Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex
- Department of Molecular Genetics and Microbiology, Duke University Medical Center, United States
- Broad Institute of MIT and Harvard, United States
- Westerdijk Fungal Biodiversity Institute, Netherlands
- Institute of Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Netherlands
- Génomique Métabolique, CNRS, University Evry, Université Paris-Saclay, France
- Lehrstuhl für Molekulare und Zelluläre Botanik, Ruhr-Universität Bochum, Germany
Figures
Figure 1 with 5 supplements
Genome-wide comparison between the two C. depauperatus strains and phylogenetic placement of C. depauperatus.
(A) Circos plot comparing the genome assemblies of C. depauperatus CBS7841 and CBS7855. The two assemblies are overall syntenic, except for five inversions (labelled ‘a’ to ‘e’; see Figure 1—figure …
Figure 1—figure supplement 1
Statistics of the genome assemblies of CBS7841 and CBS7855 and electrophoretic karyotyping.
(A) Genome sequencing data generated, final genome assembly statistics, and (B) genome completeness assessment upon aligning C. depauperatus gene sets to 4284 Benchmarking Universal Single-Copy …
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Figure 1—figure supplement 1—source data 1
Raw images of clamped homogeneous electrical field (CHEF) gels.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Inversions between the two C. depauperatus isolates.
(A) Synteny maps of C. depauperatus CBS7841 and CBS7855 highlighting two large and three small inversions detected between the two strains, with each inversion containing more than two genes …
Figure 1—figure supplement 3
Centromere length comparison between CBS7841 and CBS7855.
(A) Box plots depicting the predicted centromere length. Each dot represents one centromere, and the horizontal red lines depict mean centromere lengths in the corresponding strain. Shaded boxes …
Figure 1—figure supplement 4
Genome-wide divergence (k, with Jukes–Cantor correction) of C. depauperatus CBS7855 relative to CBS7841 genome reference.
This analysis shows no evidence of introgression (which would appear as regions with zero or nearly zero divergence) between the two strains (y-axis values represent percentages; x-axis values …
Figure 1—figure supplement 5
Tree topology inferred from the coalescence-based approach.
(A) Tree topology derived from the coalescence approach is fully congruent with the maximum likelihood (ML) tree derived from a concatenation-based approach. Numerical values on the branches …
Figure 2 with 3 supplements
The predicted MAT locus of C. depauperatus.
(A) Circos plot depicting the distribution of BLASTN hits between the C. amylolentus chromosomes containing the P/R (Chr 10; links colored in teal blue) and HD (Chr 11; links colored in gold) MAT …
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Figure 2—source data 1
Genes within or adjacent to the C. neoformans H99 MAT locus or the P/R and HD MAT loci of C. amylolentus CBS6039, and corresponding orthologs in C. depauperatus.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Individual genealogies of genes within the MAT locus of C. neoformans exhibiting different level of trans-specific polymorphism.
Of the analyzed genes, STE3 and MYO2 are both found within the predicted ancestral MAT region of C. depauperatus, and group with strong support with the a/A2alleles from other Cryptococcus species. …
Figure 2—figure supplement 2
Genome comparison showing the distribution of BLASTN hits (>0.5kb) represented as links between Chr 4 of C. depauperatus, on which the predicted MAT locus is located, and all 14 chromosomes of (A) C. amylolentus CBS6039 and (B) C. neoformans H99.
Tracks ‘A’ to ‘E’ depict genomic features as given in the legend of Figure 1.
Figure 2—figure supplement 3
The MAT region of C. depauperatus contains genes that are flanking the centromeres of MAT-containing chromosomes in C. neoformans and C. amylolentus.
(A) Linear chromosome plots depicting syntenic regions between the MAT chromosomes of C. depauperatus (Chr 4), C. neoformans (Chr 5) and C. amylolentus (Chrs 10 and 11). In C. neoformans, CEN5 …
Figure 3
C. depauperatus displays upregulation of key mating and meiotic genes in sporulating conditions.
(A) Heatmap of gene expression analysis of mating and meiosis-related genes for CBS7841 and CBS7855 (see Figure 3—source data 1 and Figure 3—source data 2 for the complete list of genes). Log2 fold …
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Figure 3—source data 1
Mating and meiosis genes and their expression under sporulation (solid) vs. non-sporulation (liquid) conditions.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig3-data1-v2.xlsx
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Figure 3—source data 2
C. depauperatus gene expression analysis comparing sporulation (solid) vs. non-sporulation (liquid) conditions.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig3-data2-v2.xlsx
Figure 4
Ectopic expression of the C. depauperatus MFα gene in C. neoformans induces self-filamentation through Ste3 and Ste6.
(A) The C. depauperatus MFα gene was introduced ectopically into C. neoformans MATα (H99α crg1Δ) and MATa(KN99a crg1Δ) strains, and transformants harboring the transgene were assessed for their …
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Figure 4—source data 1
Raw images of gels validating C. neoformans WT, ste3Δ and ste6Δ strains expressing the C. depauperatus pheromone.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig4-data1-v2.zip
Figure 5 with 2 supplements
C. depauperatus mfαΔ and ste3Δ mutants display defects in basidia maturation and sporulation, and the dmc1Δ mutant shows impaired sporulation despite achieving basidial maturation.
