232888 (AtPGD, Arabidopsis thaliana); 46098078 (Um02189, Ustilago maydis 521) and 71014537 (Um02577, Ustilago maydis 521). B. Conserved motifs. *, ∧ and > indicate functionally important residues [4].

https://doi.org/10.1371/journal.pone.0016438.g004

Biochemical properties of Tsr1

To determine the biochemical function of Um02592, we expressed the cDNA in E. coli and purified the recombinant enzyme as a hexa-histidine-tagged protein. Soluble recombinant protein (rTsr1) was produced when the induction temperature was lowered to 25°C. The protein was readily enriched more than 8-fold to higher than 95% homogeneity by a single cycle of affinity chromatography in a Ni-chelating column (Support information Table S1). The purified enzyme exhibited a single dominant band on SDS-PAGE, migrating with an apparent molecular weight of about 40 kDa (Support information Figure S2A). This in good agreement with the predicted size of 41 kDa and protein band was able to cross-react with anti-hexa-histidine antibodies (Support information Figure S2B). Gel filtration result showed that rTsr1 has a molecular weight of 85 kDa in its natural state, suggesting that it exists as a dimer in aqueous solution (Support information Figure S2A and 2C). The purified rTsr1 was highly unstable in Tris-Cl buffer (pH 8.0) and rapidly precipitated during storage (<12 h at 4°C). However, enzyme stability and solubility could be improved in phosphate buffer at pH 7.0 (Support information Figure S3D).

The purified rTsr1 was able to oxidize _DL-glyceric acid using NAD(P)⁺ as cofactor. The test of four different β-hydroxyacid substrates showed that rTsr1 was highly specific to glyceric acid and exhibited little activity towards 6-phosphogluconic acid, β-hydroxybutyric acid and _D-threonine (Figure 5A). Although NAD⁺ could also be used as a cofactor, the enzyme showed approximately 26% lower activity compared with NADP⁺ in the oxidation of _DL-glyceric acid (Figure 5B). rTsr1 showed an optimal activity at 40°C and pH 8.5, although it had better stability around pH 7.0 (Support information Figure S3A, 3B and 3D). An abrupt drop in enzyme stability and activity was observed at pH 6.5, presumably due to precipitation of the enzyme as this coincides with its predicted isoelectric point (Support information Figure S3C and S3D). In the reduction reaction, rTsr1 showed an optimal pH of 5.5 (Support information Figure S3B). rTsr1 activity seemed to require metal ions as cofactor as EDTA severely inhibited its activity (Support information Table S2).

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Figure 5. Tsr1-catalyzed reactions.

A. Oxidation of various substrates. β-HBA: β-hydroxybutyric acid; 6-PGA: 6-phosphogluconate acid; D-thr: _D-threonine. B. Effects of cofactors on reduction and oxidation reactions. Standard deviation (SD) derived from triplicates. * and ** indicate significant differences in the group (p<0.05 and 0.001 respectively) based on one-way ANOVA test.

https://doi.org/10.1371/journal.pone.0016438.g005

Using non-linear regression method, the Michaelis constants K_m and V_max of rTsr1 on _D- and _L-glycerate was determined. The enzyme had a K_m of 17.7 mM and 123.2 mM with _D-glycerate and _L-glycerate respectively, indicating a much higher affinity to _D-glycerate (Table 1). In the reduction reaction with tartronic semialdehyde and NADH as the substrate and cofactor respectively, the K_m was dramatically reduced (0.19±0.03 mM) (Table 1). This suggests that the metabolic flow to glycerate production is favored under normal conditions.

