Ploidy determination by FACS for blastospores produced during unisexual development.

<p>Ploidy determination by FACS for blastospores produced during unisexual development.</p

<i>PRM1</i> and <i>KAR5</i> function in cell-cell fusion and karyogamy to drive distinct bisexual and unisexual cycles in the <i>Cryptococcus</i> pathogenic species complex

<div><p>Sexual reproduction is critical for successful evolution of eukaryotic organisms in adaptation to changing environments. In the opportunistic human fungal pathogens, the <i>Cryptococcus</i> pathogenic species complex, <i>C</i>. <i>neoformans</i> primarily undergoes bisexual reproduction, while <i>C</i>. <i>deneoformans</i> undergoes both unisexual and bisexual reproduction. During both unisexual and bisexual cycles, a common set of genetic circuits regulates a yeast-to-hyphal morphological transition, that produces either monokaryotic or dikaryotic hyphae. As such, both the unisexual and bisexual cycles can generate genotypic and phenotypic diversity <i>de novo</i>. Despite the similarities between these two cycles, genetic and morphological differences exist, such as the absence of an opposite mating-type partner and monokaryotic instead of dikaryotic hyphae during <i>C</i>. <i>deneoformans</i> unisexual cycle. To better understand the similarities and differences between these modes of sexual reproduction, we focused on two cellular processes involved in sexual reproduction: cell-cell fusion and karyogamy. We identified orthologs of the plasma membrane fusion protein Prm1 and the nuclear membrane fusion protein Kar5 in both <i>Cryptococcus</i> species, and demonstrated their conserved roles in cell fusion and karyogamy during <i>C</i>. <i>deneoformans</i> α-α unisexual reproduction and <i>C</i>. <i>deneoformans</i> and <i>C</i>. <i>neoformans</i> <b>a</b>-α bisexual reproduction. Notably, karyogamy occurs inside the basidum during bisexual reproduction in <i>C</i>. <i>neoformans</i>, but often occurs earlier following cell fusion during bisexual reproduction in <i>C</i>. <i>deneoformans</i>. Characterization of these two genes also showed that cell fusion is dispensable for solo unisexual reproduction in <i>C</i>. <i>deneoformans</i>. The blastospores produced along hyphae during <i>C</i>. <i>deneoformans</i> unisexual reproduction are diploid, suggesting that diploidization occurs early during hyphal development, possibly through either an endoreplication pathway or cell fusion-independent karyogamy events. Taken together, our findings suggest distinct mating mechanisms for unisexual and bisexual reproduction in <i>Cryptococcus</i>, exemplifying distinct evolutionary trajectories within this pathogenic species complex.</p></div

Sexual cycles in <i>Cryptococcus</i>.

<p>During <i>C</i>. <i>neoformans</i> bisexual reproduction, <b>a</b>-α cell-cell fusion generates dikaryotic hyphae and karyogamy occurs inside the basidia. During <i>C</i>. <i>deneoformans</i> bisexual reproduction, karyogamy takes place at different stages and generates both dikaryotic and monokaryotic diploid hyphae. During <i>C</i>. <i>deneoformans</i> unisexual reproduction, diploidization in the hyphae is achieved early during differentiation through either endoreplication or cell fusion-independent karyogamy events. Cell fusion plays less significant roles during solo unisexual reproduction.</p

Deletion of <i>PRM1</i> blocks cell-cell fusion and clamp cell fusion during <i>Cryptococcus neoformans</i> bisexual reproduction.

