pph22Δ mutants exhibit reduced mating efficiency and abnormal sexual structures

We previously showed that deletion of PPH22 causes severe mating defects during α-a sexual reproduction, as well as in a self-fertile α/a diploid strain, leading to significantly delayed sexual development [30]. To further investigate the dynamics of pph22Δ mutants during mating, pph22Δ strains were crossed with a wild-type partner of the opposite mating type and incubated on MS medium. To overcome the growth defects of pph22Δ on nutrient-limited MS media, mating was facilitated by pre-growing mutant cells before adding a smaller amount of wild-type cells, as described previously [30]. After six weeks of incubation, wild-type crosses displayed extensive hyphal growth, basidia formation, and basidiospore production. In contrast, pph22Δ x wild type mating patches showed only limited signs of sexual development (Fig 1A). Most patches lacked visible sexual structures, though occasional hyphae and basidia were observed. Basidia were often bald or produced few spores, and many spores appeared collapsed rather than forming the elongated spore chains characteristic of wild-type mating.

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Fig 1. pph22Δ x wild-type crosses exhibit unusual sexual development.

A) Mating efficiency of pph22Δ strains crossed with wild-type of the opposite mating type (H99α or KN99a). Images were taken between 4 to 6 weeks of incubation on MS plates. Scale bars in the middle and bottom panels are 200 μm and 20 μm, respectively. B) Scanning electron microscopy (SEM) of sexual structures in WT x WT and pph22Δ x WT crosses showing examples of a hyphal filament terminating in a basidial head and basidiospores emerging from a basidium. The clamp cells (white arrowheads) in the wild-type cross are fused to the subapical cell in the elongating hypha. In pph22Δ x WT crosses, both fused and unfused clamp cells were often observed along the same hypha. Scale bars represent 5 μm.

https://doi.org/10.1371/journal.pgen.1011774.g001

To observe morphological differences in sexual structures between pph22Δ and wild-type crosses at a higher resolution, scanning electron microscopy (SEM) of the mated strains was performed. In pph22Δ x wild type crosses, many basidia were considerably rounded and enlarged compared to the wild-type cross and did not produce spores (Fig 1B). When spores were observed, they often had a collapsed appearance, similar to what was seen by light microscopy (Fig 1A). During α-a sexual reproduction in the wild type x wild type cross, the clamp cells, which are critical for faithful segregation of nuclei in the dikaryotic hyphae [41,42], were fused to the subapical cell in the elongating hyphal branch. In mated pph22Δ x wild type, both fused and unfused clamp cells could be observed along the same hypha, suggesting a potential defect in nuclear distribution or clamp cell fusion. Due to the observed defects in mating, we quantified cell–cell fusion frequency by incubating crosses between pph22Δ::NAT and wild-type::NEO strains on MS medium for three days. After incubation, cells were harvested and plated on YPD supplemented with either NAT, NEO, or both NAT and NEO. Crosses between wild-type::NAT and wild-type::NEO strains were included as a control. Fusion events were quantified by counting colony-forming units (CFUs) on YPD + NAT + NEO plates, which represent dual-resistant fusion products. Fusion frequency was calculated as the number of CFUs on YPD + NAT + NEO divided by the sum of CFUs on YPD + NAT and YPD + NEO, representing the total recovered population. A significant reduction in fusion frequency was observed in pph22Δa × WTα and pph22Δα × WTa crosses, corresponding to 5.1% and 5.7%, respectively, of the fusion frequency in the wild-type x wild-type control (S1A Fig). Taken together, these results suggest that PPH22 is critical for α-a sexual mating and proper sexual development.

Progeny from pph22Δ x wild-type crosses are produced exclusively through pseudosexual reproduction

To further investigate the reproductive mode of pph22Δ x WT crosses, spores were dissected from individual basidia for phenotypic and genotypic analysis. pph22Δ mutant strains have severe growth defects and form significantly smaller colonies compared to wild type. Germinated spores from pph22Δ x WT crosses formed colonies that were fast growing and uniform in size on YPD (S1B Fig). Streaking onto YPD and YPD + NAT (selecting for the pph22Δ::NAT mutation) showed that all dissected spores were NAT-sensitive, indicating the absence of pph22Δ mutants among the recovered progeny, and potentially suggesting that the spores originated from unisexual reproduction of the wild-type parental strain, in which a single cell can undergo diploidization, filamentation, and sporulation to produce recombinant haploid progeny [43]. To investigate this possibility, spores were dissected from individual basidia of two independent pph22Δa × WTα crosses and one reciprocal pph22Δα × WTa cross to yield viable progeny. PCR genotyping of the STE20 gene in the mating-type locus (MAT) revealed that all progeny from each individual basidium contained either the MATa or the MATα allele, which strictly corresponded to the allele from the wild-type parent (Table 1 and S1C Fig). Importantly, the vast majority of basidia showed high germination rates (>75%, Table 1), suggesting the absence of the pph22Δ allele among the progeny was not due to failure of mutant progeny to germinate.