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Table 1. Kinetic parameters of rTsr1^a.

https://doi.org/10.1371/journal.pone.0016438.t001

Over-expression and gene deletion of tsr1

To confirm that tsr1 is involved in glycerol catabolism in U. maydis, gain-of-function (tsr1^gpd) and loss-of-function (tsr1Δ) mutants were generated using ATMT technique. As expected, tsr1Δ strain displayed a 45.2% reduction in glycolipids production and the utilization of glycerol was also reduced. The opposite effect was observed with the tsr1^gpd strain, which had an approximate 40% increase in glycolipids accumulation (Table 2). Analysis of Tsr1 activities of the cell extract of the strains that were cultured for 24 hours in glycerol-containing medium showed the tsr1Δ strain had essentially abolished Tsr1 activity whereas the tsr1^gpd strain had approximately 100% higher activity over the WT (Figure 6A). Northern blot analysis confirmed the enhanced mRNA expression in the tsr1^gpd strain and total absence of expression in the tsr1Δ strain (Figure 6B). tsr1Δ cells showed no obvious abnormality except that accumulation of black pigment was observed when cultured in MinG medium (Figure 6C). These results confirm that tsr1 play a major role in the bioconversion and assimilation of glycerol in U. maydis.

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Figure 6. Effects of tsr1 knockout and over-expression.

A. Relative TSR activity in various strains. Tsr1 activity was assayed using _DL-glycerate as substrate and NADP⁺ as cofactor according to the standard enzyme assay method. The activity of WT is set at 100%. tsr1Δ and tsr1^gpd are the knockout and over-expression strains in L8 background, respectively. Tsr1 activity was assayed in triplicates. B. Expression of tsr1 mRNA in various strains. Strains were cultured in YPD medium to late exponential phase (time zero) before shifted to MinG medium for the duration indicated. The blot was probed with digoxigenin-labeled tsr1 cDNA. In tsr1^gpd, the upper band is the overexpressed tsr1-egfp mRNA. The rRNA bands were stained with Methylene Blue. C. Cell morphology and glycolipids accumulation. Cells of WT, tsr1^gpd and tsr1Δ strains that were cultured for 14 days in MinG medium. The needle-like or fibrous material around the cells is UA. The scale bar represents 10 µm.

https://doi.org/10.1371/journal.pone.0016438.g006

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Table 2. Effects of knockout (tsr1Δ) and over-expression (tsr1^gpd) strains of tsr1^a.

https://doi.org/10.1371/journal.pone.0016438.t002

Restoration of glycolipids production by supplementation of glycerate

Since the loss-of-function strain tsr1Δ produced considerably less glycolipids than WT in MinG medium, we explored to see whether this defect could be complemented with glycerate. As expected, WT and tsr1Δ showed comparable glycolipids profiles when _DL-glycerate was supplemented to MinG medium (Figure 7A). As shown in Figure 7B, glycerol utilization in WT was only slightly improved by the supplementation of glycerate. On the other hand, glycerol utilization was restored to WT level in tsr1Δ when glycerate was supplemented. These results suggest that glycerate production catalyzed by Tsr1 is a rate-limiting step in the bioconversion of glycerol to glycolipids in U. maydis.

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Figure 7. Effects of glycerate supplementation.

A. Glycolipid profiles of WT and tsr1Δ. Single colonies of WT and tsr1Δ were cultured in MinG (10 g/l glycerol) with (+) or without (-) _DL-glycerate (1%, v/v) at 30°C, 250 rpm for 5 days. B. Glycerol utilization in WT and tsr1Δ. Residual glycerol was quantified by HPLC after a 5-day culture in MinG medium and the relative glycerol utilization was calculated from three independent repeats. ** indicates statistical significance (p<0.001) by one-way ANOVA test.

https://doi.org/10.1371/journal.pone.0016438.g007

Promoter analysis of tsr1

To investigate the transcriptional regulation of tsr1, eGFP [21] encoding sequence was fused to the upstream sequences of the Tsr1-encoding region (Figure 8A). The strongest fluorescence was observed in the longest promoter (-1 to -926) and shortening the promoter towards the 3′ region steadily reduced the promoter activity. Notably, inclusion of the sequence from -179 to -393, the region in which the T-DNA was inserted in GUM1, led to a nearly 9-fold decrease in promoter activity (Figure 8B). These results suggest that the region from -393 to -179 contain a negative cis-acting DNA element with an important role in the regulation of tsr1 expression. A binding motif (AGGGG) for yeast Gis1p, a transcriptional factor with known functions in reprogramming of carbon metabolism in yeast [22], [23], was identified through scanning the above 214 bp sequence region using the YEASTRACT program (http://www.yeastract.com/formtfsbindingsites.php). Further studies on the -393 to -179 region should shed more light on the regulatory control of tsr1 in U. maydis.