<p><b>(A)</b> Mating phenotypes for a wild type cross between H99α and KN99<b>a</b> and two independent <i>prm1</i> bilateral mutant crosses between CF30 and CF448, and between CF56 and CF562. All mating patches were spotted on MS medium and incubated in the dark at room temperature. The top row shows hyphal growth on the edge of mating patches five days after inoculation. The scale bar is 100 μm. The bottom row shows the basidium and spore chain morphology (indicated by arrows)10 days after inoculation. The scale bar equals 20 μm. <b>(B)</b> Unilateral and bilateral <i>prm1</i> mutant cell fusion frequency compared to wild type. <b>(C)</b> Scanning electron microscopy of clamp cell morphology of wild type cross (H99α X KN99<b>a</b>) and <i>prm1</i> bilateral mutant cross (CF56 X CF562). The scale bar is 5 μm. <b>(D)</b> DAPI staining of mature hyphae and nuclei inside hyphae and basidia of <i>C</i>. <i>neoformans</i> wild type (left panel) and <i>prm1</i> (right panel) bisexual crosses. Arrows in the <i>prm1</i> column indicate nuclei trapped in unfused clamp cells. The scale bar is 5 μm. Gene expression patterns for <b>(E)</b> <i>PRM1</i> and <b>(F)</b> <i>MF</i>α were examined by RT-PCR (* indicates <i>p</i> <0.05 and ** indicates <i>p</i> <0.005 for each pairwise comparison). A wild type cross (H99α X KN99<b>a</b>) was grown on YPD medium for 36 hours, and on V8 medium for 36 hours or one week. The <i>prm1</i> bilateral mutant cross (CF56 X CF562) was grown on V8 medium for 36 hours. The Y axis for panel F is in base-2 log scale. The error bars represent the standard deviation of the mean for the three biological replicates.</p

Karyogamy occurs at different stages during <i>C</i>. <i>deneoformans</i> bisexual reproduction.

<p>GFP labeled nucleolar marker Nop1 and mCherry labeled nuclear marker Histone H3 protein were used to study the formation of monokaryotic hyphae during <i>C</i>. <i>denoeformans</i> bisexual mating (CF830 α <i>NOP1-GFP-NAT</i> X CF1076 <b>a</b> <i>H3-mCherry-NAT</i>). <b>(A)</b> Nuclear congression occurs in <b>a</b>-α fused cells. <b>(B)</b> Nuclear fusion occurs in <b>a</b>-α fused cells. Arrow points to the fused nucleus, as indicated by the mixing of the fluorescent signals. <b>(C-D)</b> <i>C</i>. <i>deneoformans</i> bisexual reproduction produces both <b>(C)</b> dikaryotic hyphae with fused clamp cells and <b>(D)</b> monokaryotic hyphae with unfused clamp cells. Single hyphal compartments are marked with dotted circles. Arrows point to nuclei labeled with both GFP and mCherry. Arrowheads point to a fused clamp cell in panel C and an unfused clamp cell in panel D. The scale bar is 5 μm. <b>(E-F)</b> Quantification of monokaryon and dikaryon fusion products <b>(E)</b> or mature hyphae <b>(F)</b> for wild type (CF830 α <i>NOP1-GFP-NAT</i> X CF1076 <b>a</b> <i>H3-mCherry-NAT</i>) and <i>kar5</i> mutant (CF1185 α <i>kar5</i>Δ::<i>NEO H3-mCherry-NAT</i> X CF723 <b>a</b> <i>kar5</i>Δ::<i>NEO NOP1-GFP-NAT</i>) crosses. Representative dikaryon and monokaryon fusion products are shown on the right. Single hyphal compartments are marked with dotted circles, and each arrow points to one nucleus. The scale bar is 5 μm.</p

Ploidy determination by FACS for blastospores produced during unisexual reproduction.

<p>The upper left panel is the diagram for dissection of blastospores. The circle at left is before and the circle at right is after the blastospores were removed for dissection. The upper middle and right panels are FACS results for haploid control XL280α and diploid control MN142.6 α/α. The middle and lower panels are representative FACS results for blastospores produced by the indicated strains. Wild type XL280α, <i>prm1</i>Δ, <i>prm1</i>Δ <i>spo11</i>Δ, and <i>kar5</i>Δ produced diploid blastospores, whereas, <i>kar5</i>Δ <i>spo11</i>Δ produced both haploid and diploid blastospores.</p

<i>prm1</i> mutants are defective in plasma membrane fusion during <i>C</i>. <i>deneoformans</i> bisexual reproduction.

