Identification and characterization of the Komagataella phaffii mating pheromone genes

Identification and characterization of the Komagataella phaffii mating pheromone genes Abstract The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is a haploid yeast that is able to form diploid cells by mating once nitrogen becomes limiting. Activation of the mating response requires the secretion of a- and α-factor pheromones, which bind to G-protein coupled receptors on cells of opposite mating type. In K. phaffii, the genes coding for the α-factor (MFα), the pheromone surface receptors and the conserved a-factor biogenesis pathway have been annotated previously. Initial homology-based search failed to identify potential a-factor genes (MFA). By using transcriptome data of heterothallic strains under mating conditions, we found two K. phaffiia-factor genes. Deletion of both MFA genes prevented mating of a-type cells. MFA single mutants were still able to mate and activate the mating response pathway in α-type cells. A reporter assay was used to confirm the biological activity of synthetic a- and α-factor peptides. The identification of the a-factor genes enabled the first characterization of the role and regulation of the mating pheromone genes and the response of K. phaffii to synthetic pheromones and will help to gain a better understanding of the mating behavior of K. phaffii. Komagataella phaffii, Pichia pastoris, yeast, mating, mating factor, pheromone INTRODUCTION Yeast mating is mediated by secreted peptide pheromones binding to surface receptors on cells of the opposite mating type. In ascomycetes, one cell type secretes unmodified hydrophilic peptides (known as α-factor), whereas cells of the other mating type produce hydrophobic peptides with a characteristic C-terminal farnesyl and carboxylmethyl modification (a-factor). Secretion of these small peptides is essential for mating partner recognition and activation of the genes required for diploid cell formation. The different aspects of the mating process are well understood in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe but are much less characterized in other relevant yeast species. The S. cerevisiae α-factor pheromone is an unmodified hydrophilic peptide of 13 amino acids secreted exclusively by haploid cells of the α-mating type. It is encoded by MFα1 and MFα2 and translated as precursor containing four and two copies of the mature α-factor, respectively (Kurjan and Herskowitz 1982; Singh et al. 1983). In S. cerevisiaea-type cells, the MFA1 and MFA2 genes are constitutively transcribed and translated into precursor proteins of 36 and 38 amino acids, respectively (Michaelis and Herskowitz 1988). While the α-factor is processed and secreted via the classical secretory pathway, the a-factor is modified in the cytosol. Following translation, the C-terminal CAAX motif (C is cysteine, A is usually aliphatic, X can be any amino acid) is recognized by a cytosolic farnesyl transferase consisting of two subunits (Ram1 and Ram2) resulting in farnesylation of the C-terminal cysteine residue. Subsequently, the three C-terminal amino acids (AAX) are cleaved by the proteases Rce1 or Ste24 and a carboxylmethyl group is added to the farnesylcystein by the isoprenyl cysteine carboxyl methyltransferase Ste14. The N-terminus of a-factor is processed in two consecutive proteolytic cleavage steps by zinc proteases Ste24 and Axl1, resulting in mature a-factor peptides with a length of only 12 amino acids differing solely in a single residue (Betz et al. 1987; Michaelis and Barrowman 2012). Mature a-factor is exported from the cytosol by Ste6, an ATP-binding cassette (ABC) transporter located in the plasma membrane (Kuchler, Sterne and Thorner 1989). Once secreted, a-factor binds to the G-protein coupled receptor Ste3 on the surface of α-type cells, activating a mitogen-activated protein kinase (MAPK) signaling cascade resulting in the activation of genes required for mating, growth of mating projections (shmoos) and arrest in the G1 phase of the cell cycle (Jones and Bennett 2011; Merlini, Dudin and Martin 2013). The enzymes of the a-factor biogenesis pathway are conserved in yeasts and higher eukaryotes. However, due to their short length and poor sequence conservation, the genes encoding a-factor pheromones are hard to identify. Besides the well-characterized S. cerevisiae mating factors, a-factor pheromones have been investigated in a number of other yeast species like Sc. pombe (M-factor), Kluyveromyces lactis and Candida albicans (Davey 1992; Kjaerulff, Davey and Nielsen 1994; Dignard et al. 2007; Ongay-Larios et al. 2007). All the identified proteins contain a C-terminal CAAX motif for farnesylation and were found to be essential for mating of a-type cells. The number of genes encoding the a-factor varies in different species, from only one gene copy in C. albicans or Eremothecium gossypii to up to 12 predicted MFA genes in C. tropicalis (Dignard et al. 2007; Wendland, Dunkler and Walther 2011). It has been hypothesized that the redundancy in the MFA genes might be necessary to allow evolution of pheromones required for the development of new reproductive groups while ensuring mating within a species (Seike, Nakamura and Shimoda 2015). The methylotrophic yeast Komagataella phaffii (also referred to as Pichia pastoris) is a haploid yeast that is able to form diploids by mating if nitrogen becomes limiting. Wild-type strains are homothallic and can undergo mating-type switching by inversion of the chromosomal region between two mating-type (MAT) loci located on chromosome 4. While the expression of the MAT genes at MAT locus 1 is downregulated by telomere position effects, the mating type of a cell is determined by the MAT genes actively transcribed from locus 2. Both MAT loci are flanked by large inverted repeat regions which have been proposed as homologous regions involved in mating-type switching (Hanson, Byrne and Wolfe 2014). Deletion of the ‘outer’ repeat region at MAT locus 2 (including DIC1–2) prevents mating-type switching and allowed the generation of K. phaffii strains with stable mating types (Heistinger, Gasser and Mattanovich 2018). The K. phaffii α-factor is encoded by a single MFα gene (CBS7435_Chr2–0522) and is translated as a precursor peptide consisting of a pre-pro leader sequence and nine copies of the mature α-factor flanked by Kex2 and Ste13 processing sites (Kuberl et al. 2011). Experiments using the pre-pro leader sequence have shown that it can be used for efficient secretion of recombinant proteins (Kuberl et al. 2011) but no further studies characterizing the K. phaffii α-factor peptide itself have been conducted. Orthologs of α-and a-factor receptor genes (STE2 and STE3, respectively) as well as all the genes essential for a-factor processing and secretion in other yeasts can be found in the K. phaffii genome using homology-based search (Kuberl et al. 2011; Valli et al. 2016). However, so far no gene coding for the a-factor pheromone has been identified. Within this study we could identify two putative MFA genes in the genomes of K. phaffii and K. pastoris by using a combination of transcriptomics data analysis and homology search. The role of the newly identified peptides in K. phaffii mating was confirmed using MFA deletion mutants and fully modified synthetic pheromones. Furthermore, we analyzed the mating phenotype of mfαΔ mutants and the response of K. phaffii cultures to synthetic α-factor. MATERIALS AND METHODS Yeast strains and vectors All K. phaffii strains described in this study (Table 1) are based on the homothallic CBS2612 wild-type strain. Generation of the dic1–2Δ strains with stable mating types was described in detail in Heistinger, Gasser and Mattanovich (2018). To obtain the mating-factor deletion strains, the whole coding sequence of the target gene was replaced by an antibiotic resistance marker using the split-marker technique (Fairhead et al. 1996; Gasser et al. 2013). Homologous flanking regions of around 1000 bp for integration were PCR amplified from K. phaffii genomic DNA with primers containing BsaI restriction sites and fusion sites (FS) for Golden Gate cloning (FS AB and CD for the 5΄ and 3΄ region, respectively) using the previously published GoldenPiCS system (Weber et al. 2011; Prielhofer et al. 2017; Sarkari et al. 2017). Marker sequences were amplified from available plasmid vectors to introduce FS BC. In the next step, a vector (BB3) containing the marker gene flanked by the two homologous regions was assembled by Golden Gate cloning using BsaI. This vector was used as template for PCR-based amplification of the two knockout cassettes overlapping in the marker gene sequence. For the construction of the PFUS1_eGFP reporter construct, the sequence upstream of the FUS1 start codon (Chr4:560823..560339 [FR839631.1]) was amplified from genomic DNA of K. phaffii CBS2612 using primers containing FS AB and BsaI restriction sites for cloning into a BB1 GoldenPiCS vector. In the next step, the expression cassette consisting of the enhanced green fluorescent protein (eGFP) coding sequence under control of the FUS1 promoter and S. cerevisiae CYC1 terminator was assembled from the available BB1 constructs into a BB3 vector using BpiI. The final plasmid also contained a NatMX resistance cassette for selection with nourseothricin and the AOX1 terminator sequence as homologous region for genomic integration after linearization with AscI. Empty pPUZZLE vectors were used for the integration of additional marker genes required for the selection of diploid cells into the TDH3 promoter or AOX1 terminator region, respectively (Stadlmayr et al. 2010). All primers used in this study are listed in Table S1 (Supporting Information). Table 1. List of strains used in this study. Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study aThe dic1–2Δ(a) strain contains one loxP sequence between SLA2–2 and MATα1 in MAT locus 1 and another loxP sequence in MAT locus 2 between and SLA2–1 and MATa1. View Large Table 1. List of strains used in this study. Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study aThe dic1–2Δ(a) strain contains one loxP sequence between SLA2–2 and MATα1 in MAT locus 1 and another loxP sequence in MAT locus 2 between and SLA2–1 and MATa1. View Large Cultivation conditions Yeast cells were grown in standard YP medium (yeast extract 10 g/l, soy peptone 20 g/l) containing 2% glucose as carbon source. All liquid cultures were grown at 25°C. After transformation, cells were plated on YPD agar (2% agar-agar) supplemented with the appropriate antibiotics (zeocin 50 μg/ml, geneticin 500 μg/ml, nourseothricin 100 μg/ml). Increased antibiotic concentrations of up to 100 μg/ml zeocin and 1 mg/ml geneticin were used for the selection of diploid cells on mating plates. Mating conditions were defined as cultivation in nitrogen-free acetate medium (mating medium, 0.5% sodium acetate, 1% potassium chloride, 1% glucose). RNA extraction Cultivations and sampling for RNA extractions were performed as described in Heistinger, Gasser and Mattanovich (2018). Three colonies of each strain were cultivated in parallel. RNA was isolated according to the TRI reagent (Sigma-Aldrich) protocol. Residual genomic DNA was digested using the Ambion DNA- free kit (Invitrogen), and RNA was precipitated overnight (1/10 volume 3M sodium acetate, 2.5 volumes absolute ethanol, 1 μl glycogen) at –20°C to remove traces of phenol. RNA concentrations and integrity were analyzed using a Nanodrop spectrophotometer and a Bioanalyzer (Agilent) using the RNA 6000 Nano Kit. RNA sequencing data analysis Solexa Sequencing of poly(A)-enriched cDNA libraries was performed at the VBCF NGS Unit (www.vbcf.ac.at). The unsorted, unaligned BAM files were converted and split into paired-end FASTQ files with the TopHat2 tool bam2fastx v2.1.0 (Kim et al. 2013) and subsequently trimmed and quality checked using Trim-Galore v0.4.2 (Babraham Bioinformatics 2016) using default parameters. As preparation for the count quantification, a transcript index was created using the latest K. phaffii CBS7435 annotation (FR839628.1, FR839629.1, FR839630.1, FR839631.1, FR839632.1; Valli et al. 2016) and the kallisto v0.43.0 subprogram index (Bray et al. 2016). Subsequently, the read count for each sample was determined using kallisto subprogram quant (Bray et al. 2015). For differential expression analysis (Love et al. 2015) with R (R Development Core Team 2016), the packages tximport and tximportData (Soneson, Love and Robinson 2015), readr (Wickham et al. 2017) and DESeq2 (Gentleman et al. 2004; Huber et al. 2015; Love, Anders and Huber 2017) were used for each comparison. Visualization of the expression analysis results was also carried out with R. Transcripts with a log2 fold-change of below –1 and above 1 combined with an adjusted p-value below 0.05 were considered to be significantly regulated. For the detection of new, so far unannotated transcripts, the RNA sequencing data were analyzed with the HISAT-StringTie-Ballgown suite (Pertea et al. 2016). Coverage histograms were visualized with JBrowse (Skinner et al. 2009). The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (Edgar, Domrachev and Lash 2002) and are accessible through GEO Series accession number GSE111973. Quantitative PCR For quantitative PCR, RNA samples were treated with the Ambion DNA-free kit (Invitrogen) and RNA integrity was analyzed by agarose gel electrophoresis. All cDNA was synthesized using oligo(dT)23 primers (NEB) and the Biozym cDNA synthesis kit. PCR reactions were performed using the Sensi Mix SYBR Hi-Rox kit (Bioline) on a Rotor-Gene Q instrument (Qiagen). Purified PCR products of the analyzed genes were used to generate standard curves for quantification. Transcript levels were normalized to ACT1 (PP7435_Chr3–0993) expression. Mating protocols Qualitative mating experiments were performed similarly to Tolstorukov and Cregg (2007). Strains to be mated were streaked out in parallel on YPD agar plates directly from glycerol stocks and incubated at 30°C. After approximately 24 h, cells were replicated twice at an angle of 90°C onto mating agar (0.5% sodium acetate, 1% potassium chloride, 1% glucose, 2% agar) and incubated at 25°C for 3 days. Diploid cells were selected by replica plating onto YPD agar containing the appropriate antibiotics. Cell growth at crossing points was evaluated after incubation at 30°C for 3 days. For the semiquantitative mating assay, fresh colonies grown on YPD agar were used to inoculate non-selective YPD medium. After 18–20 h, approximately 6.5 × 107 cells of each strain were mixed, plated on mating agar plates and incubated at 25°C for 3 days. Cell numbers were estimated by measuring the optical density at 600 nm (OD600), with an OD600 of 1 corresponding to 1 × 107 cells. Mated cells were washed from the plates with phosphate buffered saline (PBS) (KH2PO4 0.24 g/l, Na2HPO4*2 H2O 1.8 g/l, KCl 0.2 g/l, NaCl 8 g/l) and appropriate dilutions were plated on YPD agar plates containing either one or both of the antibiotics used for the selection of diploid cells. Colonies were counted after incubation at 30°C for 3 days. The mating efficiency was calculated as the percentage of cells growing on the plates containing both antibiotics in relation to the number of cells growing in the presence of one of the antibiotics (using the cell count on the antibiotic plates with the lower number of cells). Synthetic pheromones The K. phaffiia- and α-factor peptides were synthesized by Thermo Fisher Scientific with a purity >95%. Both a-factor peptides were obtained with the C-terminal farnesyl and carboxymethyl modifications. Aliquots were dissolved in dimethyl sulfoxide and diluted to working concentrations with sterile H2O + 0.5 mg/ml BSA. The α-factor peptide was dissolved directly in sterile H2O + 0.5 mg/ml BSA. Saccharomyces cerevisiaea-factor was purchased from ZYMO Research. All synthetic peptides were added to the cultures at final concentrations of 10–20 μg/ml. Enhanced GFP reporter assays For assays with the PFUS1_eGFP reporter strains, precultures were grown in 24-well deep well plates in YP medium without glucose. After 24 h, cells were harvested and resuspended in yeast nitrogen base (YNB) medium (YNB without amino acids and ammonium sulfate 3.4 g/l, potassium phosphate buffer 0.1M, pH 6). For the main cultures, 2 ml YNB medium supplemented with two glucose release feed beads (6 mm diameter, Kuhner AG, glucose release rate: 0.79*time0.66 = mg glucose released per feed bead, theoretical glucose concentration without consumption after 40 h: 9 g/l) were inoculated at a starting OD600 of 2. To test the induction of the FUS1 promoter by a second strain of opposite mating type, equal numbers of cells from the separate precultures were mixed to reach the same starting OD600. Synthetic pheromones and solvent controls were added around 18 h