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Figure 8. Deletion analysis of tsr1 promoter.

A. Schematic illustration of reporter constructs. The location of T-DNA insertion site in GUM1 is indicated. Tnos: transcriptional terminator of A. tumefaciens nopaline synthase gene. B. Relative fluorescence intensity of reporter constructs. The normalized fluorescence intensity of the -926 promoter was set at 100% and results were average of triplicates.

https://doi.org/10.1371/journal.pone.0016438.g008

Strains, growth conditions and chemicals

U. maydis L8 is an a1 b1 strain isolated from China [19]. The strain was cultured at 28°C in YPD broth (1% yeast extract, 2% peptone, 2% glucose) or on solid potato-dextrose agar (PDA). Agrobacterium tumefaciens AGL-1 [29] was grown at 28°C in either liquid or solid 2YT medium. ATMT of U. maydis was performed as described [19]. Escherichia coli strain XL10 was cultured in LB medium and used for routine plasmid manipulation while BL21(DE3) (Novagen, USA) was used for recombinant protein expression.

_DL-glyceric acid was purchased from CP Chemicals (Japan) and _D-glycerate, _L-glycerate, β-hydroxybutyric acid, 6-phosphogluconate acid, _D-threonine and other chemicals were from Sigma-Aldrich (USA). The tartronic semialdehyde was prepared by the enolization of lithium hydroxypyruvate in alkaline medium with a conversion rate of approximately 50% [30] and the concentration was determined by HPLC. For production of glycolipids, U. maydis was cultured in MinAB medium [31] with 50 g/l of glucose (MinD) or 50 g/l of glycerol (MinG) at 30°C with constant shaking (200 rpm).

DNA constructs

Binary plasmids pTHPR1 has been described previously [19] and the structures of pEX1GPD-GUS, pEX1GPD-eGFP and pEX2tk can be found in Support information Figure S4.

Gene knockout construct pKOtsr1 was constructed by joining, in a single ligation, four DNA fragments, i.e., the 8668 bp NcoI-KpnI-cut and dephosphorylated pEX2tk, the 1994 bp HindIII-SpeI-cut fragment containing the Pgpd::hpt::T_35S gene cassette, the 1577 bp NcoI-HindIII-cut 5′ flanking sequence and the 2688 bp SpeI-KpnI-cut 3′ flanking sequence of um02592 gene. Oligos Tsr1L-Nf and Tsr1L-r were used for PCR of the 5′ flanking region while Tsr1R-f and Tsr1R-Br were used for PCR of the 3′ flanking region of um02592. Candidate constructs were identified by colony PCR, restriction digestion mapping and further confirmation by sequencing. ATMT was used to generate transformants and fungal colony PCR was used to identify candidate knockout strains, which were further confirmed by Southern blot analysis.

For overexpression of tsr1 in U. maydis, oligos Tsr1-Bsf and Tsr1-Br were used for RT-PCR. The BspHI-BamHI double-digested PCR product was ligated to NcoI-BamHI double-digested pEX1GPD-EGFP to create pEX1GPD-Tsr1EGFP, and gain-of-function mutant tsr1^gpd was generated by ATMT technique. All sequence information of the oligos used can be found in Support information Table S3.

Analysis of T-DNA insertion site

KpnI or SpeI digested genomic DNA of GUM1 was self-ligated before being used as templates for inverse PCR [32] with IP1 and IP2 as primers. Semi-nested PCR was performed using a 100-fold dilution of the inverse PCR product as template and IP1 and RBtail1 as primers. PCR was carried out using Taq DNA polymerase (Qiagen, Germany) in a PTC-200^TM Programmable Thermal Controller (BioRad, USA). PCR products were gel-purified (Qiagen kit), cloned into pGEM-T Easy vector (Promega, USA) and sequenced using BigDye Terminator sequencing method (ABI, USA).

