Regulation of Yeast-to-Hyphae Transition in Yarrowia lipolytica

smooth strains have mutations in repetitive regions of the genome. Coverage from high-throughput 150-bp paired-end Illumina sequencing from strain FKP355 (wild type) and five smooth mutant strains. Colored bases indicate polymorphic loci where reads align with SNPs at a rate greater than that expected from incorrect base calls. (A) Regions with no coverage are detected in smooth-17, smooth-33, and smooth-43 mutants at the end of scaffold 14 after alignment to the FKP355 reference genome. (B) Raw PacBio reads with homology to the single-copy region at the end of scaffold 14 (from 1 to 12 kb) were reassembled and analyzed for mutations not detected from the curated genome assembly. An example of an alternative assembly of the region detects a deletion in smooth-19 not seen in the reference assembly. (C) All five smooth mutants exhibit a different polymorphism rate than the wild-type rate at a transition point between a high-copy-number transposon-containing region and a moderate-copy-number region of short, tandem repeats.

smooth strains have reduced repetitive DNA content. (A) Illumina 150-bp sequencing reads from strain FKP355 were systematically analyzed for the presence of all possible tandem duplications with a repeat unit length of 1 to 75 bp and quantified. Identification of phased repeat units with similar coverage was used to infer arrays of tandem repeats longer than a simple duplication. Colors indicate overlapping sequence motifs found in similar repeat sequences. (B) The fraction of 150-bp sequencing reads from the wild-type and smooth strains containing high-frequency tandem duplications of 10, 12, 28, and 40 bp in length. (C) The fraction of 150-bp sequencing reads from the wild-type and smooth strains that align to the FKP355 rDNA repeat.

Effect of smooth-33 on expression of genes with specific DNA motifs near their transcription start site. The number of ACGCG and CCCCT motifs on each strand of DNA was determined (from 0 to 2 sites) between the transcription start site (labeled 0) and a given distance. The given distances shown are 200 to 2,000 bp in 200-bp intervals, both up- and downstream of the transcription start site. For each interval, the average difference in expression between FKP514 (smooth-33) and FKP391 (wild type) during chemostat cultivation is shown. Note that the presence of more CCCCT motifs close to the transcription start site is generally associated with decreased expression in the smooth-33 mutant, while the presence of more than one ACGCG site very near and 3′ of the transcription start site is associated with increased expression in the smooth-33 mutant.

Ylmsn2p and the MBP complex regulate formation of hyphae. Ylmsn2p is predicted to interact with CCCCT promoter motifs, while the MBF complex (composed of Ylswi6p and Ylmbp1p) is predicted to interact with ACGCG motifs. Ylmsn2 was overexpressed in a smooth-33 background and deleted in the parental hyphal background used for mutagenesis (FKP355). Conversely, Ylswi6 and Ylmbp1 were independently deleted in a smooth-33 background and overexpressed in the parental background. Strains were cultured on YNB agar for 3 days at 28°C prior to examination of hyphae formation and imaging. Detailed genotypes are listed in Table 4.

Mutants with a hyphal reversion phenotype in smooth-33. FKP514 (smooth-33) was mutagenized, and colonies exhibiting a transition to hyphal growth were isolated and sequenced. Mutant strains were plated on YNB agar, and isolated single colonies were imaged after 48 h at 28°C. Gene names shown are based on orthologs from S. cerevisiae and C. albicans. Mutations shown are the highest likelihood candidate identified after sequencing of each mutant.

Histidine kinases in Y. lipolytica. (A) Phylogenetic reconstruction of selected histidine kinases from ascomycete fungi. Protein sequences from the histidine kinases of Y. lipolytica with similarity to Sln1p (FKP355 JGI protein ID 128287, 113409, 109080, 128802, and 126630) were used as bait to BlastP search the proteomes of Y. lipolytica, S. cerevisiae, C. albicans, Lipomyces starkeyi, Schizosaccharomyces pombe, Taphrina deformans, Ascobolus immerses, Monacrosporium haptotylum, Aspergillus nidulans, Stagnospora nodorum, Cladonia grayi, Botrytis cinerea, Neurospora crassa, and Xylona heveae. The BlastP hits were aligned using MUSCLE and analyzed by the maximum likelihood method with 200 bootstrap replicates to define the relationships between the Y. lipolytica genes and those from other species. (B) Protein domains from Ylnik1p were predicted by InterProScan (90). The kinase domain in Ylnik1p is predicted to be an S/T protein kinase. Note that all the mutations recovered occur in the HAMP domain. The sites of mutations are indicated by asterisks.