after the start of the main culture. After 40 h in YNB medium, cells were analyzed by flow cytometry and microscopy. Flow cytometry For flow cytometry cells were diluted in PBS (KH2PO4 0.24 g/l, Na2HPO4*2 H2O 1.8 g/l, KCl 0.2 g/l, NaCl 8 g/l) and green fluorescence (FL1 channel, 505–545 nm), forward and side scatter of 20 000 cells per sample were measured on a Gallios Flow Cytometer (Beckman Coulter). Data were analyzed with the Kaluza analysis software (Beckman Coulter). GFP-positive cells were gated using the dic1–2Δ PFUS1_eGFP α-type strain in the absence of pheromone as reference. Microscopy Microscopy was performed with a Zeiss Axio Observer.Z1/7 microscope using a LCI Plan-Neofluar 63x/1.3 water immersion objective. Cell morphology was analyzed in bright-field mode. For fluorescence microscopy of eGFP-expressing cells, the 38 HE eGFP shift free (E) filter set (Zeiss) was used. Pictures were processed with the Zen 2.3 lite (blue edition) software (Carl Zeiss Microscopy GmbH). RESULTS Identification of two putative MFA sequences Due to their short coding sequences and poor sequence conservation, genes encoding a-factor mating pheromones are hard to identify by homology search (Dignard et al. 2007; OhEigeartaigh et al. 2011). To annotate possible MFA genes in the genome of K. phaffii, we used RNA sequencing data of K. phaffii CBS2612 (the homothallic wild-type strain) as well as of stable a- and α-type strains derived from it (Heistinger, Gasser and Mattanovich 2018). Sequencing reads of all strains grown in rich medium and under mating conditions were mapped to the available genome sequence of the CBS7435 strain (Kuberl et al. 2011; Valli et al. 2016). Note that no full genome sequence of the K. phaffii CBS2612 type strain is available at the moment. However, the sequences of all relevant regions investigated in this study were verified to be identical to the CBS7435 sequence. All so far not annotated open reading frames (ORFs) shorter than 200 bp that were expressed only by a-type or wild-type cells under mating conditions were analyzed. By this method, one candidate gene (MFA1) with an ORF of 123 bp (Chr1:1587540..1587662 in CBS7435 [FR839628.1]), encoding a 40 amino acid protein containing the conserved C-terminal CAAX motif, was identified. Using this sequence, a second potential a-factor gene was identified by homology search (tBLASTn). Like MFA1, the MFA2 ORF (Chr2:1840284..1840406 in CBS7435 [FR839629.1]) encodes a 40 amino acid protein containing a C-terminal CAAX motif. The ORF is part of an unknown gene annotated as CBS7435_Chr2–1002, which, according to the RNA sequencing data analysis, was upregulated to similar levels in all strains under mating conditions. However, a mating-type-specific expression pattern could be observed in the enriched coverage histograms when only the relevant sequence region was considered (Figs S1 and S2, Supporting Information). An alignment of the K. phaffiia-factor sequences is shown in Fig. 1A. The two identified proteins have an overall sequence identity of 63%. Before secretion of the mature mating factors via the Ste6 transporter, yeast a-factor peptides are modified by a number of conserved enzymes. The genome of K. phaffii encodes orthologs of all proteins known to be required for a-factor maturation in S. cerevisiae. Therefore, the N-terminal cleavage site on the K. phaffiia-factor sequences was predicted by comparison of the amino acid sequences to sequences of known a-factor peptides from other yeasts (Fig. 1B). In S. cerevisiae, the N-terminus is processed in two consecutive steps carried out by Ste24 and Axl1, with AxlI cleaving after KDN in the sequence (Michaelis and Barrowman 2012). Cleavage site sequences vary between different yeast species; however, sequence analysis showed that many of the described sites contain lysine residues and have a conserved asparagine at the final position. In K. phaffii, cleavage of the N-termini after EDN and ADN, respectively, would result in mature peptides of 17 amino acids with four residues difference between MFA1 and MFA2. Of the genes required for mating-factor cleavage, farnesylation and export, only transcript levels of RAM1 (PP7435_Chr3–0565), encoding the β-subunit of the CAAX farnesyltransferase, and STE6–1 (PP7435_Chr3–0320) were significantly upregulated under mating conditions. For RAM1, transcript levels were higher in the wild type than in pure cultures of a- or α-type cells, which points to an additional induction of gene expression by pheromone (log2 fold-changes: wild type 3.56, a-type 1.82, α-type 2.05). The transcription of the STE6–1 gene was found to be mating type specifically upregulated only in a-type and wild-type cells (log2 fold-changes: wild type 1.62, a-type 1.66). Figure 1. View largeDownload slide Alignments of yeast a-factor sequences. Protein sequences were aligned using Clustal Omega (Sievers et al. 2011). Identical amino acids are marked with an asterisk, a colon indicates residues with highly similar properties and residues with weakly similar properties are indicated by a point. Sequences of the mature peptides are shown in bold. (A) Alignment of the newly identified K. phaffiia-factor sequences. (B) Alignment of a-factor peptides from different yeast species. All sequences contain the conserved C-terminal CAAX motif (highlighted in gray). (C) Sequences of the K. pastorisa-factor peptides. The predicted mature peptides are identical to the K. phaffiia-factor peptide sequences. Figure 1. View largeDownload slide Alignments of yeast a-factor sequences. Protein sequences were aligned using Clustal Omega (Sievers et al. 2011). Identical amino acids are marked with an asterisk, a colon indicates residues with highly similar properties and residues with weakly similar properties are indicated by a point. Sequences of the mature peptides are shown in bold. (A) Alignment of the newly identified K. phaffiia-factor sequences. (B) Alignment of a-factor peptides from different yeast species. All sequences contain the conserved C-terminal CAAX motif (highlighted in gray). (C) Sequences of the K. pastorisa-factor peptides. The predicted mature peptides are identical to the K. phaffiia-factor peptide sequences. Using the K. phaffii sequences to perform homology-based search also allowed the identification of two MFA genes in K. pastoris. As in K. phaffii, the two genes are located on two different chromosomes (MFA1 Chr1:1743827..1743708 [CP014584.1], MFA2 Chr4:570171..570290 [CP014587.1], ATCC 28485 (Love et al. 2016)) and encode 40 amino acid proteins, differing from their K. phaffii orthologs only in 1 or 4 amino acids, respectively (Fig. 1C). No further a-factor sequences of other closely related yeast species with available genome sequences could be identified. Only simultaneous deletion of both MFA genes prevents mating of a-type cells To confirm the role of the newly identified sequences and to analyze the effect of MFA deletions on the mating behavior of K. phaffii, different knockout strains were generated. Genes were deleted in the homothallic CBS2612 wild-type strain as well as the previously published dic1–2Δ(a) or dic1–2Δ(α) strains with stable mating types (Heistinger, Gasser and Mattanovich 2018). In all cases, the whole gene was replaced by an antibiotic resistance marker gene. The mfa1Δ mfa2Δ double mutants were generated by consecutive transformation of two deletion cassettes. Furthermore, strains with deletion of MFα (CBS7435_Chr2–0522) or the a-factor transporter STE6–1 (PP7435_Chr3–0320) were generated. To analyze the influence of the mating-factor gene deletions on the mating behavior, all strains were mated against the unmodified a- and α-type strains using a standard mating protocol. Both of the MFA single mutants (mfa1Δ(a) and mfa2Δ(a)) were still able to mate with an α-type mating partner. Only deletion of both MFA genes from the a-type strain resulted in a complete loss of mating ability, indicating that the two candidate genes encode functionally redundant proteins (Fig. 2A). Mating experiments with the homothallic wild-type strain consisting of mutants of both mating types gave similar results (Fig. 2B). While the single mfaΔ mutants were able to mate with a- and α-type cells, MFA double mutants and the ste6–1Δ strain (Fig. 2C) were unable to mate with α-type cells. However, mating with the dic1–2Δ(a) strain was still possible for both the mfa1Δ mfa2Δ and the ste6–1Δ strain, showing that the effect of the mutations is mating type specific. The same behavior was also observed for the mfαΔ strain, where mating was only possible with the dic1–2Δ(α) strain (Fig. 2D). Figure 2. View largeDownload slide Mating phenotype of mating-factor deletion strains. Diploid cells were selected on YPD agar containing the appropriate antibiotics. All different mutants were mated against the dic1–2Δ a- and α-type strain indicated by a framed a and α. Note that the two tester strains carried the same resistance gene, preventing the selection of diploid cells formed by these strains in the positions indicated by an asterisk. (A) Diploid cell formation by mfaΔ a-type strains. Deletion of both MFA genes abolished mating with α-type cells. (B) Diploid cell formation by homothallic mfaΔ strains. Deletion of both MFA genes prevented mating of a-type cells, but did not affect α-cell mating. (C) Deletion of the a-factor transporter gene STE6–1 abolished mating of a-type cells. The cell type of the ste6–1Δ strains is given in parentheses. (D) Diploid cell formation by homothallic mfαΔ cells. Only a-type cells were able to mate and form diploids with the dic1–2Δ(α) strain. Figure 2. View largeDownload slide Mating phenotype of mating-factor deletion strains. Diploid cells were selected on YPD agar containing the appropriate antibiotics. All different mutants were mated against the dic1–2Δ a- and α-type strain indicated by a framed a and α. Note that the two tester strains carried the same resistance gene, preventing the selection of diploid cells formed by these strains in the positions indicated by an asterisk. (A) Diploid cell formation by mfaΔ a-type strains. Deletion of both MFA genes abolished mating with α-type cells. (B) Diploid cell formation by homothallic mfaΔ strains. Deletion of both MFA genes prevented mating of a-type cells, but did not affect α-cell mating. (C) Deletion of the a-factor transporter gene STE6–1 abolished mating of a-type cells. The cell type of the ste6–1Δ strains is given in parentheses. (D) Diploid cell formation by homothallic mfαΔ cells. Only a-type cells were able to mate and form diploids with the dic1–2Δ(α) strain. MFA single mutants mate with reduced efficiency To assess the contribution of the individual a-factor peptides to the overall mating efficiency of the K. phaffiia-type strain, the mating efficiency of the different mutants was analyzed by a semiquantitative mating assay. In this assay, MFA single and double mutants were mated against the unmodified dic1–2Δ(α) strain. The mating efficiency of the unmodified dic1–2Δ strains was found to be around 10% (Fig. 3). Mating with strains deleted in MFA1 and MFA2 led to a dramatic decrease in efficiency to 0.9% and 0.1%, respectively. This indicated a synergistic effect of the two a-factor pheromones in K. phaffii and showed that the expression of both MFA genes is required for efficient mating. A few diploid cells could also be obtained when crossing the mfa1Δ mfa2Δ(a) double mutant with the dic1–2Δ(α) strain, resulting in a mating efficiency of 0.01%. Figure 3. View largeDownload slide Mating efficiency of mfaΔ a-type strains. Single and double mutants as well as the dic1–2Δ(a) strain were mated with the dic1–2Δ(α) strain. Error bars represent the standard deviation of four independent mating experiments. A Student’s t-test showed significant differences between all strains (p-value < 0.02). Figure 3. View largeDownload slide Mating efficiency of mfaΔ a-type strains. Single and double mutants as well as the dic1–2Δ(a) strain were mated with the dic1–2Δ(α) strain. Error bars represent the standard deviation of four independent mating experiments. A Student’s t-test showed significant differences between all strains (p-value < 0.02). Expression of one MFA gene is sufficient for mating pathway activation Binding of mating pheromones to the pheromone surface receptors Ste2 and Ste3 (for α- and a-factor, respectively) activates a MAPK signaling cascade resulting in the activation of the genes necessary for shmoo formation and cell fusion (Jones and Bennett 2011; Merlini, Dudin and Martin 2013). The membrane protein Fus1 localizes at the shmoo tip and is required for efficient fusion of two mating cells. In S. cerevisiae, FUS1 expression is strongly induced by mating pheromones (McCaffrey et al. 1987; Trueheart, Boeke and Fink 1987). A similar pattern of regulation could be found in K. phaffii, where FUS1 (PP7435_Chr4–0336) was only expressed under mating conditions and significantly upregulated in the wild-type cultures containing cells of both mating types compared to the a- and α-type samples (log2 fold-changes under mating conditions: a-type/wild type –2.65, α-type/wild type –2.39). Fusion of the FUS1 promoter to the β-galactosidase or GFP gene has previously been used as a tool to study FUS1 regulation and the induction of the pheromone response pathway in other yeasts (Trueheart, Boeke and Fink 1987; Marcus et al. 1991; Fujimura 1992; Houser et al. 2012). To investigate the induction of mating in response to the different mating factors, the K. phaffii FUS1 promoter (Chr4:560823..560339 [FR839631.1]) was used to generate an eGFP reporter construct. This construct was integrated into the K. phaffii genome at the AOX1 terminator locus of the unmodified a- and α-type strain, resulting in the reporter strains dic1–2Δ(a) PFUS1_eGFP and dic1–2Δ(α) PFUS1_eGFP. To analyze the induction of the mating response in the reporter strains in the presence of the different mating-factor deletion strains, equal numbers of cells were mixed and cultivated under nitrogen and glucose limiting conditions, as shmoo formation and FUS1 expression were strongest under these conditions. After around 40 h, the fraction of fluorescent cells was measured by flow cytometry. Changes in cell morphology were assessed by microscopy. Flow cytometry analysis (Fig. 4A) showed no eGFP expression in the cultures of the reporter strains alone, confirming that nitrogen and glucose limitation alone is not sufficient for induction of FUS1 expression in haploid K. phaffii cells. Only after co-cultivation with cells of opposite mating type, an eGFP-positive population was observed. Mixing the a-type reporter strain with mfαΔ cells failed to induce eGFP expression, showing that the generated reporter strains are sensitive to the presence of mating pheromone. As expected from the mating experiments, the mfaΔ single mutants, secreting only one of the a-factor peptides, could still induce eGFP expression in the α-type reporter strain. No eGFP expression was observed when α-type reporter cultures were mixed with either the mfa1Δ mfa2Δ double mutant or the ste6–1Δ strain. Representative flow cytometry plots of cultures containing the mfaΔ strains are shown in Fig. 4B. Note that in the mixed cultures the theoretical maximum of eGFP-positive cells is 50%. Shmoo formation could only be observed in cultures containing the dic1–2Δ(α) PFUS1_eGFP reporter strain together with one of the mfaΔ(a) single mutants (Fig. 4C). Interestingly, no green cells forming shmoos could be observed with the mfa1Δ(a) strain, while mfa2Δ(a) cells were able to induce shmoo formation in dic1–2Δ(α) PFUS1_eGFP cells. Figure 4. View largeDownload slide Induction of the mating response by mating factor deletion strains. The different a- and α-factor deletion strains were co-cultivated with the respective dic1–2Δ PFUS1_eGFP reporter strain of the opposite mating type under nitrogen and glucose limiting conditions. (A) The proportion of eGFP-positive cells in a culture was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after co-cultivation with different mfaΔ strains. A distinct population of fluorescent cells was observed in all cultures with the mfaΔ single mutants, while the mfa1Δ mfa2Δ double mutant did not induce eGFP expression. (C) Microscopy pictures of the α-type reporter strain alone and after co-cultivation with the mfaΔ and ste6–1Δ strains. Only the mfaΔ single mutants were able to induce eGFP expression and a clear shmoo phenotype. Arrows indicate shmoo formation. Bar, 5 μm. Figure 4. View largeDownload slide Induction of the mating response by mating factor deletion strains. The different a- and α-factor deletion strains were co-cultivated with the respective dic1–2Δ PFUS1_eGFP reporter strain of the opposite mating type under nitrogen and glucose limiting conditions. (A) The proportion of eGFP-positive cells in a culture