<p><b>(A)</b> Mating phenotypes for a wild type cross between JEC21α and JEC20<b>a</b> and two independent <i>prm1</i> bilateral mutant crosses between CF1 and CF313, and CF316 and CF517. The scale bars are 100 μm and 20 μm for top row and bottom row, respectively. <b>(B)</b> Unilateral and bilateral <i>prm1</i> mutant cell fusion frequency compared to wild type. <b>(C)</b> CF712 (JEC21α <i>prm1Δ</i>::<i>NAT mCherry-NEO</i>) was mated with CF768 (JEC20<b>a</b> <i>prm1Δ</i>::<i>NEO NOP1-GFP-NAT</i>) on V8 medium for 24 hours. Confocal microscopy showed that both fused and unfused cell fusion pairs were present during bilateral <i>prm1</i> mutant mating based on the presence or absence of fluorescent signal intermixing between fusion partners. The scale bar is 5 μm. <b>(D)</b> Wild type mating between CF830 (JEC21α <i>NOP1-GFP-NAT</i>) and JEC20<b>a</b>, unilateral mating between JEC21α and CF768 (JEC20<b>a</b> <i>prm1Δ</i>::<i>NEO NOP1-GFP-NAT</i>), bilateral mating between CF1 (JEC21α <i>prm1Δ</i>::<i>NEO</i>) and CF768 (JEC20<b>a</b> <i>prm1Δ</i>::<i>NEO NOP1-GFP-NAT</i>), and bilateral mating between CF487 (JEC21α <i>kar5</i>Δ::<i>NEO</i>) and CF723 (JEC20<b>a</b> <i>kar5</i>Δ::<i>NEO NOP1-GFP-NAT</i>) were conducted and the cell fusion frequency was determined based on GFP fluorescence signal intermixing between fusion partners. <b>(E)</b> Gene expression patterns for <i>PRM1</i> were examined by RT-PCR (* indicates <i>p</i> <0.05 and ** indicates <i>p</i> <0.005 for each pairwise comparison). Wild type cross (JEC21α X JEC20<b>a</b>) was grown on YPD medium for 36 hours, and on V8 medium for 36 hours or one week. The error bars represent the standard deviation of the mean for the three biological replicates. <b>(F)</b> Fusion pairs between CF830 (JEC21α <i>NOP1-GFP-NAT</i>) and JEC20<b>a</b>, and between CF1 (JEC21α <i>prm1Δ</i>::<i>NEO</i>) and CF768 (JEC20<b>a</b> <i>prm1Δ</i>::<i>NEO NOP1-GFP-NAT</i>) were stained with FM4-64 to show the plasma membrane structures at the conjugation sites. The scale bar is 20 μm. <b>(G)</b> Fused wild type cell fusion pair (top panel) between CF830 (JEC21α <i>NOP1-GFP-NAT</i>) and CF1076 (JEC20<b>a</b> <i>H3-mCherry-NAT</i>) and unfused cell fusion pair (bottom panel) between CF712 (JEC21α <i>prm1Δ</i>::<i>NAT mCherry-NEO</i>) and CF768 (JEC20<b>a</b> <i>prm1Δ</i>::<i>NEO NOP1-GFP-NAT</i>) were examined by transmission electron microscopy. Membrane structures at the conjugation sites were further examined at higher magnification. In the left panels, the scale bars are 2 μm, and in the right panels, the scale bars are 0.5 μm.</p

Deletion of <i>KAR5</i> causes a sporulation defect during <i>C</i>. <i>neoformans</i> bisexual reproduction.

<p><b>(A)</b> Mating phenotypes for a wild type cross between H99α and KN99<b>a</b> and two independent <i>kar5</i> bilateral mutant crosses between CF57 and CF549, and CF208 and CF305. The scale bars are 100 μm and 20 μm for top row and bottom row, respectively. <b>(B)</b> Unilateral and bilateral <i>kar5</i> mutant cell fusion frequency compared to wild type. <b>(C)</b> Scanning electron microscopy of basidium morphology and sporulation patterns (indicated by arrows) for wild type cross (H99α X KN99<b>a</b>) and <i>kar5</i> bilateral mutant cross (CF57 X CF549). The scale bar is 5 μm. <b>(D)</b> DAPI staining of nuclei inside basidia from <i>C</i>. <i>neoformans</i> wild type (left panel) and <i>kar5</i> mutant (right panel) bisexual crosses. Basidia indicated by white boxes were magnified to show nuclei morphology. The scale bar is 5 μm. <b>(E)</b> Quantification of pre-karyogamy and post-karyogamy events for wild type and <i>kar5</i> mutant crosses based on DAPI staining of nuclei inside basidia. Representative pre-karyogamy (two nuclei) and post-karyogamy (one nucleus and post meiosis) events were shown on the right. The scale bar is 5 μm. <b>(F)</b> Gene expression patterns for <i>KAR5</i> were examined by RT-PCR (* indicates <i>p</i> <0.05 and ** indicates <i>p</i> <0.005 for each pairwise comparison). Wild type cross (H99α X KN99<b>a</b>) was grown on YPD medium for 36 hours, and on V8 medium for 36 hours or one week. <i>prm1</i> bilateral mutant cross (CF56 X CF562) and <i>kar5</i> bilateral mutant cross (CF57 X CF549) were grown on V8 medium for 36 hours. The error bars represent the standard deviation of the mean for the three biological replicates. <b>(G)</b> Proposed <i>C</i>. <i>neoformans</i> bisexual reproduction model. <i>PRM1</i> is required for cell-cell fusion and clamp cell fusion, and <i>KAR5</i> is required for karyogamy inside the basidium.</p