Quantification of glycolipids and glycerol

Extraction and thin-layer chromatographic identification of glycolipids were performed essentially as described previously [16]. For quantification of glycolipids, samples were diluted 10 times and separated in a Silica C18 Column (Φ5 µm, 4.6×150 mm) (Waters, USA) using methanol and 0.05% formic acid as the mobile phase. The samples were run at a flow rate of 1.0 ml/min. A column heater (Shimadzu, Japan) was used to maintain the running temperature at 45°C. Glycolipids were detected at 40°C with an evaporative light scattering (ELSD) detector (Shimadzu, Japan). Data was acquired and analyzed with LCsolution (ver1.2) (Shimadzu, Japan). The hexane-purified glycolipids from U. maydis L8 strain were used as the control and the amount of glycolipids was the average of triplicates.

For quantification of glycerol, samples (10 µl) were injected and run in an Aminex 87H column (Φ5 µm, 7.0×300 mm) (Bio-Rad, USA) at a flow rate of 0.7 ml/min using 5 mM sulfuric acid as the mobile phase. The column was maintained at 50°C and glycerol was detected with a Refractive Index Detector (RID, Shimadzu, Japan).

Northern blot analysis

Cells were cultured in YPD medium at 28°C until late logarithmic phase and collected by centrifugation. The cells were washed with sterile water and resuspended in an equal volume of yeast extract-peptone-glycerol (YPG) medium. Total RNA was extracted at various time points in YPG medium and the concentrations were determined with a Nanodrop photospectrometer (Nanodrop Technologies, USA). A 553-bp internal fragment of the gene was amplified by PCR using primers Tsr1f and Tsr1r and used as a probe for Northern analysis of tsr1 expression. Expression of um10280 mRNA was probed with a 416-bp fragment, which was amplified by PCR using primers Vas1f and Vas1r. Northern hybridization was performed as described [33]. The hybridization membrane was striped and re-hybridized against the peptidylprolyl isomerase encoding gene ppi (um03726) [34]. The 456 bp ppi probe of was amplified by PCR using primers Ppif and Ppir.

Expression and purification of recombinant Tsr1

To express Tsr1 in E. coli, tsr1 was amplified from total RNA of wild-type L8 strain by RT-PCR using oligos Tsr1-Ef and Tsr1-Xr. The EcoRI/XhoI double-digested PCR product was ligated to the similarly digested pET-28a (Novagen, USA) to create pETTsr1, which was subsequently transformed into E. coli strain BL21(DE3). Kanamycin and Chloramphenicol resistant transformants were cultured overnight at 37°C in 25 ml Luria broth (LB) supplemented with 25 µg/ml Kanamycin and 34 µg/ml Chloramphenicol. The culture was scaled up until OD₆₀₀ reached 0.7 unit before induction was initiated by adding isopropyl-D-thiogalactoside (IPTG) to a final concentration of 0.4 mM. After approximately 20 h induction at 25°C, the culture was harvested, washed with water and the cell pellet was suspended in 15 ml buffer A (50 mM Tris-Cl, 0.3 mM PMSF, 1 mM DTT, pH 7.0). Cells were disrupted by sonication and the insoluble materials were sedimented by centrifugation at 4°C, 14,000 g for 30 min. Protein was purified with an ÄKTA^TM Purifier (GE Healthcare, USA) at 4°C using a HisTrap_FF column (5 ml), which was equilibrated with buffer A supplemented with 500 mM NaCl and 20 mM imidazole. The His-tag fusion protein was eluted with buffer A supplemented with 500 mM NaCl and 200 mM imidazole. The concentration of protein was quantified using the Bradford method [35].

To prepare cell extract for TSR activity assay, cells were harvested after 24 h culture in MinG medium, resuspended in PBS buffer (pH 7.4) supplemented with 5 mM DTT and 1 mM PMSF. Total protein was recovered after sonication and centrifugation. The protein contents were determined by the Bradford method [35] and normalized to 1.0 mg/ml.