was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after co-cultivation with different mfaΔ strains. A distinct population of fluorescent cells was observed in all cultures with the mfaΔ single mutants, while the mfa1Δ mfa2Δ double mutant did not induce eGFP expression. (C) Microscopy pictures of the α-type reporter strain alone and after co-cultivation with the mfaΔ and ste6–1Δ strains. Only the mfaΔ single mutants were able to induce eGFP expression and a clear shmoo phenotype. Arrows indicate shmoo formation. Bar, 5 μm. Activation of the mating response by synthetic pheromones The same PFUS1_eGFP reporter assay was used to test the activity of synthetic mating pheromones. To ensure full activity, the proposed mature a-factor peptides were synthesized including the C-terminal methylation and farnesylation. The effect of synthetic K. phaffii α-factor was also investigated. The peptides were added to cultures of the dic1–2Δ PFUS1_eGFP a- or α-type strains after around 18 h in nitrogen and glucose limiting conditions. After another 24 h, the percentage of eGFP-positive cells was determined by flow cytometry. Changes in cell morphology were assessed by microscopy. Addition of either a-factor peptide to a final concentration of 10 μg/ml led to a clear induction of eGFP expression in around 30% of the cells of the PFUS1_eGFP reporter strain (Fig. 5A). Representative flow cytometry plots of cultures after a-factor addition are shown in Fig. 5B. The proportion of eGFP-positive cells increased only slightly when the peptide concentration was increased to 20 μg/ml and the addition of both a-factor peptides to the same culture did not result in a higher number of induced cells. At the same concentration, synthetic K. phaffii α-factor was able to induce the mating response in around 20% of the cells (Fig. 5A). No response could be observed when the S. cerevisiaea-factor peptide was used. Overall, the absolute portion of induced reporter cells after addition of the pheromones was about half compared to induction by pheromones secreted by cells of opposite mating type in the mixed cultures, which contained only 50% reporter cells. Synthetic a-factor 1 at a concentration of 10 μg/ml led to shmoo formation to a similar extent as in the control (Fig. 5C). This was also observed for a-type cells after the addition of α-factor. Addition of the same amount of a-factor 2 did not induce shmoo formation. Figure 5. View largeDownload slide Induction of the mating response by synthetic a- and α-factor (Mfa and Mfα). The synthetic peptides were added to cultures of the dic1–2Δ PFUS1_eGFP reporter strains cultivated under nitrogen and glucose limiting conditions. (A) The portion of eGFP-positive cells was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after addition of the a-factor peptides. Addition of the two peptides alone and in combination led to the formation of a distinct population of eGFP-positive cells. (C) Bright-field and fluorescence microscopy pictures of the α-type reporter strain alone and after co-cultivation with dic1–2Δ(a) cells or addition of synthetic a-factor. Clear shmoo formation was only observed in mixed cultures with dic1–2Δ(a) cells and after addition of a-factor 1. Synthetic α-factor induced shmoo formation in dic1–2Δ(a) PFUS1_eGFP cells. Bar, 5 μm. Figure 5. View largeDownload slide Induction of the mating response by synthetic a- and α-factor (Mfa and Mfα). The synthetic peptides were added to cultures of the dic1–2Δ PFUS1_eGFP reporter strains cultivated under nitrogen and glucose limiting conditions. (A) The portion of eGFP-positive cells was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after addition of the a-factor peptides. Addition of the two peptides alone and in combination led to the formation of a distinct population of eGFP-positive cells. (C) Bright-field and fluorescence microscopy pictures of the α-type reporter strain alone and after co-cultivation with dic1–2Δ(a) cells or addition of synthetic a-factor. Clear shmoo formation was only observed in mixed cultures with dic1–2Δ(a) cells and after addition of a-factor 1. Synthetic α-factor induced shmoo formation in dic1–2Δ(a) PFUS1_eGFP cells. Bar, 5 μm. Expression of MFA genes is induced by pheromone To confirm the transcriptional regulation of the mating-factor genes observed in the RNA sequencing data, samples from cultures during exponential growth in rich medium and after 24 h in mating medium were analyzed by quantitative PCR. To obtain mixed mating-type cultures, equal numbers of a- and α-type cells from the precultures were inoculated into mating medium. As expected, the transcript levels of MFA1, MFA2, MFα and the surface receptor genes STE2 and STE3 were very low in rich medium (YPD), with expression levels below 1% of the reference gene ACT1 (Fig. 6). MFA1 and MFA2 were both upregulated in a-type cells under mating conditions (Fig. 6A and B). The highest a-factor transcript levels were detected in the mixed cultures containing a- and α-type cells, indicating induction of MFA gene expression by α-factor. However, even in the mixed mating-type cultures, MFA1 transcript levels were around eight times higher than levels of MFA2. The expression of MFα was upregulated only in α-type cells under mating conditions (Fig. 6C). Interestingly, no further induction of gene expression was observed in the presence of a-type cells and transcript levels were around five times lower than the MFA1 levels in the same cultures. To emphasize the strong differences in expression levels between the three genes, a direct comparison of the mating-factor transcript levels in the different strains under mating conditions is shown in Fig. 6D. Transcript levels of the α-factor receptor STE2 were strongly upregulated in a-type cells under mating conditions (Fig. 6E). The absolute levels detected in the mixed cultures were similar to the a-type samples. However, as these cultures contained only 50% of a-type cells, this likely resulted from an additional 2-fold increase in transcript level per cell in the presence of α-factor. The same expression pattern was also observed for the a-factor receptor STE3 (Fig. 6F). Figure 6. View largeDownload slide Transcript levels of mating factor and pheromone surface receptor genes. Cultures of dic1–2Δ a- and α-type cells were analyzed during exponential growth in rich medium (YPD) and under mating condition. Mixed mating-type cultures consisted of an equal number of cells of both mating types grown under mating conditions. Transcript levels were normalized to ACT1 expression. Error bars represent the standard deviation of three biological replicates. Differences in gene expression between the cultures in mating medium were analyzed using a Student’s t-test. Significant differences with a p-value < 0.05 are indicated by an asterisk. (A) MFA1 was highly expressed in a-type cells under mating conditions. An additional increase in transcript level was measured in the mixed mating-type cultures. (B) Transcription of MFA2 was induced in a-type cells under mating conditions and further increased significantly in the mixed mating-type cultures. (C) Mating-type-specific expression of MFα under mating conditions. (D) Direct comparison of the mating-factor transcript levels in the different cultures in mating medium. (E) Expression levels of STE2 under mating conditions were similar in pure a-type and mixed cultures. (F) STE3 transcript levels are upregulated in α-type cells and mixed cultures under mating conditions. Figure 6. View largeDownload slide Transcript levels of mating factor and pheromone surface receptor genes. Cultures of dic1–2Δ a- and α-type cells were analyzed during exponential growth in rich medium (YPD) and under mating condition. Mixed mating-type cultures consisted of an equal number of cells of both mating types grown under mating conditions. Transcript levels were normalized to ACT1 expression. Error bars represent the standard deviation of three biological replicates. Differences in gene expression between the cultures in mating medium were analyzed using a Student’s t-test. Significant differences with a p-value < 0.05 are indicated by an asterisk. (A) MFA1 was highly expressed in a-type cells under mating conditions. An additional increase in transcript level was measured in the mixed mating-type cultures. (B) Transcription of MFA2 was induced in a-type cells under mating conditions and further increased significantly in the mixed mating-type cultures. (C) Mating-type-specific expression of MFα under mating conditions. (D) Direct comparison of the mating-factor transcript levels in the different cultures in mating medium. (E) Expression levels of STE2 under mating conditions were similar in pure a-type and mixed cultures. (F) STE3 transcript levels are upregulated in α-type cells and mixed cultures under mating conditions. DISCUSSION The preferentially haploid yeast K. phaffii can undergo mating and meiosis under nitrogen limitation conditions, and orthologs of many of the genes involved in the MAPK pathway leading to activation of the mating response in other yeasts are found in the genome. This includes the pheromone surface receptor genes STE2 and STE3 as well as the genes coding for α-factor and the conserved biogenesis pathway of the a-factor pheromone. However, no gene coding for the K. phaffiia-factor itself has been identified so far. Annotation of yeast a-factor genes by standard homology-based search is difficult because of their short sequence length and relatively poor sequence conservation. Furthermore, as the mating genes of K. phaffii are only expressed under nitrogen limitation conditions, relevant transcripts would not be detected in transcriptome analyses of strains under standard growth conditions (Valli et al. 2016). Starting from RNA sequencing data of previously generated heterothallic K. phaffii strains (Heistinger, Gasser and Mattanovich 2018) under mating conditions, we were able to identify two independent MFA genes. The systematic search for short ORFs expressed in a mating-type-specific manner and containing a C-terminal CAAX motif yielded MFA1. MFA2 was subsequently identified using MFA1 homology search. The MFA2 ORF is part of a predicted gene (CBS7435_Chr2–1002), probably wrongly annotated by automated search to code for a non-conserved hypothetical protein. It is likely that MFA2 was not considered during the first analysis because the average expression over the whole annotated gene was similar in both mating types. However, mating-type-specific expression could be observed in the short MFA2 region and was also confirmed by quantitative PCR. Both identified K. phaffii MFA sequences code for 40 amino acid long proteins with an overall sequence identity of 63% and contain the conserved C-terminal CAAX motif required for a-factor modification. The proposed N-terminal AxlI cleavage site after the asparagine at position 20 was predicted by comparison to other known a-factor sequences. The resulting mature peptides of 17 amino acids are slightly longer than the well-studied a-factor peptides of S. cerevisiae and the Sc. pombe M-factor, which have a confirmed length of 12 and 9 amino acids, respectively (Betz et al. 1987; Davey 1992). In our experiments, synthetic a-factor peptides with the predicted N-termini were able to activate the mating response pathway and to induce shmoo formation in α-type cells. The sequences of the N-terminal processing sites need to be confirmed by future experiments. Analysis of the mating phenotype of MFA mutants showed that only mfa1Δ mfa2Δ double deletion strains were unable to mate or induce the mating response pathway in α-type cells. MFA single mutants were still able to induce the mating response in a similar number of α-type cells as the unmodified a-type strain. They were also able to mate with α-type cells, although with lower efficiency. These results indicated that the two newly identified genes both encode a variant of the a-factor and that there are no further a-factor genes in the genome of K. phaffii. For both of the single mutants, the mating efficiency was reduced to below 10% of the mating efficiency of the unmodified a-type strain. This strong reduction in efficiency might be due to a synergistic effect of the two peptide variants. However, no such effect could be observed after simultaneous addition of both pheromones in comparison to addition of one of the synthetic peptides alone. Another possibility is that with only one of the genes being expressed, the total a-factor concentration might simply be too low for an efficient induction of the mating response. Saccharomyces cerevisiae mfaΔ single mutants were shown to mate with a similar efficiency as wild-type cells while secreting only about half the amount of a-factor (Michaelis and Herskowitz 1988). Similarly, the deletion of one of the three M-factor gene copies of Sc. pombe did not affect mating, even though pheromone production was found to be reduced. In M-factor double mutants, the mating efficiency was reduced to 25–30% compared to the wild type (Kjaerulff, Davey and Nielsen 1994). Deletion of the gene encoding the a-factor transporter Ste6 has been shown to prevent a-factor secretion and mating of S. cerevisiaea-type cells (Kuchler, Sterne and Thorner 1989). The same mating-type-specific non-mating phenotype was observed for K. phaffii ste6–1Δ strains, indicating that STE6–1 is the only gene coding for the a-factor transporter. The additionally annotated STE6–2 (PP7435_Chr1–1574) and STE6–3 (PP7435_Chr2–1257) genes most likely encode other closely related ABC transporters. As expected, the deletion of the single MFα gene abolished mating of α-type cells. In K. phaffii, the expression levels of mating specific genes are very low during growth in rich medium and increase only under nitrogen starvation conditions (Heistinger, Gasser and Mattanovich 2018). The same pattern of gene expression was also observed for the mating-factor genes, with transcript levels below 1% of the ACT1 control in rich medium and strong induction to up to 2500% of ACT1 expression for MFA1 under mating conditions. Furthermore, gene expression was found to be mating type specific, with the MFA genes being expressed only in a-type and MFα being expressed only in α-type cells. It is likely that the expression of the MFA and MFα genes is activated by Mata2 and Matα1, respectively, as it has been observed for the corresponding surface receptor genes, although this remains to be shown in further experiments (Heistinger, Gasser and Mattanovich 2018). Under mating conditions, transcript levels of MFA1 were up to 50 times higher than levels of MFA2. This difference in transcript levels was less pronounced in the mixed cultures containing both mating types where MFA1 levels were only around eight times higher. In these cultures, the presence of α-type cells led to an additional increase in the MFA1 and MFA2 transcript levels. An additional induction of pheromone expression in the presence of a pheromone of the opposite mating type has been described for several other yeasts like S. cerevisiae, Sc. pombe and C. albicans (Strazdis and MacKay 1983; Achstetter 1989; Imai and Yamamoto 1994; Kjaerulff, Davey and Nielsen 1994; Zhao et al. 2005; Dignard et al. 2007). It likely serves as a feedback mechanism to ensure high enough local pheromone levels to induce the mating process. Interestingly, in our experiments no additional induction of MFα expression was observed in mixed mating-type cultures. The fact that MFα levels are already strongly induced by nitrogen limitation and one transcript encodes nine copies of the mature α-factor could be a possible reason why further induction of α-factor signal might not be necessary for efficient mating. The measured expression levels of the corresponding pheromone receptor genes in the mixed cultures were equal to the levels obtained for samples containing only one mating type, indicating a 2-fold increase in receptor expression per cell in the presence of pheromone of the opposite mating type. In a homothallic K. phaffii wild-type culture containing a higher portion of α-type cells, a-factor receptor STE2 was expressed at lower levels than in a pure a-type culture while the transcript levels of the α-factor receptor STE3 were highest in the mixed cultures (Heistinger, Gasser and Mattanovich 2018 and RNA sequencing data). Addition of synthetic mating pheromones to cultures under mating conditions resulted in an induction of the mating response pathway in about 20–30% of the population, confirming that the peptides are able to act as mating pheromones. Interestingly, even after addition of cells of opposite mating type only around 60% of the reporter cells expressed eGFP. The missing induction of the mating response in a rather large portion of the population might be one of the reasons for the low mating efficiency of K. phaffii. Other indicators for the