<i>kar5</i> mutants are defective in cell fusion and basidium sporulation during <i>C</i>. <i>deneoformans</i> bisexual reproduction.

<p><b>(A)</b> Mating phenotypes for a wild type cross between JEC21α and JEC20<b>a</b> and two independent <i>kar5</i> bilateral mutant crosses between CF226 and CF364, and CF487 and CF464. The scale bars are 100 μm and 20 μm for top row and bottom row, respectively. <b>(B)</b> Scanning electron microscopy of basidia morphology and sporulation patterns (indicated by arrows) for wild type cross (JEC21α X JEC20<b>a</b>) and <i>kar5</i> bilateral mutant cross (CF226 X CF364). The scale bar is 5 μm. <b>(C)</b> Unilateral and bilateral <i>kar5</i> mutant cell fusion frequency compared to wild type. Gene expression patterns for <b>(D)</b> <i>KAR5</i> and <b>(E)</b> <i>MF</i>α were examined by RT-PCR (* indicates <i>p</i> <0.05 and ** indicates <i>p</i> <0.005 for each pairwise comparison). Wild type cross (JEC21α X JEC20<b>a</b>) was grown on YPD medium for 36 hours, and on V8 medium for 36 hours or one week. <i>prm1</i> bilateral mutant cross (CF1 X CF313) and <i>kar5</i> bilateral mutant cross (CF226 X CF364) were grown on V8 medium for 36 hours. The error bars represent the standard deviation of the mean for the three biological replicates. <b>(F)</b> The GFP labeled nucleolar marker Nop1 was used to study <i>C</i>. <i>neoformans</i> bisexual cross (CF830 α <i>NOP1-GFP-NAT</i> X JEC20<b>a</b>) hyphal nuclear morphology. Arrows indicate instances of both monokaryotic and dikaryotic hyphae during early stages of bisexual reproduction, and the area within the box was magnified to highlight two nuclei in close contact.</p

<i>PRM1</i> and <i>KAR5</i> are largely dispensable for unisexual reproduction.

<p><b>(A)</b> Mating phenotypes for wild type XL280α, two independent <i>prm1</i> mutants (CF317 and CF659), and two independent <i>kar5</i> mutants (CF150 and CF260) during <i>C</i>. <i>deneoformans</i> unisexual reproduction. The scale bars are 100 μm and 20 μm for top row and bottom row, respectively. <b>(B)</b> Gene expression patterns for <i>PRM1</i>, <i>KAR5</i>, and <i>MF</i>α were examined by RT-PCR (* indicates <i>p</i> <0.05 and ** indicates <i>p</i> <0.005 for each pairwise comparison). Wild type (XL280α) was grown on YPD medium for 36 hours, and on V8 medium for 36 hours or one week. <i>prm1</i> mutant (CF317) and <i>kar5</i> mutant (CF150) were grown on V8 medium for 36 hours. The error bars represent the standard deviation of the mean for the three biological replicates. <b>(C)</b> Comparison of wild type cell-cell fusion frequency among three different sexual cycles between <i>C</i>. <i>neoformans</i> and <i>C</i>. <i>deneoformans</i>. <b>(D)</b> Unilateral and bilateral <i>prm1</i> mutant and <i>kar5</i> mutant cell fusion frequency compared to wild type. <b>(E)</b> Scanning electron microscopy of basidium morphology and sporulation patterns (indicated by arrows) of the wild type (XL280α) and the <i>kar5</i> mutant (CF150). The scale bar is 5 μm.</p