Enzyme assay

TSR activity was determined as described previously [4] with some modifications. Enzyme preparation (10 µl) was added to 190 µl pre-warmed (40°C) reaction mixture consisting of 50 mM glycine, 100 µM β-NAD⁺ or β-NADP⁺, and 2 mM _DL-glyceric acid (pH 8.5). The absorbance at 340 nm was monitored using a Tecan Infinite® M200 plate reader and data was collected using I-Control^TM software (Tecan, Germany). The kinetic parameters were calculated as described previously [3]. One unit of activity is defined by the production of 1 µmol of NADH or NADPH per minute at 40°C. Enzyme activity was calculated using following equation: Tsr1 unit  =  v * s/(ε * l), wherein v is the volume of reaction system (200 µl), s is the increase of OD₃₄₀ value per minute, ε is the molar extinction coefficient of NADH or NADPH at 340 nm (ε_NADH  = 6220 µl/µmol cm; ε_NADPH  = 6270 µl/µmol cm), l is the depth of a 200 µl reaction system in one well (l  = 0.6 cm in the Nunc^TM transparent 96-well plate).

Enzyme stability and optima

The optimum temperature and pH for activity of rTsr1 were determined by varying the assay temperature and pH values in the reaction mixture, respectively. Thermal stability was obtained after pre-incubation of rTsr1 at different temperature for 30 min. pH stability was investigated after pre-incubation of rTsr1 in various pH buffers for 30 min before the assay at pH 7.0. Pre-treated samples were centrifuged at 4°C, 14, 000 g for 5 min to remove the denatured protein before the assay. In all cases, overlaps were obtained when buffers were changed to correct the spurious buffer effects.

Substrate and cofactor specificity

Activity of purified rTsr1 (1.0 µg/assay) was assayed using various substrates, i.e., _DL-glycerate (25 mM), _D-glycerate (25 mM), _L-glycerate (25 mM), 6-phosphogluconic acid (0.5 mM), β-hydroxybutyric acid (0.5 mM) and _D-threonine (0.5 mM). NAD⁺ and NADP⁺ (10 mM) were supplemented individually to determine the preference for cofactor with _DL-glycerate as the substrate.

Analysis of promoter activity

To generate the 926 bp promoter fusion construct with enhanced green fluorescence protein encoding gene, the upstream sequence of tsr1 was amplified using WT genomic DNA as template and oligo pair Vas1r/Ptsr1-Nr. The PCR products were end-blunted with T4 DNA polymerase, digested with NcoI and ligated with the PmeI/NcoI double-digested pEX1GPD-eGFP. To generate 658 bp, 393 bp and 178 bp promoter-eGFP gene cassettes, the above 926 bp PCR product was double-digested with NcoI and PvuI, SacI or SalI, respectively (Figure 7A). All transformants were obtained by ATMT technique. To minimize positional effect of reporter gene expression, at least 1000 transformants on the membrane were collected to create a mixed population of transformants, which were cultured and assayed for the strength of GFP florescence. Sporidia culture at mid-exponential phase was pelleted by centrifugation and thoroughly washed with water. After diluted to 0.2 OD₆₀₀ in MinG medium, the cells were cultured at 30°C for 2 h. An aliquot (100 µl) was loaded to a well in a 96-well plate. Flat transparent plates (Nunc, USA) and MicroFLUOR round-bottom plates (Dynatech, USA) were used for determination of cell optical density and GFP fluorescence intensity in a Tecan Infinite® M200 plate reader (Tecan, Germany), respectively. Cell optical density was read at 600 nm while GFP fluorescence was measured with the excitation and emission wavelength set at 488 nm and 508 nm, respectively. The fluorescence intensity was normalized against the cell density (OD₆₀₀).

Light microscopy

U. maydis cells were observed with a Nikon Eclipse 80i microscope equipped with CFI Plan Apochromat objectives (Nikon, Japan), and images were acquired with a Nikon DS camera and Nikon ACT-2U software.