biological activity of mating pheromones are arrest of the cells in the G1 phase of the cell cycle and changes in cell morphology to the so-called shmoo phenotype (Merlini, Dudin and Martin 2013). Many of the K. phaffii mating genes are only activated under nitrogen starvation conditions in which hardly any mitotic growth is observed. Therefore, it is difficult to use cell cycle arrest to assess the response to pheromone. In Sc. pombe, deletion of the adenylate cyclase (CYR1) to mimic nitrogen starvation was used to induce mating gene expression in growing cultures, enabling the demonstration of G1 arrest in response to mating pheromone (Maeda, Mochizuki and Yamamoto 1990; Imai and Yamamoto 1994). It remains to be tested if this strategy could also be applied in K. phaffii. Under the conditions used, shmoo formation could always be observed in mixed mating-type cultures of the unmodified strains. It was also induced by synthetic α-factor and, to a lesser extent, by a-factor 1. However, no shmoo formation could be observed after the addition of a-factor 2 or in fluorescent cells of the α-type reporter strain in cultures with the mfa1Δ strain. It is unclear what causes this difference in the response to the a-factor peptides. Actual shmoo formation probably requires higher concentrations of a-factor than the activation of the FUS1 promoter, which was similar for both a-factor peptides and only slightly higher with the mfa2Δ strain than in the cultures with the mfa1Δ strain. As MFA2 expression levels are lower, the amounts of active a-factor secreted by mfa1Δ cells could be just too low to induce the shmoo phenotype. It is possible that local areas of increased pheromone concentration close to actively secreting cells are responsible for the more efficient induction in mixed mating-type cultures, although cultures were well mixed and no cell aggregation was observed. It has been described for S. cerevisiae that shmoo formation after addition of a-factor was less pronounced than for α-factor (Betz, MacKay and Duntze 1977). Another study with S. cerevisiae found that although lower concentrations of synthetic a-factor than α-factor were necessary to induce G1 arrest, only little shmoo formation was observed at concentrations where α-factor could efficiently induce shmoo formation (O’Reilly et al. 2012). Generally, concentrations of pheromones required to induce a response in K. phaffii were a little higher than described for S. cerevisiae. At least 10 μg/ml synthetic pheromone was necessary to achieve mating pathway activation in more than 10% of the cells. In comparison, similar concentrations of 5–10 μg/ml synthetic α-factor can be used to induce G1 arrest in S. cerevisiae cultures, leading to a shmoo phenotype in >95% of the cells (Breeden 1997; Wu, Liu and Huang 2011). For S. cerevisiaea-factor, growth inhibition and shmoo formation were observed with as little as 40–200 ng/ml of purified or synthetic pheromone (Anderegg et al. 1988; Xue et al. 1989; Marcus et al. 1991; O’Reilly et al. 2012). The need for high local pheromone concentrations might be a reason why K. phaffii requires rather high cell densities for efficient mating. In S. cerevisiae, the barrier proteases Bar1 and Afb1 produced by a- and α-type cells, respectively, are required for fast recovery from the response to pheromone (Sprague and Herskowitz 1981; MacKay et al. 1988; Huberman and Murray 2013). In K. phaffii, a potential protease showing a-type specific expression under mating conditions is the aspartyl protease Yps1–5 (PP7435_Chr3–0313). Interestingly, no α-specific genes coding for a potential a-factor degrading enzyme could be found in our dataset. Within this study we identified the two MFA genes of K. phaffii. Both a-factor variants contribute to efficient mating of a-type cells and show a mating-type-specific expression pattern under nitrogen-limiting conditions. Furthermore, we could confirm that the α-factor is encoded by a single MFα gene which is essential for α-cell mating. Expression level analysis indicated an additional induction of a-factor and surface receptor gene expression upon pheromone binding. It remains to be investigated why this additional layer of activation could not be detected for the MFα gene. Using synthetic peptides we could demonstrate the biological role of the identified sequences in mating pathway activation. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. Acknowledgements The authors thank Petra Cazzanelli and Stefan Ergoth for their practical support during strain generation and the mating assays, and Verena Puxbaum for her help with microscopy and image processing. We also want to thank Prof. Jürgen Wendland, Vrije Universiteit Brussel, for valuable discussion. FUNDING This work was supported by the Christian Doppler Research Association (Christian Doppler Laboratory for Innovative Immunotherapeutics), Merck KGaA, as well as the Austrian Science Fund (FWF W1224—Doctoral Program on Biomolecular Technology of Proteins—BioToP). JM, NET, and MV were supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and Business Agency Vienna through the COMET Funding Program managed by the Austrian Research Promotion Agency FFG. Conflicts of interest. None declared. REFERENCES Achstetter T. Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes. Mol Cell Biol 1989 ; 9 : 4507 – 14 . Google Scholar CrossRef Search ADS PubMed Anderegg RJ , Betz R , Carr SA et al. Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component . J Biol Chem 1988 ; 263 : 18236 – 40 . Google Scholar PubMed Betz R , Crabb JW , Meyer HE et al. Amino acid sequences of a-factor mating peptides from Saccharomyces cerevisiae . J Biol Chem 1987 ; 262 : 546 – 8 . Google Scholar PubMed Betz R , MacKay VL , Duntze W . a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a . J Bacteriol 1977 ; 132 : 462 – 72 . Google Scholar PubMed Babraham Bioinformatics . Trim Galore , 2016 . http://www.bioinformatics.babraham.ac.uk/projects/trim_galore (2 May 2018, date last accessed) . Bray N , Pimentel H , Melsted P et al. Near-Optimal RNA-Seq Quantification . 2015 . https://arxiv.org/abs/1505.02710 (2 May 2018, date last accessed) . Bray NL , Pimentel H , Melsted P et al. Near-optimal probabilistic RNA-seq quantification . Nat Biotechnol 2016 ; 34 : 525 – 7 . Google Scholar CrossRef Search ADS PubMed Breeden LL . α-factor synchronization of budding yeast . Methods Enzymol 1997 ; 283 : 332 – 41 . Google Scholar CrossRef Search ADS PubMed Davey J. Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone . EMBO J 1992 ; 11 : 951 – 60 . Google Scholar PubMed Dignard D , El-Naggar AL , Logue ME et al. Identification and characterization of MFA1, the gene encoding Candida albicans a-factor pheromone . Eukaryot Cell 2007 ; 6 : 487 – 94 . Google Scholar CrossRef Search ADS PubMed Edgar R , Domrachev M , Lash AE . Gene Expression Omnibus: NCBI gene expression and hybridization array data repository . Nucleic Acids Res 2002 ; 30 : 207 – 10 . Google Scholar CrossRef Search ADS PubMed Fairhead C , Llorente B , Denis F et al. New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination . Yeast 1996 ; 12 : 1439 – 57 . Google Scholar CrossRef Search ADS PubMed Fujimura H . Molecular monitoring of the transcriptional activation of the yeast Saccharomyces kluyveri mating pheromone signal transduction by using FUS1-lacZ fusion gene . FEMS Microbiol Lett 1992 ; 73 : 23 – 6 . Google Scholar CrossRef Search ADS PubMed Gasser B , Prielhofer R , Marx H et al. Pichia pastoris : protein production host and model organism for biomedical research . Future Microbiol 2013 ; 8 : 191 – 208 . Google Scholar CrossRef Search ADS PubMed Gentleman RC , Carey VJ , Bates DM et al. Bioconductor: open software development for computational biology and bioinformatics . Genome Biol 2004 ; 5 : R80 . Google Scholar CrossRef Search ADS PubMed Hanson SJ , Byrne KP , Wolfe KH . Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system . Proc Natl Acad Sci USA 2014 ; 111 : E4851 – 8 . Google Scholar CrossRef Search ADS PubMed Heistinger L , Gasser B , Mattanovich D . Creation of stable heterothallic strains of Komagataella phaffii enables dissection of mating gene regulation . Mol Cell Biol 2018 ; 38 : e00398 – 17 . Google Scholar PubMed Houser JR , Ford E , Chatterjea SM et al. An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae . Yeast 2012 ; 29 : 519 – 30 . Google Scholar CrossRef Search ADS PubMed Huber W , Carey VJ , Gentleman R et al. Orchestrating high-throughput genomic analysis with Bioconductor . Nat Methods 2015 ; 12 : 115 – 21 . Google Scholar CrossRef Search ADS PubMed Huberman LB , Murray AW . Genetically engineered transvestites reveal novel mating genes in budding yeast . Genetics 2013 ; 195 : 1277 – 90 . Google Scholar CrossRef Search ADS PubMed Imai Y , Yamamoto M . The fission yeast mating pheromone P-factor: its molecular structure, gene structure, and ability to induce gene expression and G1 arrest in the mating partner. Genes Dev 1994 ; 8 : 328 – 38 . Google Scholar CrossRef Search ADS PubMed Jones SK Jr , Bennett RJ . Fungal mating pheromones: choreographing the dating game . Fungal Genet Biol 2011 ; 48 : 668 – 76 . Google Scholar CrossRef Search ADS PubMed Kim D , Pertea G , Trapnell C et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions . Genome Biol 2013 ; 14 : R36 . Google Scholar CrossRef Search ADS PubMed Kjaerulff S , Davey J , Nielsen O . Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3. , Mol Cell Biol 1994 ; 14 : 3895 – 905 . Google Scholar CrossRef Search ADS PubMed Kuberl A , Schneider J , Thallinger GG et al. High-quality genome sequence of Pichia pastoris CBS7435 . J Biotechnol 2011 ; 154 : 312 – 20 . Google Scholar CrossRef Search ADS PubMed Kuchler K , Sterne RE , Thorner J . Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells . EMBO J 1989 ; 8 : 3973 – 84 . Google Scholar PubMed Kurjan J , Herskowitz I . Structure of a yeast pheromone gene (MFα): a putative α-factor precursor contains four tandem copies of mature α-factor . Cell 1982 ; 30 : 933 – 43 . Google Scholar CrossRef Search ADS PubMed Love KR , Shah KA , Whittaker CA et al. Comparative genomics and transcriptomics of Pichia pastoris . BMC Genomics 2016 ; 17 : 550 . Google Scholar CrossRef Search ADS PubMed Love MI , Anders S , Huber W . DESeq2: Differential gene expression analysis based on the negative binomial distribution . 2017 . https://rdrr.io/bioc/DESeq2 (8 May 2018, date last accessed). Love MI , Anders S , Kim V et al. RNA-Seq workflow: gene-level exploratory analysis and differential expression . F1000Res 2015 ; 4 : 1070 . Google Scholar CrossRef Search ADS PubMed McCaffrey G , Clay FJ , Kelsay K et al. Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae. Mol Cell Biol 1987 ; 7 : 2680 – 90 . Google Scholar CrossRef Search ADS PubMed MacKay VL , Welch SK , Insley MY et al. The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. Proc Natl Acad Sci USA 1988 ; 85 : 55 – 9 . Google Scholar CrossRef Search ADS PubMed Maeda T , Mochizuki N , Yamamoto M . Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe . Proc Natl Acad Sci USA 1990 ; 87 : 7814 – 8 . Google Scholar CrossRef Search ADS PubMed Marcus S , Caldwell GA , Miller D et al. Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. Mol Cell Biol 1991 ; 11 : 3603 – 12 . Google Scholar CrossRef Search ADS PubMed Merlini L , Dudin O , Martin SG . Mate and fuse: how yeast cells do it . Open Biol 2013 ; 3 : 130008 . Google Scholar CrossRef Search ADS PubMed Michaelis S , Barrowman J . Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease . Microbiol Mol Biol R 2012 ; 76 : 626 – 51 . Google Scholar CrossRef Search ADS Michaelis S , Herskowitz I . The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol 1988 ; 8 : 1309 – 18 . Google Scholar CrossRef Search ADS PubMed O’Reilly N , Charbin A , Lopez-Serra L et al. Facile synthesis of budding yeast a-factor and its use to synchronize cells of α mating type . Yeast 2012 ; 29 : 233 – 40 . Google Scholar CrossRef Search ADS PubMed OhEigeartaigh SS , Armisen D , Byrne KP et al. Systematic discovery of unannotated genes in 11 yeast species using a database of orthologous genomic segments . BMC Genomics 2011 ; 12 : 377 . Google Scholar CrossRef Search ADS PubMed Ongay-Larios L , Navarro-Olmos R , Kawasaki L et al. Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to α-factor . FEMS Yeast Res 2007 ; 7 : 740 – 7 . Google Scholar CrossRef Search ADS PubMed Pertea M , Kim D , Pertea GM et al. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown . Nat Protoc 2016 ; 11 : 1650 – 67 . Google Scholar CrossRef Search ADS PubMed Prielhofer R , Barrero JJ , Steuer S et al. GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris . BMC Syst Biol 2017 ; 11 : 123 . Google Scholar CrossRef Search ADS PubMed R Development Core Team . R: A Language and Environment for Statistical Computing . Vienna, Austria : R Foundation for Statistical Computing . 2016 . Sarkari P , Marx H , Blumhoff ML et al. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger . Bioresour Technol 2017 ; 245 : 1327 – 33 . Google Scholar CrossRef Search ADS PubMed Seike T , Nakamura T , Shimoda C . Molecular coevolution of a sex pheromone and its receptor triggers reproductive isolation in Schizosaccharomyces pombe . Proc Natl Acad Sci USA 2015 ; 112 : 4405 – 10 . Google Scholar CrossRef Search ADS PubMed Sievers F , Wilm A , Dineen D et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega . Mol Syst Biol 2014 ; 7 : 539 . Google Scholar CrossRef Search ADS Singh A , Chen EY , Lugovoy JM et al. Saccharomyces cerevisiae contains two discrete genes coding for the α-factor pheromone . Nucleic Acids Res 1983 ; 11 : 4049 – 63 . Google Scholar CrossRef Search ADS PubMed Skinner ME , Uzilov AV , Stein LD et al. JBrowse: a next-generation genome browser . Genome Res 2009 ; 19 : 1630 – 8 . Google Scholar CrossRef Search ADS PubMed Soneson C , Love MI , Robinson MD . Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences . F1000Res 2015 ; 4 : 1521 . Google Scholar CrossRef Search ADS PubMed Sprague GF Jr , Herskowitz I . Control of yeast cell type by the mating type locus . J Mol Biol 1981 ; 153 : 305 – 21 . Google Scholar CrossRef Search ADS PubMed Stadlmayr G , Mecklenbrauker A , Rothmuller M et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production . J Biotechnol 2010 ; 150 : 519 – 29 . Google Scholar CrossRef Search ADS PubMed Strazdis JR , MacKay VL . Induction of yeast mating pheromone a-factor by α cells . Nature 1983 ; 305 : 543 – 5 . Google Scholar CrossRef Search ADS PubMed Tolstorukov I , Cregg JM . Classical Genetics . In: Cregg JM (ed.) Pichia Protocols: Methods in Molecular Biology , 389 : 2nd edn . Totowa, NJ : Humana Press , 2007 , 189 – 201 . Trueheart J , Boeke JD , Fink GR . Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol 1987 ; 7 : 2316 – 28 . Google Scholar CrossRef Search ADS PubMed Valli M , Tatto NE , Peymann A et al. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function . FEMS Yeast Res 2016 ; 16 : fow051 . Google Scholar CrossRef Search ADS PubMed Weber E , Engler C , Gruetzner R et al. A modular cloning system for standardized assembly of multigene constructs . PLoS One 2011 ; 6 : e16765 . Google Scholar CrossRef Search ADS PubMed Wendland J , Dunkler A , Walther A . Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii . FEMS Yeast Res 2011 ; 11 : 418 – 29 . Google Scholar CrossRef Search ADS PubMed Wickham H , Hester J , Francois R . readr: Read Rectangular Text Data . 2017 . https://CRAN.R-687 project.org/package = readr (2 May 2018, date last accessed) . Wu X , Liu L , Huang M . Analysis of changes in protein level and subcellular localization during cell cycle progression using the budding yeast Saccharomyces cerevisiae . Methods Mol Biol 2011 ; 782 : 47 – 57 . Google Scholar CrossRef Search ADS PubMed Xue CB , Caldwell GA , Becker JM et al. Total synthesis of the lipopeptide a-mating factor of Saccharomyces cerevisiae . Biochem Bioph Res Co 1989 ; 162 : 253 – 7 . Google Scholar CrossRef Search ADS Zhao R , Daniels KJ , Lockhart SR et al. Unique aspects of gene expression during Candida albicans mating and possible G1 dependency . Eukaryot Cell 2005 ; 4 : 1175 – 90 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Yeast Research Oxford University Press

Identification and characterization of the Komagataella phaffii mating pheromone genes

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Abstract

