Inversion of the chromosomal region between two mating type loci switches the mating type in Hansenula polymorpha

Abstract

Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci.

Figure 1. Schematic of phylogenetic relationships among yeast species and conservation of silent mating type cassettes and the HO gene.

Information on silent cassettes and the HO gene is based on ref. 13. The tree is not drawn to scale.

Figure 2. Two mating type loci in H. polymorpha.

Schematic of the chromosomal region surrounding MAT1 and MAT2. The chromosome is represented by a thick grey line; α2, α1, a 1* and a 1 genes are indicated by pink, red, and light and dark blue arrows, respectively. MAT loci are indicated by yellow. Predicted ORFs are indicated by blue arrows above the chromosome. Chromosomal positions proximal and distal to SLA2 are marked as positions 1 and 2, respectively.

Figure 3. Functions of mating type genes in sexual development.

(A) Two mating pheromone receptors are required for mating. Wild-type, ste2Δ and ste3Δ H. polymorpha strains of ura3-1 (BY4330, HPH555, and HPH582 respectively) and leu1-1 (HPH22, HPH553, and HPH581 respectively) genotypes were combined on MEMA mating medium and incubated at 30°C. After 24 h, cells were spread on SD plates to select for Leu+Ura+ diploids. Colony number was counted after 2 days at 37°C. Shown is the average of three independent matings. Error bars indicate SD. (B) Mating assay of wild-type, α1Δ, α2Δ, a 1*Δ, and a 1Δ strains. Wild-type (HPH22 and BY4330), α1Δ(HPH546 and HPH548), α2Δ (HPH329 and HPH331), a 1*Δ (HPH517 and HPH521), and a 1Δ (HPH675 and HPH678) strains were treated as described in (A). Shown is the average of three independent matings. Error bars indicate SD. (C) Mating assays of wild-type (HPH22 and BY4330), α1Δ (HPH546 and HPH548), α2Δ (HPH329 and HPH331), a 1*Δ (HPH517 and HPH521), and a 1Δ (HPH675 and HPH678) strains with ste2Δ (HPH553 and HPH555) and ste3Δ (HPH581 and HPH582) strains. Cells were treated as described in (A). Shown is the average of three independent matings. Note that ste2Δ and ste3Δ strains behave as heterothallic α or a strains, respectively. Error bars indicate SD. (D) Mating assay for the α1Δ ste2Δ strain. Wild-type (BY4330), α1 (HPH548), ste2Δ (HPH555), and α1Δ ste2Δ (HPH642) strains of the ura3-1 genotype were combined with a wild-type strain of the leu1-1 (HPH22) genotype as described in (A). Shown is the average of three independent matings. Error bars indicate SD. (E) a 1* and a 1 are functionally distinct. Logarithmically growing wild-type diploid (HPH723) and a 1Δ homozygous diploid (a 1Δ/ a 1Δ; HPH724) cells carrying the indicated plasmid were spotted on MEMA plates and incubated at 30°C for 24 h. Plasmids used were pHM850 (P_TEF1), pHM848 (P_TEF1-a 1*), and pHM849 (P_TEF1-a 1). Shown are merged brightfield and DAPI epifluorescence images. Yellow arrows indicate spores. Bar, 2 µm. (F) Functions of a 1 and α2 are essential for meiosis and sporulation. Cells were prepared as described in (E). Shown are merged brightfield and DAPI epifluorescence images. Yellow arrows indicate spores. Bar, 5 µm.

Figure 4. Inversion of the MAT intervening region alters the expression status of mating type genes.