(A) Scanning electron microscopy images of wild-type CBS7841, mfαΔ (SEC831), ste3Δ (SEC836), and dmc1Δ (SEC866) deletion mutants. Cells were imaged following 1week of incubation on V8 medium at room …
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Figure 5—source data 1
Frequency of basidia defective in sporulation (bald basidia) and basidia with spores in C. depauperatus wild-type (CBS7841) and mutant strains, and one-way ANOVA and Tukey’s HSD post hoc statistical tests for frequencies of bald basidia.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig5-data1-v2.pdf
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Figure 5—source data 2
Diameter of unsporulated and sporulating basidia in C. depauperatus wild-type (CBS7841) and mutant strains (mfαΔ, ste3Δ, and dmc1Δ) following incubation on Murashige–Skoog (MS) medium for 25days at room temperature in the dark.
Statistical tests for differences in basidial diameter (one-way ANOVA followed by Tukey’s HSD post hoc; when comparing two groups of observations, the results of Tukey’s HSD test are equivalent to Student’s t-test).
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
Schematic of the transformation system optimized for C. depauperatus and genotypic analysis of C. depauperatus transformants.
(A) The efficient homologous recombination system of S. cerevisiae was employed to construct plasmids for A. tumefaciens-mediated transformation of C. depauperatus as illustrated in panel (B).(C) …
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Figure 5—figure supplement 1—source data 1
Raw images of gels validating the C. depauperatus deletion mutant strains.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
Representative microscopy images of (A) wild-type CBS7841, (B) dmc1Δ (SEC866), mfαΔ (SEC831), (C) ste3Δ-1 (SEC836), (D), ste3Δ-2 (SEC853), and (E) dmc1Δ (SEC866) deletion mutants.
Plates were imaged following 25days of incubation on Murashige–Skoog (MS) medium at room temperature in the dark. Images were taken at ×2.5 (bars = 200μm) and ×12.5magnification (bars = 50μm) from …
Figure 6 with 1 supplement
Sporulation is partially restored in mfαΔ × ste3Δ co-cultures.
(A) Illustration and light microscopy images of the C. depauperatus mfαΔ mutant (left), ste3Δ mutant (right), and a co-culture of the mfαΔ and ste3Δ mutants (middle). Images were taken following …
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Figure 6—source data 1
Frequency of basidia defective in sporulation (bald basidia) and basidia with spores in C. depauperatus wild-type (CBS7841), mfαΔ and ste3Δ single mutants, and the mfαΔ × ste3αΔ co-cultures mutant strains and one-way ANOVA and Tukey’s HSD post hoc statistical tests for frequencies of sporulating basidia.
Related to Figure 6B.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig6-data1-v2.pdf
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Figure 6—source data 2
Phenotyping and genotyping analyses of the progeny derived from mfαΔ × ste3Δ co-cultures.
Related to Figure 6C. Progeny from each basidium were plated on YPD and YPD+ NATto test for NAT resistance or sensitivity and subjected to PCR and gel electrophoresis with primers targeting the wild-type MFα and STE3 alleles, as well as the 3′ junction of the mfαΔ::NAT and ste3Δ::NAT deletion loci. Phenotyping and genotyping observations were compiled to determine whether exogenous pheromone from the ste3Δ mutant may have stimulated sporulation of the mfαΔ mutant, or mating occurred between the mfαΔ and ste3Δ mutants. Each basidium was scored as parental ditype (PD), nonparental ditype (NPD), tetratype (TT), and uniparental (Uni.) based upon the genotype of parental and recombinant spores they comprised.
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
C. depauperatus mutant strains do not undergo sporulation in confrontation assays.
Spores and/or hyphae from wild-type and mutant strains were struck~2mm apart on Murashige–Skoog (MS) medium and incubated at room temperature for 2weeks. Sporulating basidia (a few examples …
Figure 7 with 1 supplement
Analysis of C. depauperatus can1 and fur1 mutants, and mating in intra- and inter-strain crosses.
Phenotypic and genotypic analysis of (A) can1 and (B) fur1 mutants. Top panels: wild-type and UV-induced can1 mutants on YNB and YNB+ 60µg/mL canavanine (Can), and wild-type (WT) and spontaneous fur1…
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Figure 7—source data 1
Source raw data for Figure 7A and B (raw images of gels).
- https://cdn.elifesciences.org/articles/79114/elife-79114-fig7-data1-v2.zip
Figure 7—figure supplement 1
Sanger sequencing of CAN1 and FUR1 loci from double-drug-resistant isolates recovered from C. depauperatus intra- and inter-strain crosses.
Isolates marked in green did not inherit both parental mutant alleles; rather, they gained de novo spontaneous mutations in CAN1 or FUR1 loci, or in other genes.
Figure 8
Meiotic mapping of UV-induced mutations in recombinant progeny.
(A) Chromosome locations of the FUR1 and CAN1 genes and the three new mutations induced by UV irradiation (UV1, UV2, and UV3). Two of the mutations (UV1 and UV2) are spaced~715kb apart on Chr 3. (B) …
Figure 9
Proposed models for the evolution of MAT and homothallism in C. depauperatus.
(A) Following cell fusion of two haploid cells with opposite mating types with linked P/R and HD loci (bipolar configuration), a self-filamentous diploid cell was formed. During meiosis, one set of …
Author response image 1
Additional files
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Supplementary file 1
Strains used in this study.
- https://cdn.elifesciences.org/articles/79114/elife-79114-supp1-v2.xlsx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/79114/elife-79114-supp2-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79114/elife-79114-mdarchecklist1-v2.docx