Abstract The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is a haploid yeast that is able to form diploid cells by mating once nitrogen becomes limiting. Activation of the mating response requires the secretion of a- and α-factor pheromones, which bind to G-protein coupled receptors on cells of opposite mating type. In K. phaffii, the genes coding for the α-factor (MFα), the pheromone surface receptors and the conserved a-factor biogenesis pathway have been annotated previously. Initial homology-based search failed to identify potential a-factor genes (MFA). By using transcriptome data of heterothallic strains under mating conditions, we found two K. phaffiia-factor genes. Deletion of both MFA genes prevented mating of a-type cells. MFA single mutants were still able to mate and activate the mating response pathway in α-type cells. A reporter assay was used to confirm the biological activity of synthetic a- and α-factor peptides. The identification of the a-factor genes enabled the first characterization of the role and regulation of the mating pheromone genes and the response of K. phaffii to synthetic pheromones and will help to gain a better understanding of the mating behavior of K. phaffii. Komagataella phaffii, Pichia pastoris, yeast, mating, mating factor, pheromone INTRODUCTION Yeast mating is mediated by secreted peptide pheromones binding to surface receptors on cells of the opposite mating type. In ascomycetes, one cell type secretes unmodified hydrophilic peptides (known as α-factor), whereas cells of the other mating type produce hydrophobic peptides with a characteristic C-terminal farnesyl and carboxylmethyl modification (a-factor). Secretion of these small peptides is essential for mating partner recognition and activation of the genes required for diploid cell formation. The different aspects of the mating process are well understood in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe but are much less characterized in other relevant yeast species. The S. cerevisiae α-factor pheromone is an unmodified hydrophilic peptide of 13 amino acids secreted exclusively by haploid cells of the α-mating type. It is encoded by MFα1 and MFα2 and translated as precursor containing four and two copies of the mature α-factor, respectively (Kurjan and Herskowitz 1982; Singh et al. 1983). In S. cerevisiaea-type cells, the MFA1 and MFA2 genes are constitutively transcribed and translated into precursor proteins of 36 and 38 amino acids, respectively (Michaelis and Herskowitz 1988). While the α-factor is processed and secreted via the classical secretory pathway, the a-factor is modified in the cytosol. Following translation, the C-terminal CAAX motif (C is cysteine, A is usually aliphatic, X can be any amino acid) is recognized by a cytosolic farnesyl transferase consisting of two subunits (Ram1 and Ram2) resulting in farnesylation of the C-terminal cysteine residue. Subsequently, the three C-terminal amino acids (AAX) are cleaved by the proteases Rce1 or Ste24 and a carboxylmethyl group is added to the farnesylcystein by the isoprenyl cysteine carboxyl methyltransferase Ste14. The N-terminus of a-factor is processed in two consecutive proteolytic cleavage steps by zinc proteases Ste24 and Axl1, resulting in mature a-factor peptides with a length of only 12 amino acids differing solely in a single residue (Betz et al. 1987; Michaelis and Barrowman 2012). Mature a-factor is exported from the cytosol by Ste6, an ATP-binding cassette (ABC) transporter located in the plasma membrane (Kuchler, Sterne and Thorner 1989). Once secreted, a-factor binds to the G-protein coupled receptor Ste3 on the surface of α-type cells, activating a mitogen-activated protein kinase (MAPK) signaling cascade resulting in the activation of genes required for mating, growth of mating projections (shmoos) and arrest in the G1 phase of the cell cycle (Jones and Bennett 2011; Merlini, Dudin and Martin 2013). The enzymes of the a-factor biogenesis pathway are conserved in yeasts and higher eukaryotes. However, due to their short length and poor sequence conservation, the genes encoding a-factor pheromones are hard to identify. Besides the well-characterized S. cerevisiae mating factors, a-factor pheromones have been investigated in a number of other yeast species like Sc. pombe (M-factor), Kluyveromyces lactis and Candida albicans (Davey 1992; Kjaerulff, Davey and Nielsen 1994; Dignard et al. 2007; Ongay-Larios et al. 2007). All the identified proteins contain a C-terminal CAAX motif for farnesylation and were found to be essential for mating of a-type cells. The number of genes encoding the a-factor varies in different species, from only one gene copy in C. albicans or Eremothecium gossypii to up to 12 predicted MFA genes in C. tropicalis (Dignard et al. 2007; Wendland, Dunkler and Walther 2011). It has been hypothesized that the redundancy in the MFA genes might be necessary to allow evolution of pheromones required for the development of new reproductive groups while ensuring mating within a species (Seike, Nakamura and Shimoda 2015). The methylotrophic yeast Komagataella phaffii (also referred to as Pichia pastoris) is a haploid yeast that is able to form diploids by mating if nitrogen becomes limiting. Wild-type strains are homothallic and can undergo mating-type switching by inversion of the chromosomal region between two mating-type (MAT) loci located on chromosome 4. While the expression of the MAT genes at MAT locus 1 is downregulated by telomere position effects, the mating type of a cell is determined by the MAT genes actively transcribed from locus 2. Both MAT loci are flanked by large inverted repeat regions which have been proposed as homologous regions involved in mating-type switching (Hanson, Byrne and Wolfe 2014). Deletion of the ‘outer’ repeat region at MAT locus 2 (including DIC1–2) prevents mating-type switching and allowed the generation of K. phaffii strains with stable mating types (Heistinger, Gasser and Mattanovich 2018). The K. phaffii α-factor is encoded by a single MFα gene (CBS7435_Chr2–0522) and is translated as a precursor peptide consisting of a pre-pro leader sequence and nine copies of the mature α-factor flanked by Kex2 and Ste13 processing sites (Kuberl et al. 2011). Experiments using the pre-pro leader sequence have shown that it can be used for efficient secretion of recombinant proteins (Kuberl et al. 2011) but no further studies characterizing the K. phaffii α-factor peptide itself have been conducted. Orthologs of α-and a-factor receptor genes (STE2 and STE3, respectively) as well as all the genes essential for a-factor processing and secretion in other yeasts can be found in the K. phaffii genome using homology-based search (Kuberl et al. 2011; Valli et al. 2016). However, so far no gene coding for the a-factor pheromone has been identified. Within this study we could identify two putative MFA genes in the genomes of K. phaffii and K. pastoris by using a combination of transcriptomics data analysis and homology search. The role of the newly identified peptides in K. phaffii mating was confirmed using MFA deletion mutants and fully modified synthetic pheromones. Furthermore, we analyzed the mating phenotype of mfαΔ mutants and the response of K. phaffii cultures to synthetic α-factor. MATERIALS AND METHODS Yeast strains and vectors All K. phaffii strains described in this study (Table 1) are based on the homothallic CBS2612 wild-type strain. Generation of the dic1–2Δ strains with stable mating types was described in detail in Heistinger, Gasser and Mattanovich (2018). To obtain the mating-factor deletion strains, the whole coding sequence of the target gene was replaced by an antibiotic resistance marker using the split-marker technique (Fairhead et al. 1996; Gasser et al. 2013). Homologous flanking regions of around 1000 bp for integration were PCR amplified from K. phaffii genomic DNA with primers containing BsaI restriction sites and fusion sites (FS) for Golden Gate cloning (FS AB and CD for the 5΄ and 3΄ region, respectively) using the previously published GoldenPiCS system (Weber et al. 2011; Prielhofer et al. 2017; Sarkari et al. 2017). Marker sequences were amplified from available plasmid vectors to introduce FS BC. In the next step, a vector (BB3) containing the marker gene flanked by the two homologous regions was assembled by Golden Gate cloning using BsaI. This vector was used as template for PCR-based amplification of the two knockout cassettes overlapping in the marker gene sequence. For the construction of the PFUS1_eGFP reporter construct, the sequence upstream of the FUS1 start codon (Chr4:560823..560339 [FR839631.1]) was amplified from genomic DNA of K. phaffii CBS2612 using primers containing FS AB and BsaI restriction sites for cloning into a BB1 GoldenPiCS vector. In the next step, the expression cassette consisting of the enhanced green fluorescent protein (eGFP) coding sequence under control of the FUS1 promoter and S. cerevisiae CYC1 terminator was assembled from the available BB1 constructs into a BB3 vector using BpiI. The final plasmid also contained a NatMX resistance cassette for selection with nourseothricin and the AOX1 terminator sequence as homologous region for genomic integration after linearization with AscI. Empty pPUZZLE vectors were used for the integration of additional marker genes required for the selection of diploid cells into the TDH3 promoter or AOX1 terminator region, respectively (Stadlmayr et al. 2010). All primers used in this study are listed in Table S1 (Supporting Information). Table 1. List of strains used in this study. Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study aThe dic1–2Δ(a) strain contains one loxP sequence between SLA2–2 and MATα1 in MAT locus 1 and another loxP sequence in MAT locus 2 between and SLA2–1 and MATa1. View Large Table 1. List of strains used in this study. Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study Strain name Genotype Mating type Source CBS2612 Wild type mixed CBS2612 dic1–2Δ(α) dic1–2Δ::loxP MATα Heistinger, Gasser and Mattanovich (2018) CBS2612 dic1–2Δ(a) dic1–2Δ::loxP, MATα-loxP a MATa Heistinger, Gasser and Mattanovich (2018) CBS2612 mfa1Δ mfa1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa2Δ mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfa1Δ mfa2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP mixed This study CBS2612 mfαΔ mfαΔ:: loxP-kanMX-loxP mixed This study CBS2612 ste6–1Δ ste6–1Δ::loxP-natMX-loxP mixed This study CBS2612 mfa1Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP MATa This study CBS2612 mfa2Δ(a) dic1–2Δ mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 mfa1Δ mfa2Δ(a) dic1–2Δ mfa1Δ::loxP-natMX-loxP mfa2Δ::loxP-ZeoR-loxP MATa This study CBS2612 ste6–1Δ(a) ste6–1Δ::loxP-natMX-loxP MATa This study CBS2612 dic1–2Δ(α) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATα This study CBS2612 dic1–2Δ(a) PFUS1_eGFP dic1–2Δ 5΄-AOX1tt-PFUS1-eGFP-ScCYC1tt-natMX-AOX1tt-3΄ MATa This study aThe dic1–2Δ(a) strain contains one loxP sequence between SLA2–2 and MATα1 in MAT locus 1 and another loxP sequence in MAT locus 2 between and SLA2–1 and MATa1. View Large Cultivation conditions Yeast cells were grown in standard YP medium (yeast extract 10 g/l, soy peptone 20 g/l) containing 2% glucose as carbon source. All liquid cultures were grown at 25°C. After transformation, cells were plated on YPD agar (2% agar-agar) supplemented with the appropriate antibiotics (zeocin 50 μg/ml, geneticin 500 μg/ml, nourseothricin 100 μg/ml). Increased antibiotic concentrations of up to 100 μg/ml zeocin and 1 mg/ml geneticin were used for the selection of diploid cells on mating plates. Mating conditions were defined as cultivation in nitrogen-free acetate medium (mating medium, 0.5% sodium acetate, 1% potassium chloride, 1% glucose). RNA extraction Cultivations and sampling for RNA extractions were performed as described in Heistinger, Gasser and Mattanovich (2018). Three colonies of each strain were cultivated in parallel. RNA was isolated according to the TRI reagent (Sigma-Aldrich) protocol. Residual genomic DNA was digested using the Ambion DNA- free kit (Invitrogen), and RNA was precipitated overnight (1/10 volume 3M sodium acetate, 2.5 volumes absolute ethanol, 1 μl glycogen) at –20°C to remove traces of phenol. RNA concentrations and integrity were analyzed using a Nanodrop spectrophotometer and a Bioanalyzer (Agilent) using the RNA 6000 Nano Kit. RNA sequencing data analysis Solexa Sequencing of poly(A)-enriched cDNA libraries was performed at the VBCF NGS Unit (www.vbcf.ac.at). The unsorted, unaligned BAM files were converted and split into paired-end FASTQ files with the TopHat2 tool bam2fastx v2.1.0 (Kim et al. 2013) and subsequently trimmed and quality checked using Trim-Galore v0.4.2 (Babraham Bioinformatics 2016) using default parameters. As preparation for the count quantification, a transcript index was created using the latest K. phaffii CBS7435 annotation (FR839628.1, FR839629.1, FR839630.1, FR839631.1, FR839632.1; Valli et al. 2016) and the kallisto v0.43.0 subprogram index (Bray et al. 2016). Subsequently, the read count for each sample was determined using kallisto subprogram quant (Bray et al. 2015). For differential expression analysis (Love et al. 2015) with R (R Development Core Team 2016), the packages tximport and tximportData (Soneson, Love and Robinson 2015), readr (Wickham et al. 2017) and DESeq2 (Gentleman et al. 2004; Huber et al. 2015; Love, Anders and Huber 2017) were used for each comparison. Visualization of the expression analysis results was also carried out with R. Transcripts with a log2 fold-change of below –1 and above 1 combined with an adjusted p-value below 0.05 were considered to be significantly regulated. For the detection of new, so far unannotated transcripts, the RNA sequencing data were analyzed with the HISAT-StringTie-Ballgown suite (Pertea et al. 2016). Coverage histograms were visualized with JBrowse (Skinner et al. 2009). The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (Edgar, Domrachev and Lash 2002) and are accessible through GEO Series accession number GSE111973. Quantitative PCR For quantitative PCR, RNA samples were treated with the Ambion DNA-free kit (Invitrogen) and RNA integrity was analyzed by agarose gel electrophoresis. All cDNA was synthesized using oligo(dT)23 primers (NEB) and the Biozym cDNA synthesis kit. PCR reactions were performed using the Sensi Mix SYBR Hi-Rox kit (Bioline) on a Rotor-Gene Q instrument (Qiagen). Purified PCR products of the analyzed genes were used to generate standard curves for quantification. Transcript levels were normalized to ACT1 (PP7435_Chr3–0993) expression. Mating protocols Qualitative mating experiments were performed similarly to Tolstorukov and Cregg (2007). Strains to be mated were streaked out in parallel on YPD agar plates directly from glycerol stocks and incubated at 30°C. After approximately 24 h, cells were replicated twice at an angle of 90°C onto mating agar (0.5% sodium acetate, 1% potassium chloride, 1% glucose, 2% agar) and incubated at 25°C for 3 days. Diploid cells were selected by replica plating onto YPD agar containing the appropriate antibiotics. Cell growth at crossing points was evaluated after incubation at 30°C for 3 days. For the semiquantitative mating assay, fresh colonies grown on YPD agar were used to inoculate non-selective YPD medium. After 18–20 h, approximately 6.5 × 107 cells of each strain were mixed, plated on mating agar plates and incubated at 25°C for 3 days. Cell numbers were estimated by measuring the optical density at 600 nm (OD600), with an OD600 of 1 corresponding to 1 × 107 cells. Mated cells were washed from the plates with phosphate buffered saline (PBS) (KH2PO4 0.24 g/l, Na2HPO4*2 H2O 1.8 g/l, KCl 0.2 g/l, NaCl 8 g/l) and appropriate dilutions were plated on YPD agar plates containing either one or both of the antibiotics used for the selection of diploid cells. Colonies were counted after incubation at 30°C for 3 days. The mating efficiency was calculated as the percentage of cells growing on the plates containing both antibiotics in relation to the number of cells growing in the presence of one of the antibiotics (using the cell count on the antibiotic plates with the lower number of cells). Synthetic pheromones The K. phaffiia- and α-factor peptides were synthesized by Thermo Fisher Scientific with a purity >95%. Both a-factor peptides were obtained with the C-terminal farnesyl and carboxymethyl modifications. Aliquots were dissolved in dimethyl sulfoxide and diluted to working concentrations with sterile H2O + 0.5 mg/ml BSA. The α-factor peptide was dissolved directly in sterile H2O + 0.5 mg/ml BSA. Saccharomyces cerevisiaea-factor was purchased from ZYMO Research. All synthetic peptides were added to the cultures at final concentrations of 10–20 μg/ml. Enhanced GFP reporter assays For assays with the PFUS1_eGFP reporter strains, precultures were grown in 24-well deep well plates in YP medium without glucose. After 24 h, cells were harvested and resuspended in yeast nitrogen base (YNB) medium (YNB without amino acids and ammonium sulfate 3.4 g/l, potassium phosphate buffer 0.1M, pH 6). For the main cultures, 2 ml YNB medium supplemented with two glucose release feed beads (6 mm diameter, Kuhner