(A) Schematics of the chromosomal region surrounding MAT1 and MAT2. α1, α2, a 1*, and a 1 genes are indicated by pink, red, and light and dark blue arrows, respectively. IR regions are shown as thick orange arrows. DNA fragments used as probes for Southern blot analysis in (B) are shown as dark blue (probe A) or red (probe C) bars. The upper schematic shows the draft genome sequence. The lower schematic shows the predicted DNA sequences after the inversion between IR regions. Upper and lower panels show restriction enzyme sites deduced from the DNA sequences and the size of the DNA fragment hybridized by each probe. X, XhoI; P, PstI; E, EcoRI; B, BamHI. (B) Two types of chromosome configuration in different wild-type strains. Genomic DNA of HPH22 (indicated as 1) and BY4330 (indicated as 2) were prepared from logarithmically growing cells in YPDS medium and analyzed by Southern blotting using probes A and C (upper panel). The lower panel shows the results predicted from Upper (U) and Lower (L) schematics in (A). (C) PCR amplification of the I- or A-type MAT1 locus (reaction I with Primer_I/Primer_D or reaction A with Primer_A/Primer_D). The presence of the I product and absence of the A product for HPH22 indicates that the chromosome is in the I-type orientation. HPH22i and BY4330 have an A-type chromosome. (D) Mating type genes are transcriptionally active at position 1 and repressed at position 2. The expression of α1, α2, a 1*, and a 1 genes was examined by RT-PCR. RNA samples were prepared from logarithmically growing wild-type cells in YPDS medium at 30°C (HPH22, HPH22i, and BY4330). HPH22i is a clone isolated from HPH22 (Fig. S4B; see text).

Figure 5. Inversion of the MAT intervening region is induced during mating.

(A) Mating assay between I (HPH22 and HPH719)- and A (HPH22i and BY4330)-type strains. Cells were treated as described in Fig. 3A. Shown is the average of three independent matings. Error bars indicate SD. (B) RT-PCR analysis of α1 and a 1 genes. RNA samples were prepared from I (HPH22)- or A (BY4330)-type wild-type cells incubated on MEMA medium for the indicated times. Primers used for PCR are listed in Table S2. (C) Quantitative digital PCR analysis of α1 and a 1 genes. RNA samples in (B) were subjected to digital PCR analysis. α1 and a 1 RNA levels were normalized to that of ACT1 RNA. Shown are the averages of two independent PCR reactions. Error bars indicate SD. (D) PCR amplification of the I- or A-type MAT1 locus. PCR reactions are as described in Fig. 4C. Genomic DNA samples were prepared from three wild-type strains (HPH22, HPH22i, and BY4330) after incubation on MEMA for the indicated times. The appearance of the I product in the reaction with BY4330 (A-type) after 9 and 24 h indicates a switch to the I-type in a subset of the population. (E) Meiosis in a 1Δ/+ heterozygous diploid cells. a 1Δ (I-type)/+ (A-type) is defective in meiosis. Cells were prepared as described in Fig. 4E. Shown are merged brightfield and DAPI epifluorescence images. Bar, 5 µm.

Figure 6. Inversion of the MAT intervening region is essential for homothallism.

(A) Schematic of the strategy for IR₂ deletion on the A-type chromosome. (B) IR₂Δ cells are defective for the inversion. Logarithmically growing wild-type (HPH22i) and IR₂Δ (HPH833) cells in YPDS medium (+N, nutrient plus) were transferred to YPDS (+N → +N) or MEMA (+N → −N, nutrient minus) and incubated for 20 h. Genomic DNA samples were prepared and inversion was detected by two PCR reactions, A and Ai, using the primer sets Primer_D/Primer_A and Primer_E/Primer_A, respectively. (C) Cells of the A-type strain carrying the IR₂Δ allele are incapable of mating with each other and with ste2Δ. Wild-type (HPH22 and SH4330), ste2Δ (HPH553), ste3Δ (HPH581), and IR₂Δ (HPH833 and HPH835) cells were treated as described in Fig. 3A. Shown is the average of three independent matings. Error bars indicate SD.

Figure 7. Model of mating type regulation in H. polymorpha.

(A) Function of mating type genes in establishing mating type identity. (B) Model of homothallism in H. polymorpha.