AG, glucose release rate: 0.79*time0.66 = mg glucose released per feed bead, theoretical glucose concentration without consumption after 40 h: 9 g/l) were inoculated at a starting OD600 of 2. To test the induction of the FUS1 promoter by a second strain of opposite mating type, equal numbers of cells from the separate precultures were mixed to reach the same starting OD600. Synthetic pheromones and solvent controls were added around 18 h after the start of the main culture. After 40 h in YNB medium, cells were analyzed by flow cytometry and microscopy. Flow cytometry For flow cytometry cells were diluted in PBS (KH2PO4 0.24 g/l, Na2HPO4*2 H2O 1.8 g/l, KCl 0.2 g/l, NaCl 8 g/l) and green fluorescence (FL1 channel, 505–545 nm), forward and side scatter of 20 000 cells per sample were measured on a Gallios Flow Cytometer (Beckman Coulter). Data were analyzed with the Kaluza analysis software (Beckman Coulter). GFP-positive cells were gated using the dic1–2Δ PFUS1_eGFP α-type strain in the absence of pheromone as reference. Microscopy Microscopy was performed with a Zeiss Axio Observer.Z1/7 microscope using a LCI Plan-Neofluar 63x/1.3 water immersion objective. Cell morphology was analyzed in bright-field mode. For fluorescence microscopy of eGFP-expressing cells, the 38 HE eGFP shift free (E) filter set (Zeiss) was used. Pictures were processed with the Zen 2.3 lite (blue edition) software (Carl Zeiss Microscopy GmbH). RESULTS Identification of two putative MFA sequences Due to their short coding sequences and poor sequence conservation, genes encoding a-factor mating pheromones are hard to identify by homology search (Dignard et al. 2007; OhEigeartaigh et al. 2011). To annotate possible MFA genes in the genome of K. phaffii, we used RNA sequencing data of K. phaffii CBS2612 (the homothallic wild-type strain) as well as of stable a- and α-type strains derived from it (Heistinger, Gasser and Mattanovich 2018). Sequencing reads of all strains grown in rich medium and under mating conditions were mapped to the available genome sequence of the CBS7435 strain (Kuberl et al. 2011; Valli et al. 2016). Note that no full genome sequence of the K. phaffii CBS2612 type strain is available at the moment. However, the sequences of all relevant regions investigated in this study were verified to be identical to the CBS7435 sequence. All so far not annotated open reading frames (ORFs) shorter than 200 bp that were expressed only by a-type or wild-type cells under mating conditions were analyzed. By this method, one candidate gene (MFA1) with an ORF of 123 bp (Chr1:1587540..1587662 in CBS7435 [FR839628.1]), encoding a 40 amino acid protein containing the conserved C-terminal CAAX motif, was identified. Using this sequence, a second potential a-factor gene was identified by homology search (tBLASTn). Like MFA1, the MFA2 ORF (Chr2:1840284..1840406 in CBS7435 [FR839629.1]) encodes a 40 amino acid protein containing a C-terminal CAAX motif. The ORF is part of an unknown gene annotated as CBS7435_Chr2–1002, which, according to the RNA sequencing data analysis, was upregulated to similar levels in all strains under mating conditions. However, a mating-type-specific expression pattern could be observed in the enriched coverage histograms when only the relevant sequence region was considered (Figs S1 and S2, Supporting Information). An alignment of the K. phaffiia-factor sequences is shown in Fig. 1A. The two identified proteins have an overall sequence identity of 63%. Before secretion of the mature mating factors via the Ste6 transporter, yeast a-factor peptides are modified by a number of conserved enzymes. The genome of K. phaffii encodes orthologs of all proteins known to be required for a-factor maturation in S. cerevisiae. Therefore, the N-terminal cleavage site on the K. phaffiia-factor sequences was predicted by comparison of the amino acid sequences to sequences of known a-factor peptides from other yeasts (Fig. 1B). In S. cerevisiae, the N-terminus is processed in two consecutive steps carried out by Ste24 and Axl1, with AxlI cleaving after KDN in the sequence (Michaelis and Barrowman 2012). Cleavage site sequences vary between different yeast species; however, sequence analysis showed that many of the described sites contain lysine residues and have a conserved asparagine at the final position. In K. phaffii, cleavage of the N-termini after EDN and ADN, respectively, would result in mature peptides of 17 amino acids with four residues difference between MFA1 and MFA2. Of the genes required for mating-factor cleavage, farnesylation and export, only transcript levels of RAM1 (PP7435_Chr3–0565), encoding the β-subunit of the CAAX farnesyltransferase, and STE6–1 (PP7435_Chr3–0320) were significantly upregulated under mating conditions. For RAM1, transcript levels were higher in the wild type than in pure cultures of a- or α-type cells, which points to an additional induction of gene expression by pheromone (log2 fold-changes: wild type 3.56, a-type 1.82, α-type 2.05). The transcription of the STE6–1 gene was found to be mating type specifically upregulated only in a-type and wild-type cells (log2 fold-changes: wild type 1.62, a-type 1.66). Figure 1. View largeDownload slide Alignments of yeast a-factor sequences. Protein sequences were aligned using Clustal Omega (Sievers et al. 2011). Identical amino acids are marked with an asterisk, a colon indicates residues with highly similar properties and residues with weakly similar properties are indicated by a point. Sequences of the mature peptides are shown in bold. (A) Alignment of the newly identified K. phaffiia-factor sequences. (B) Alignment of a-factor peptides from different yeast species. All sequences contain the conserved C-terminal CAAX motif (highlighted in gray). (C) Sequences of the K. pastorisa-factor peptides. The predicted mature peptides are identical to the K. phaffiia-factor peptide sequences. Figure 1. View largeDownload slide Alignments of yeast a-factor sequences. Protein sequences were aligned using Clustal Omega (Sievers et al. 2011). Identical amino acids are marked with an asterisk, a colon indicates residues with highly similar properties and residues with weakly similar properties are indicated by a point. Sequences of the mature peptides are shown in bold. (A) Alignment of the newly identified K. phaffiia-factor sequences. (B) Alignment of a-factor peptides from different yeast species. All sequences contain the conserved C-terminal CAAX motif (highlighted in gray). (C) Sequences of the K. pastorisa-factor peptides. The predicted mature peptides are identical to the K. phaffiia-factor peptide sequences. Using the K. phaffii sequences to perform homology-based search also allowed the identification of two MFA genes in K. pastoris. As in K. phaffii, the two genes are located on two different chromosomes (MFA1 Chr1:1743827..1743708 [CP014584.1], MFA2 Chr4:570171..570290 [CP014587.1], ATCC 28485 (Love et al. 2016)) and encode 40 amino acid proteins, differing from their K. phaffii orthologs only in 1 or 4 amino acids, respectively (Fig. 1C). No further a-factor sequences of other closely related yeast species with available genome sequences could be identified. Only simultaneous deletion of both MFA genes prevents mating of a-type cells To confirm the role of the newly identified sequences and to analyze the effect of MFA deletions on the mating behavior of K. phaffii, different knockout strains were generated. Genes were deleted in the homothallic CBS2612 wild-type strain as well as the previously published dic1–2Δ(a) or dic1–2Δ(α) strains with stable mating types (Heistinger, Gasser and Mattanovich 2018). In all cases, the whole gene was replaced by an antibiotic resistance marker gene. The mfa1Δ mfa2Δ double mutants were generated by consecutive transformation of two deletion cassettes. Furthermore, strains with deletion of MFα (CBS7435_Chr2–0522) or the a-factor transporter STE6–1 (PP7435_Chr3–0320) were generated. To analyze the influence of the mating-factor gene deletions on the mating behavior, all strains were mated against the unmodified a- and α-type strains using a standard mating protocol. Both of the MFA single mutants (mfa1Δ(a) and mfa2Δ(a)) were still able to mate with an α-type mating partner. Only deletion of both MFA genes from the a-type strain resulted in a complete loss of mating ability, indicating that the two candidate genes encode functionally redundant proteins (Fig. 2A). Mating experiments with the homothallic wild-type strain consisting of mutants of both mating types gave similar results (Fig. 2B). While the single mfaΔ mutants were able to mate with a- and α-type cells, MFA double mutants and the ste6–1Δ strain (Fig. 2C) were unable to mate with α-type cells. However, mating with the dic1–2Δ(a) strain was still possible for both the mfa1Δ mfa2Δ and the ste6–1Δ strain, showing that the effect of the mutations is mating type specific. The same behavior was also observed for the mfαΔ strain, where mating was only possible with the dic1–2Δ(α) strain (Fig. 2D). Figure 2. View largeDownload slide Mating phenotype of mating-factor deletion strains. Diploid cells were selected on YPD agar containing the appropriate antibiotics. All different mutants were mated against the dic1–2Δ a- and α-type strain indicated by a framed a and α. Note that the two tester strains carried the same resistance gene, preventing the selection of diploid cells formed by these strains in the positions indicated by an asterisk. (A) Diploid cell formation by mfaΔ a-type strains. Deletion of both MFA genes abolished mating with α-type cells. (B) Diploid cell formation by homothallic mfaΔ strains. Deletion of both MFA genes prevented mating of a-type cells, but did not affect α-cell mating. (C) Deletion of the a-factor transporter gene STE6–1 abolished mating of a-type cells. The cell type of the ste6–1Δ strains is given in parentheses. (D) Diploid cell formation by homothallic mfαΔ cells. Only a-type cells were able to mate and form diploids with the dic1–2Δ(α) strain. Figure 2. View largeDownload slide Mating phenotype of mating-factor deletion strains. Diploid cells were selected on YPD agar containing the appropriate antibiotics. All different mutants were mated against the dic1–2Δ a- and α-type strain indicated by a framed a and α. Note that the two tester strains carried the same resistance gene, preventing the selection of diploid cells formed by these strains in the positions indicated by an asterisk. (A) Diploid cell formation by mfaΔ a-type strains. Deletion of both MFA genes abolished mating with α-type cells. (B) Diploid cell formation by homothallic mfaΔ strains. Deletion of both MFA genes prevented mating of a-type cells, but did not affect α-cell mating. (C) Deletion of the a-factor transporter gene STE6–1 abolished mating of a-type cells. The cell type of the ste6–1Δ strains is given in parentheses. (D) Diploid cell formation by homothallic mfαΔ cells. Only a-type cells were able to mate and form diploids with the dic1–2Δ(α) strain. MFA single mutants mate with reduced efficiency To assess the contribution of the individual a-factor peptides to the overall mating efficiency of the K. phaffiia-type strain, the mating efficiency of the different mutants was analyzed by a semiquantitative mating assay. In this assay, MFA single and double mutants were mated against the unmodified dic1–2Δ(α) strain. The mating efficiency of the unmodified dic1–2Δ strains was found to be around 10% (Fig. 3). Mating with strains deleted in MFA1 and MFA2 led to a dramatic decrease in efficiency to 0.9% and 0.1%, respectively. This indicated a synergistic effect of the two a-factor pheromones in K. phaffii and showed that the expression of both MFA genes is required for efficient mating. A few diploid cells could also be obtained when crossing the mfa1Δ mfa2Δ(a) double mutant with the dic1–2Δ(α) strain, resulting in a mating efficiency of 0.01%. Figure 3. View largeDownload slide Mating efficiency of mfaΔ a-type strains. Single and double mutants as well as the dic1–2Δ(a) strain were mated with the dic1–2Δ(α) strain. Error bars represent the standard deviation of four independent mating experiments. A Student’s t-test showed significant differences between all strains (p-value < 0.02). Figure 3. View largeDownload slide Mating efficiency of mfaΔ a-type strains. Single and double mutants as well as the dic1–2Δ(a) strain were mated with the dic1–2Δ(α) strain. Error bars represent the standard deviation of four independent mating experiments. A Student’s t-test showed significant differences between all strains (p-value < 0.02). Expression of one MFA gene is sufficient for mating pathway activation Binding of mating pheromones to the pheromone surface receptors Ste2 and Ste3 (for α- and a-factor, respectively) activates a MAPK signaling cascade resulting in the activation of the genes necessary for shmoo formation and cell fusion (Jones and Bennett 2011; Merlini, Dudin and Martin 2013). The membrane protein Fus1 localizes at the shmoo tip and is required for efficient fusion of two mating cells. In S. cerevisiae, FUS1 expression is strongly induced by mating pheromones (McCaffrey et al. 1987; Trueheart, Boeke and Fink 1987). A similar pattern of regulation could be found in K. phaffii, where FUS1 (PP7435_Chr4–0336) was only expressed under mating conditions and significantly upregulated in the wild-type cultures containing cells of both mating types compared to the a- and α-type samples (log2 fold-changes under mating conditions: a-type/wild type –2.65, α-type/wild type –2.39). Fusion of the FUS1 promoter to the β-galactosidase or GFP gene has previously been used as a tool to study FUS1 regulation and the induction of the pheromone response pathway in other yeasts (Trueheart, Boeke and Fink 1987; Marcus et al. 1991; Fujimura 1992; Houser et al. 2012). To investigate the induction of mating in response to the different mating factors, the K. phaffii FUS1 promoter (Chr4:560823..560339 [FR839631.1]) was used to generate an eGFP reporter construct. This construct was integrated into the K. phaffii genome at the AOX1 terminator locus of the unmodified a- and α-type strain, resulting in the reporter strains dic1–2Δ(a) PFUS1_eGFP and dic1–2Δ(α) PFUS1_eGFP. To analyze the induction of the mating response in the reporter strains in the presence of the different mating-factor deletion strains, equal numbers of cells were mixed and cultivated under nitrogen and glucose limiting conditions, as shmoo formation and FUS1 expression were strongest under these conditions. After around 40 h, the fraction of fluorescent cells was measured by flow cytometry. Changes in cell morphology were assessed by microscopy. Flow cytometry analysis (Fig. 4A) showed no eGFP expression in the cultures of the reporter strains alone, confirming that nitrogen and glucose limitation alone is not sufficient for induction of FUS1 expression in haploid K. phaffii cells. Only after co-cultivation with cells of opposite mating type, an eGFP-positive population was observed. Mixing the a-type reporter strain with mfαΔ cells failed to induce eGFP expression, showing that the generated reporter strains are sensitive to the presence of mating pheromone. As expected from the mating experiments, the mfaΔ single mutants, secreting only one of the a-factor peptides, could still induce eGFP expression in the α-type reporter strain. No eGFP expression was observed when α-type reporter cultures were mixed with either the mfa1Δ mfa2Δ double mutant or the ste6–1Δ strain. Representative flow cytometry plots of cultures containing the mfaΔ strains are shown in Fig. 4B. Note that in the mixed cultures the theoretical maximum of eGFP-positive cells is 50%. Shmoo formation could only be observed in cultures containing the dic1–2Δ(α) PFUS1_eGFP reporter strain together with one of the mfaΔ(a) single mutants (Fig. 4C). Interestingly, no green cells forming shmoos could be observed with the mfa1Δ(a) strain, while mfa2Δ(a) cells were able to induce shmoo formation in dic1–2Δ(α) PFUS1_eGFP cells. Figure 4. View largeDownload slide Induction of the mating response by mating factor deletion strains. The different a- and α-factor deletion strains were co-cultivated with the respective dic1–2Δ PFUS1_eGFP reporter strain of the opposite mating type under nitrogen and glucose limiting conditions. (A) The proportion of eGFP-positive cells in a culture was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after co-cultivation with different mfaΔ strains. A distinct population of fluorescent cells was observed in all cultures with the mfaΔ single mutants, while the mfa1Δ mfa2Δ double mutant did not induce eGFP expression. (C) Microscopy pictures of the α-type reporter strain alone and after co-cultivation with the mfaΔ and ste6–1Δ strains. Only the mfaΔ single mutants were able to induce eGFP expression and a clear shmoo phenotype. Arrows indicate shmoo formation. Bar, 5 μm. Figure 4. View largeDownload slide Induction of the mating response by mating factor deletion strains. The different a- and α-factor deletion strains were co-cultivated with the respective dic1–2Δ PFUS1_eGFP reporter strain of the opposite mating type under nitrogen and glucose limiting conditions. (A) The proportion of eGFP-positive cells in a culture was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after co-cultivation with different mfaΔ strains. A distinct population of fluorescent cells was observed in all cultures with the mfaΔ single mutants, while the mfa1Δ mfa2Δ double mutant did not induce eGFP expression. (C) Microscopy pictures of the α-type reporter strain alone and after co-cultivation with the mfaΔ and ste6–1Δ strains. Only the mfaΔ single mutants were able to induce eGFP expression and a clear shmoo phenotype. Arrows indicate shmoo formation. Bar, 5 μm. Activation of the mating response by synthetic pheromones The same PFUS1_eGFP reporter assay was used to test the activity of synthetic mating pheromones. To ensure full activity, the proposed mature a-factor peptides were synthesized including the C-terminal methylation and farnesylation. The effect of synthetic K. phaffii α-factor was also investigated. The peptides were added to cultures of the dic1–2Δ PFUS1_eGFP a- or α-type strains after around 18 h in nitrogen and glucose limiting conditions. After another 24 h, the percentage of eGFP-positive cells was determined by flow cytometry. Changes in cell morphology were assessed by microscopy. Addition of either a-factor peptide to a final concentration of 10 μg/ml led to a clear induction of eGFP expression in around 30% of the cells of the PFUS1_eGFP reporter strain (Fig. 5A). Representative flow cytometry plots of cultures after a-factor addition are shown in Fig. 5B. The proportion of eGFP-positive cells increased only slightly when the peptide concentration was increased to 20 μg/ml and the addition of both a-factor peptides to the same culture did not result in a higher number of induced cells. At the same concentration, synthetic K. phaffii α-factor was able to induce the mating response in around 20% of the cells (Fig. 5A). No response could be observed when the S. cerevisiaea-factor peptide was used. Overall, the absolute portion of induced reporter cells after addition of the pheromones was about half compared to induction by pheromones secreted by cells of opposite mating type in the mixed cultures, which contained only 50% reporter cells. Synthetic a-factor 1 at a concentration of 10 μg/ml led to shmoo formation to a similar extent as in the control (Fig. 5C). This was also observed for a-type cells after the addition of α-factor. Addition of the same amount of a-factor 2 did not induce shmoo formation. Figure 5. View largeDownload slide Induction of the mating response by synthetic a- and α-factor (Mfa and Mfα). The synthetic peptides were added to cultures of the dic1–2Δ PFUS1_eGFP reporter strains cultivated under nitrogen and glucose limiting conditions. (A) The portion of eGFP-positive cells was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after addition of the a-factor peptides. Addition of the two peptides alone and in combination led to the formation of a distinct population of eGFP-positive cells. (C) Bright-field and fluorescence microscopy pictures of the α-type reporter strain alone and after co-cultivation with dic1–2Δ(a) cells or addition of synthetic a-factor. Clear shmoo formation was only observed in mixed cultures with dic1–2Δ(a) cells and after addition of a-factor 1. Synthetic α-factor induced shmoo formation in dic1–2Δ(a) PFUS1_eGFP cells. Bar, 5 μm. Figure 5. View largeDownload slide Induction of the mating response by synthetic a- and α-factor (Mfa and Mfα). The synthetic peptides were added to cultures of the dic1–2Δ PFUS1_eGFP reporter strains cultivated under nitrogen and glucose limiting conditions. (A) The portion of eGFP-positive cells was analyzed by flow cytometry. Error bars represent the standard deviation of three independent cultivations. (B) Representative flow cytometry plots of the α-type reporter strain alone and after addition of the a-factor peptides. Addition of the two peptides alone and in combination led to the formation of a distinct population of eGFP-positive cells. (C) Bright-field and fluorescence microscopy pictures of the α-type reporter strain alone and after co-cultivation with dic1–2Δ(a) cells or addition of synthetic a-factor. Clear shmoo formation was only observed in mixed cultures with dic1–2Δ(a) cells and after addition of a-factor 1. Synthetic α-factor induced shmoo formation in dic1–2Δ(a) PFUS1_eGFP cells. Bar, 5 μm. Expression of MFA genes is induced by pheromone To confirm the transcriptional regulation of the mating-factor genes observed in the RNA sequencing data, samples from cultures during exponential growth in rich medium and after 24 h in mating medium were analyzed by quantitative PCR. To obtain mixed mating-type cultures, equal numbers of a- and α-type cells from the precultures were inoculated into mating medium. As expected, the transcript levels of MFA1, MFA2, MFα and the surface receptor genes STE2 and STE3 were very low in rich medium (YPD), with expression levels below 1% of the reference gene ACT1 (Fig. 6). MFA1 and MFA2 were both upregulated in a-type cells under mating conditions (Fig. 6A and B). The highest a-factor transcript levels were detected in the mixed cultures containing a- and α-type cells, indicating induction of MFA gene expression by α-factor. However, even in the mixed mating-type cultures, MFA1 transcript levels were around eight times higher than levels of MFA2. The expression of MFα was upregulated only in α-type cells under mating conditions (Fig. 6C). Interestingly, no further induction of gene expression was observed in the presence of a-type cells and transcript levels were around five times lower than the MFA1 levels in the same cultures. To emphasize the strong differences in expression levels between the three genes, a direct comparison of the mating-factor transcript levels in the different strains under mating conditions is shown in Fig. 6D. Transcript levels of the α-factor receptor STE2 were strongly upregulated in a-type cells under mating conditions (Fig. 6E). The absolute levels detected in the mixed cultures were similar to the a-type samples. However, as these cultures contained only 50% of a-type cells, this likely resulted from an additional 2-fold increase in transcript level per cell in the presence of α-factor. The same expression pattern was also observed for the a-factor receptor STE3 (Fig. 6F). Figure 6. View largeDownload slide Transcript levels of mating factor and pheromone surface receptor genes. Cultures of dic1–2Δ a- and α-type cells were analyzed during exponential growth in rich medium (YPD) and under mating condition. Mixed mating-type cultures consisted of an equal number of cells of both mating types grown under mating conditions. Transcript levels were normalized to ACT1 expression. Error bars represent the standard deviation of three biological replicates. Differences in gene expression between the cultures in mating medium were analyzed using a Student’s t-test. Significant differences with a p-value < 0.05 are indicated by an asterisk. (A) MFA1 was highly expressed in a-type cells under mating conditions. An additional increase in transcript level was measured in the mixed mating-type cultures. (B) Transcription of MFA2 was induced in a-type cells under mating conditions and further increased significantly in the mixed mating-type cultures. (C) Mating-type-specific expression of MFα under mating conditions. (D) Direct comparison of the mating-factor transcript levels in the different cultures in mating medium. (E) Expression levels of STE2 under mating conditions were similar in pure a-type and mixed cultures. (F) STE3 transcript levels are upregulated in α-type cells and mixed cultures under mating conditions. Figure 6. View largeDownload slide Transcript levels of mating factor and pheromone surface receptor genes. Cultures of dic1–2Δ a- and α-type cells were analyzed during exponential growth in rich medium (YPD) and under mating condition. Mixed mating-type cultures consisted of an equal number of cells of both mating types grown under mating conditions. Transcript levels were normalized to ACT1 expression. Error bars represent the standard deviation of three biological replicates. Differences in gene expression between the cultures in mating medium were analyzed using a Student’s t-test. Significant differences with a p-value < 0.05 are indicated by an asterisk. (A) MFA1 was highly expressed in a-type cells under mating conditions. An additional increase in transcript level was measured in the mixed mating-type cultures. (B) Transcription of MFA2 was induced in a-type cells under mating conditions and further increased significantly in the mixed mating-type cultures. (C) Mating-type-specific expression of MFα under mating conditions. (D) Direct comparison of the mating-factor transcript levels in the different cultures in mating medium. (E) Expression levels of STE2 under mating conditions were similar in pure a-type and mixed cultures. (F) STE3 transcript levels are upregulated in α-type cells and mixed cultures under mating conditions. DISCUSSION The preferentially haploid yeast K. phaffii can undergo mating and meiosis under nitrogen limitation conditions, and orthologs of many of the genes involved in the MAPK pathway leading to activation of the mating response in other yeasts are found in the genome. This includes the pheromone surface receptor genes STE2 and STE3 as well as the genes coding for α-factor and the conserved biogenesis pathway of the a-factor pheromone. However, no gene coding for the K. phaffiia-factor itself has been identified so far. Annotation of yeast a-factor genes by standard homology-based search is difficult because of their short sequence length and relatively poor sequence conservation. Furthermore, as the mating genes of K. phaffii are only expressed under nitrogen limitation conditions, relevant transcripts would not be detected in transcriptome analyses of strains under standard growth conditions (Valli et al. 2016). Starting from RNA sequencing data of previously generated heterothallic K. phaffii strains (Heistinger, Gasser and Mattanovich 2018) under mating conditions, we were able to identify two independent MFA genes. The systematic search for short ORFs expressed in a mating-type-specific manner and containing a C-terminal CAAX motif yielded MFA1. MFA2 was subsequently identified using MFA1 homology search. The MFA2 ORF is part of a predicted gene (CBS7435_Chr2–1002), probably wrongly annotated by automated search to code for a non-conserved hypothetical protein. It is likely that MFA2 was not considered during the first analysis because the average expression over the whole annotated gene was similar in both mating types. However, mating-type-specific expression could be observed in the short MFA2 region and was also confirmed by quantitative PCR. Both identified K. phaffii MFA sequences code for 40 amino acid long proteins with an overall sequence identity of 63% and contain the conserved C-terminal CAAX motif required for a-factor modification. The proposed N-terminal AxlI cleavage site after the asparagine at position 20 was predicted by comparison to other known a-factor sequences. The resulting mature peptides of 17 amino acids are slightly longer than the well-studied a-factor peptides of S. cerevisiae and the Sc. pombe M-factor, which have a confirmed length of 12 and 9 amino acids, respectively (Betz et al. 1987; Davey 1992). In our experiments, synthetic a-factor peptides with the predicted N-termini were able to activate the mating response pathway and to induce shmoo formation in α-type cells. The sequences of the N-terminal processing sites need to be confirmed by future experiments. Analysis of the mating phenotype of MFA mutants showed that only mfa1Δ mfa2Δ double deletion strains were unable to mate or induce the mating response pathway in α-type cells. MFA single mutants were still able to induce the mating response in a similar number of α-type cells as the unmodified a-type strain. They were also able to mate with α-type cells, although with lower efficiency. These results indicated that the two newly identified genes both encode a variant of the a-factor and that there are no further a-factor genes in the genome of K. phaffii. For both of the single mutants, the mating efficiency was reduced to below 10% of the mating efficiency of the unmodified a-type strain. This strong reduction in efficiency might be due to a synergistic effect of the two peptide variants. However, no such effect could be observed after simultaneous addition of both pheromones in comparison to addition of one of the synthetic peptides alone. Another possibility is that with only one of the genes being expressed, the total a-factor concentration might simply be too low for an efficient induction of the mating response. Saccharomyces cerevisiae mfaΔ single mutants were shown to mate with a similar efficiency as wild-type cells while secreting only about half the amount of a-factor (Michaelis and Herskowitz 1988). Similarly, the deletion of one of the three M-factor gene copies of Sc. pombe did not affect mating, even though pheromone production was found to be reduced. In M-factor double mutants, the mating efficiency was reduced to 25–30% compared to the wild type (Kjaerulff, Davey and Nielsen 1994). Deletion of the gene encoding the a-factor transporter Ste6 has been shown to prevent a-factor secretion and mating of S. cerevisiaea-type cells (Kuchler, Sterne and Thorner 1989). The same mating-type-specific non-mating phenotype was observed for K. phaffii ste6–1Δ strains, indicating that STE6–1 is the only gene coding for the a-factor transporter. The additionally annotated STE6–2 (PP7435_Chr1–1574) and STE6–3 (PP7435_Chr2–1257) genes most likely encode other closely related ABC transporters. As expected, the deletion of the single MFα gene abolished mating of α-type cells. In K. phaffii, the expression levels of mating specific genes are very low during growth in rich medium and increase only under nitrogen starvation conditions (Heistinger, Gasser and Mattanovich 2018). The same pattern of gene expression was also observed for the mating-factor genes, with transcript levels below 1% of the ACT1 control in rich medium and strong induction to up to 2500% of ACT1 expression for MFA1 under mating conditions. Furthermore, gene expression was found to be mating type specific, with the MFA genes being expressed only in a-type and MFα being expressed only in α-type cells. It is likely that the expression of the MFA and MFα genes is activated by Mata2 and Matα1, respectively, as it has been observed for the corresponding surface receptor genes, although this remains to be shown in further experiments (Heistinger, Gasser and Mattanovich 2018). Under mating conditions, transcript levels of MFA1 were up to 50 times higher than levels of MFA2. This difference in transcript levels was less pronounced in the mixed cultures containing both mating types where MFA1 levels were only around eight times higher. In these cultures, the presence of α-type cells led to an additional increase in the MFA1 and MFA2 transcript levels. An additional induction of pheromone expression in the presence of a pheromone of the opposite mating type has been described for several other yeasts like S. cerevisiae, Sc. pombe and C. albicans (Strazdis and MacKay 1983; Achstetter 1989; Imai and Yamamoto 1994; Kjaerulff, Davey and Nielsen 1994; Zhao et al. 2005; Dignard et al. 2007). It likely serves as a feedback mechanism to ensure high enough local pheromone levels to induce the mating process. Interestingly, in our experiments no additional induction of MFα expression was observed in mixed mating-type cultures. The fact that MFα levels are already strongly induced by nitrogen limitation and one transcript encodes nine copies of the mature α-factor could be a possible reason why further induction of α-factor signal might not be necessary for efficient mating. The measured expression levels of the corresponding pheromone receptor genes in the mixed cultures were equal to the levels obtained for samples containing only one mating type, indicating a 2-fold increase in receptor expression per cell in the presence of pheromone of the opposite mating type. In a homothallic K. phaffii wild-type culture containing a higher portion of α-type cells, a-factor receptor STE2 was expressed at lower levels than in a pure a-type culture while the transcript levels of the α-factor receptor STE3 were highest in the mixed cultures (Heistinger, Gasser and Mattanovich 2018 and RNA sequencing data). Addition of synthetic mating pheromones to cultures under mating conditions resulted in an induction of the mating response pathway in about 20–30% of the population, confirming that the peptides are able to act as mating pheromones. Interestingly, even after addition of cells of opposite mating type only around 60% of the reporter cells expressed eGFP. The missing induction of the mating response in a rather large portion of the population might be one of the reasons for the low mating efficiency of K. phaffii. Other indicators for the biological activity of mating pheromones are arrest of the cells in the G1 phase of the cell cycle and changes in cell morphology to the so-called shmoo phenotype (Merlini, Dudin and Martin 2013). Many of the K. phaffii mating genes are only activated under nitrogen starvation conditions in which hardly any mitotic growth is observed. Therefore, it is difficult to use cell cycle arrest to assess the response to pheromone. In Sc. pombe, deletion of the adenylate cyclase (CYR1) to mimic nitrogen starvation was used to induce mating gene expression in growing cultures, enabling the demonstration of G1 arrest in response to mating pheromone (Maeda, Mochizuki and Yamamoto 1990; Imai and Yamamoto 1994). It remains to be tested if this strategy could also be applied in K. phaffii. Under the conditions used, shmoo formation could always be observed in mixed mating-type cultures of the unmodified strains. It was also induced by synthetic α-factor and, to a lesser extent, by a-factor 1. However, no shmoo formation could be observed after the addition of a-factor 2 or in fluorescent cells of the α-type reporter strain in cultures with the mfa1Δ strain. It is unclear what causes this difference in the response to the a-factor peptides. Actual shmoo formation probably requires higher concentrations of a-factor than the activation of the FUS1 promoter, which was similar for both a-factor peptides and only slightly higher with the mfa2Δ strain than in the cultures with the mfa1Δ strain. As MFA2 expression levels are lower, the amounts of active a-factor secreted by mfa1Δ cells could be just too low to induce the shmoo phenotype. It is possible that local areas of increased pheromone concentration close to actively secreting cells are responsible for the more efficient induction in mixed mating-type cultures, although cultures were well mixed and no cell aggregation was observed. It has been described for S. cerevisiae that shmoo formation after addition of a-factor was less pronounced than for α-factor (Betz, MacKay and Duntze 1977). Another study with S. cerevisiae found that although lower concentrations of synthetic a-factor than α-factor were necessary to induce G1 arrest, only little shmoo formation was observed at concentrations where α-factor could efficiently induce shmoo formation (O’Reilly et al. 2012). Generally, concentrations of pheromones required to induce a response in K. phaffii were a little higher than described for S. cerevisiae. At least 10 μg/ml synthetic pheromone was necessary to achieve mating pathway activation in more than 10% of the cells. In comparison, similar concentrations of 5–10 μg/ml synthetic α-factor can be used to induce G1 arrest in S. cerevisiae cultures, leading to a shmoo phenotype in >95% of the cells (Breeden 1997; Wu, Liu and Huang 2011). For S. cerevisiaea-factor, growth inhibition and shmoo formation were observed with as little as 40–200 ng/ml of purified or synthetic pheromone (Anderegg et al. 1988; Xue et al. 1989; Marcus et al. 1991; O’Reilly et al. 2012). The need for high local pheromone concentrations might be a reason why K. phaffii requires rather high cell densities for efficient mating. In S. cerevisiae, the barrier proteases Bar1 and Afb1 produced by a- and α-type cells, respectively, are required for fast recovery from the response to pheromone (Sprague and Herskowitz 1981; MacKay et al. 1988; Huberman and Murray 2013). In K. phaffii, a potential protease showing a-type specific expression under mating conditions is the aspartyl protease Yps1–5 (PP7435_Chr3–0313). Interestingly, no α-specific genes coding for a potential a-factor degrading enzyme could be found in our dataset. Within this study we identified the two MFA genes of K. phaffii. Both a-factor variants contribute to efficient mating of a-type cells and show a mating-type-specific expression pattern under nitrogen-limiting conditions. Furthermore, we could confirm that the α-factor is encoded by a single MFα gene which is essential for α-cell mating. Expression level analysis indicated an additional induction of a-factor and surface receptor gene expression upon pheromone binding. It remains to be investigated why this additional layer of activation could not be detected for the MFα gene. Using synthetic peptides we could demonstrate the biological role of the identified sequences in mating pathway activation. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. Acknowledgements The authors thank Petra Cazzanelli and Stefan Ergoth for their practical support during strain generation and the mating assays, and Verena Puxbaum for her help with microscopy and image processing. We also want to thank Prof. Jürgen Wendland, Vrije Universiteit Brussel, for valuable discussion. FUNDING This work was supported by the Christian Doppler Research Association (Christian Doppler Laboratory for Innovative Immunotherapeutics), Merck KGaA, as well as the Austrian Science Fund (FWF W1224—Doctoral Program on Biomolecular Technology of Proteins—BioToP). JM, NET, and MV were supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and Business Agency Vienna through the COMET Funding Program managed by the Austrian Research Promotion Agency FFG. Conflicts of interest. None declared. REFERENCES Achstetter T. Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes. Mol Cell Biol 1989 ; 9 : 4507 – 14 . Google Scholar CrossRef Search ADS PubMed Anderegg RJ , Betz R , Carr SA et al. Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component . J Biol Chem 1988 ; 263 : 18236 – 40 . Google Scholar PubMed Betz R , Crabb JW , Meyer HE et al. Amino acid sequences of a-factor mating peptides from Saccharomyces cerevisiae . J Biol Chem 1987 ; 262 : 546 – 8 . Google Scholar PubMed Betz R , MacKay VL , Duntze W . a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a . J Bacteriol 1977 ; 132 : 462 – 72 . Google Scholar PubMed Babraham Bioinformatics . Trim Galore , 2016 . http://www.bioinformatics.babraham.ac.uk/projects/trim_galore (2 May 2018, date last accessed) . Bray N , Pimentel H , Melsted P et al. Near-Optimal RNA-Seq Quantification . 2015 . https://arxiv.org/abs/1505.02710 (2 May 2018, date last accessed) . Bray NL , Pimentel H , Melsted P et al. Near-optimal probabilistic RNA-seq quantification . Nat Biotechnol 2016 ; 34 : 525 – 7 . Google Scholar CrossRef Search ADS PubMed Breeden LL . α-factor synchronization of budding yeast . Methods Enzymol 1997 ; 283 : 332 – 41 . Google Scholar CrossRef Search ADS PubMed Davey J. Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone . EMBO J 1992 ; 11 : 951 – 60 . Google Scholar PubMed Dignard D , El-Naggar AL , Logue ME et al. Identification and characterization of MFA1, the gene encoding Candida albicans a-factor pheromone . Eukaryot Cell 2007 ; 6 : 487 – 94 . Google Scholar CrossRef Search ADS PubMed Edgar R , Domrachev M , Lash AE . Gene Expression Omnibus: NCBI gene expression and hybridization array data repository . Nucleic Acids Res 2002 ; 30 : 207 – 10 . Google Scholar CrossRef Search ADS PubMed Fairhead C , Llorente B , Denis F et al. New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination . Yeast 1996 ; 12 : 1439 – 57 . Google Scholar CrossRef Search ADS PubMed Fujimura H . Molecular monitoring of the transcriptional activation of the yeast Saccharomyces kluyveri mating pheromone signal transduction by using FUS1-lacZ fusion gene . FEMS Microbiol Lett 1992 ; 73 : 23 – 6 . Google Scholar CrossRef Search ADS PubMed Gasser B , Prielhofer R , Marx H et al. Pichia pastoris : protein production host and model organism for biomedical research . Future Microbiol 2013 ; 8 : 191 – 208 . Google Scholar CrossRef Search ADS PubMed Gentleman RC , Carey VJ , Bates DM et al. Bioconductor: open software development for computational biology and bioinformatics . Genome Biol 2004 ; 5 : R80 . Google Scholar CrossRef Search ADS PubMed Hanson SJ , Byrne KP , Wolfe KH . Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system . Proc Natl Acad Sci USA 2014 ; 111 : E4851 – 8 . Google Scholar CrossRef Search ADS PubMed Heistinger L , Gasser B , Mattanovich D . Creation of stable heterothallic strains of Komagataella phaffii enables dissection of mating gene regulation . Mol Cell Biol 2018 ; 38 : e00398 – 17 . Google Scholar PubMed Houser JR , Ford E , Chatterjea SM et al. An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae . Yeast 2012 ; 29 : 519 – 30 . Google Scholar CrossRef Search ADS PubMed Huber W , Carey VJ , Gentleman R et al. Orchestrating high-throughput genomic analysis with Bioconductor . Nat Methods 2015 ; 12 : 115 – 21 . Google Scholar CrossRef Search ADS PubMed Huberman LB , Murray AW . Genetically engineered transvestites reveal novel mating genes in budding yeast . Genetics 2013 ; 195 : 1277 – 90 . Google Scholar CrossRef Search ADS PubMed Imai Y , Yamamoto M . The fission yeast mating pheromone P-factor: its molecular structure, gene structure, and ability to induce gene expression and G1 arrest in the mating partner. Genes Dev 1994 ; 8 : 328 – 38 . Google Scholar CrossRef Search ADS PubMed Jones SK Jr , Bennett RJ . Fungal mating pheromones: choreographing the dating game . Fungal Genet Biol 2011 ; 48 : 668 – 76 . Google Scholar CrossRef Search ADS PubMed Kim D , Pertea G , Trapnell C et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions . Genome Biol 2013 ; 14 : R36 . Google Scholar CrossRef Search ADS PubMed Kjaerulff S , Davey J , Nielsen O . Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3. , Mol Cell Biol 1994 ; 14 : 3895 – 905 . Google Scholar CrossRef Search ADS PubMed Kuberl A , Schneider J , Thallinger GG et al. High-quality genome sequence of Pichia pastoris CBS7435 . J Biotechnol 2011 ; 154 : 312 – 20 . Google Scholar CrossRef Search ADS PubMed Kuchler K , Sterne RE , Thorner J . Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells . EMBO J 1989 ; 8 : 3973 – 84 . Google Scholar PubMed Kurjan J , Herskowitz I . Structure of a yeast pheromone gene (MFα): a putative α-factor precursor contains four tandem copies of mature α-factor . Cell 1982 ; 30 : 933 – 43 . Google Scholar CrossRef Search ADS PubMed Love KR , Shah KA , Whittaker CA et al. Comparative genomics and transcriptomics of Pichia pastoris . BMC Genomics 2016 ; 17 : 550 . Google Scholar CrossRef Search ADS PubMed Love MI , Anders S , Huber W . DESeq2: Differential gene expression analysis based on the negative binomial distribution . 2017 . https://rdrr.io/bioc/DESeq2 (8 May 2018, date last accessed). Love MI , Anders S , Kim V et al. RNA-Seq workflow: gene-level exploratory analysis and differential expression . F1000Res 2015 ; 4 : 1070 . Google Scholar CrossRef Search ADS PubMed McCaffrey G , Clay FJ , Kelsay K et al. Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae. Mol Cell Biol 1987 ; 7 : 2680 – 90 . Google Scholar CrossRef Search ADS PubMed MacKay VL , Welch SK , Insley MY et al. The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. Proc Natl Acad Sci USA 1988 ; 85 : 55 – 9 . Google Scholar CrossRef Search ADS PubMed Maeda T , Mochizuki N , Yamamoto M . Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe . Proc Natl Acad Sci USA 1990 ; 87 : 7814 – 8 . Google Scholar CrossRef Search ADS PubMed Marcus S , Caldwell GA , Miller D et al. Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. Mol Cell Biol 1991 ; 11 : 3603 – 12 . Google Scholar CrossRef Search ADS PubMed Merlini L , Dudin O , Martin SG . Mate and fuse: how yeast cells do it . Open Biol 2013 ; 3 : 130008 . Google Scholar CrossRef Search ADS PubMed Michaelis S , Barrowman J . Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease . Microbiol Mol Biol R 2012 ; 76 : 626 – 51 . Google Scholar CrossRef Search ADS Michaelis S , Herskowitz I . The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol 1988 ; 8 : 1309 – 18 . Google Scholar CrossRef Search ADS PubMed O’Reilly N , Charbin A , Lopez-Serra L et al. Facile synthesis of budding yeast a-factor and its use to synchronize cells of α mating type . Yeast 2012 ; 29 : 233 – 40 . Google Scholar CrossRef Search ADS PubMed OhEigeartaigh SS , Armisen D , Byrne KP et al. Systematic discovery of unannotated genes in 11 yeast species using a database of orthologous genomic segments . BMC Genomics 2011 ; 12 : 377 . Google Scholar CrossRef Search ADS PubMed Ongay-Larios L , Navarro-Olmos R , Kawasaki L et al. Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to α-factor . FEMS Yeast Res 2007 ; 7 : 740 – 7 . Google Scholar CrossRef Search ADS PubMed Pertea M , Kim D , Pertea GM et al. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown . Nat Protoc 2016 ; 11 : 1650 – 67 . Google Scholar CrossRef Search ADS PubMed Prielhofer R , Barrero JJ , Steuer S et al. GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris . BMC Syst Biol 2017 ; 11 : 123 . Google Scholar CrossRef Search ADS PubMed R Development Core Team . R: A Language and Environment for Statistical Computing . Vienna, Austria : R Foundation for Statistical Computing . 2016 . Sarkari P , Marx H , Blumhoff ML et al. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger . Bioresour Technol 2017 ; 245 : 1327 – 33 . Google Scholar CrossRef Search ADS PubMed Seike T , Nakamura T , Shimoda C . Molecular coevolution of a sex pheromone and its receptor triggers reproductive isolation in Schizosaccharomyces pombe . Proc Natl Acad Sci USA 2015 ; 112 : 4405 – 10 . Google Scholar CrossRef Search ADS PubMed Sievers F , Wilm A , Dineen D et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega . Mol Syst Biol 2014 ; 7 : 539 . Google Scholar CrossRef Search ADS Singh A , Chen EY , Lugovoy JM et al. Saccharomyces cerevisiae contains two discrete genes coding for the α-factor pheromone . Nucleic Acids Res 1983 ; 11 : 4049 – 63 . Google Scholar CrossRef Search ADS PubMed Skinner ME , Uzilov AV , Stein LD et al. JBrowse: a next-generation genome browser . Genome Res 2009 ; 19 : 1630 – 8 . Google Scholar CrossRef Search ADS PubMed Soneson C , Love MI , Robinson MD . Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences . F1000Res 2015 ; 4 : 1521 . Google Scholar CrossRef Search ADS PubMed Sprague GF Jr , Herskowitz I . Control of yeast cell type by the mating type locus . J Mol Biol 1981 ; 153 : 305 – 21 . Google Scholar CrossRef Search ADS PubMed Stadlmayr G , Mecklenbrauker A , Rothmuller M et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production . J Biotechnol 2010 ; 150 : 519 – 29 . Google Scholar CrossRef Search ADS PubMed Strazdis JR , MacKay VL . Induction of yeast mating pheromone a-factor by α cells . Nature 1983 ; 305 : 543 – 5 . Google Scholar CrossRef Search ADS PubMed Tolstorukov I , Cregg JM . Classical Genetics . In: Cregg JM (ed.) Pichia Protocols: Methods in Molecular Biology , 389 : 2nd edn . Totowa, NJ : Humana Press , 2007 , 189 – 201 . Trueheart J , Boeke JD , Fink GR . Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol 1987 ; 7 : 2316 – 28 . Google Scholar CrossRef Search ADS PubMed Valli M , Tatto NE , Peymann A et al. Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function . FEMS Yeast Res 2016 ; 16 : fow051 . Google Scholar CrossRef Search ADS PubMed Weber E , Engler C , Gruetzner R et al. A modular cloning system for standardized assembly of multigene constructs . PLoS One 2011 ; 6 : e16765 . Google Scholar CrossRef Search ADS PubMed Wendland J , Dunkler A , Walther A . Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii . FEMS Yeast Res 2011 ; 11 : 418 – 29 . Google Scholar CrossRef Search ADS PubMed Wickham H , Hester J , Francois R . readr: Read Rectangular Text Data . 2017 . https://CRAN.R-687 project.org/package = readr (2 May 2018, date last accessed) . Wu X , Liu L , Huang M . Analysis of changes in protein level and subcellular localization during cell cycle progression using the budding yeast Saccharomyces cerevisiae . Methods Mol Biol 2011 ; 782 : 47 – 57 . Google Scholar CrossRef Search ADS PubMed Xue CB , Caldwell GA , Becker JM et al. Total synthesis of the lipopeptide a-mating factor of Saccharomyces cerevisiae . Biochem Bioph Res Co 1989 ; 162 : 253 – 7 . Google Scholar CrossRef Search ADS Zhao R , Daniels KJ , Lockhart SR et al. Unique aspects of gene expression during Candida albicans mating and possible G1 dependency . Eukaryot Cell 2005 ; 4 : 1175 – 90 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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