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Table 1. Genotyping analysis of spores from individual basidia and from random spore dissection in pph22Δ x wild-type crosses. Spot refers to the individual mating spot from which those basidia were dissected in the indicated crosses, with each spot being an independent experiment. "Mito." refers to mitochondria.

https://doi.org/10.1371/journal.pgen.1011774.t001

Additionally, PCR of the COX1 gene indicated that the mitochondria was predominantly inherited from the MATa parent, consistent with mitochondrial uniparental inheritance (mito-UPI) in Cryptococcus [39,44]. MATα mitochondrial inheritance was observed in pph22Δa × WTα crosses at a frequency of 32% (7 out of 22 basidia), which is higher than the expected frequency of MATα mitochondria leakage of around 5% to 10% [45]. However, six of these basidia originated from the same mating spot, leaving open the possibility that the inherited MATα mitochondria in the progeny resulted from a single mating event. Similarly, in random spore dissection, 15 out of 65 germinated progeny possessed MATα mitochondria, but were dissected from a single mating spot. It is unclear whether the pph22Δ mutation leads to increased MATα mitochondrial inheritance. These results suggest that progeny from pph22Δ × WT inherited only the wild-type parental nuclei. Additionally, the presence of mitochondria from the MATa parent in MATα progeny indicates that these progeny are not the result of unisexual reproduction. These observations demonstrate that the mode of reproduction in pph22Δ × WT crosses is pseudosexual, during which cells of the opposite mating type fuse but the nuclear content of only one parental nucleus is transmitted to the progeny [38].

Following PCR genotyping assays, whole-genome sequence analysis was conducted to determine if there was any evidence of meiotic recombination in the F1 progeny from pph22Δa × WTα. The pph22Δ strains utilized in this study were obtained from dissection of a KN99a/α PPH22/pph22Δ heterozygous diploid mutant and are congenic with H99 [30,46]. Besides the mating-type locus on chromosome 5, there are two other genomic regions on chromosomes 11 and 14 that differ between H99α and KN99a background strains [47,48]. Twelve progeny from a single basidia (Table 1, spot D basidium 1) were subjected to Illumina whole-genome sequencing and reads were aligned to the H99α reference genome for variant calling and SNP analysis (Fig 2). The KN99a pph22Δ parental strain and the KN99a/α diploid strain (CnLC6693) from which it was derived served as controls. Variant analysis confirmed that only the H99α nuclear genome was inherited in the progeny, as evidenced by the absence of SNPs at the indicated polymorphic loci. Alignment of reads from the KN99 parental strains and progeny to the H99α mitochondrial genome also confirmed the presence of the intron in the KN99a COX1 gene, supporting the results from PCR genotyping. Collectively, the lack of meiotic recombination supports that the progeny from mating of pph22Δ × WT arose through a pseudosexual reproductive process, and not via α-a sexual reproduction or unisexual reproduction.

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Fig 2. Progeny from pph22Δ x wild type exhibit uniparental nuclear inheritance from H99α and lack evidence of meiotic recombination.

Whole-genome sequencing followed by SNP analysis revealed no contribution of the KN99a pph22Δ parental genome, as evidenced by the absence of SNPs compared to the H99α reference genome. The three known polymorphic regions distinguishing H99 and KN99 background strains are located on chromosomes 5 (the mating-type locus), 11, and 14. These regions are highlighted in the lower panels, with the chromosomal coordinates labeled accordingly. The KN99a/KN99α diploid strain (CnLC6683), from which KN99a pph22Δ was obtained, was used as a control for variant calling. The few SNPs identified in the progeny were restricted to nucleotide repeat regions. Mitochondrial haplotypes were determined based on the presence or absence of an intron in the COX1 gene from whole-genome sequencing reads. “Germ.” stands for the germination frequency of the indicated basidium and “P” stands for progeny.

https://doi.org/10.1371/journal.pgen.1011774.g002

Uniparental nuclear inheritance observed after mating via fluorescence microscopy

Previous studies with fluorescently marked nuclei provided evidence that pseudosexual reproduction in Cryptococcus results from loss of one parental nucleus during hyphal branching following mating [38]. A similar approach was taken to investigate nuclear migration during mating in pph22Δ × WT crosses. The gene encoding the nucleolar protein Nop1 was fused with GFP or RFP (mCherry) and introduced ectopically into the safe haven region of pph22Δa (NOP1-GFP-NEO), KN99a (NOP1-GFP-NEO), and H99α (NOP1-mCherry-HYG) [49]. Homologous recombination at the target locus was confirmed by PCR genotyping. pph22Δ and KN99a strains expressing Nop1-GFP were crossed with H99α expressing Nop1-RFP on MS media and monitored for signs of mating (S2A Fig). In wild-type crosses, live-cell imaging revealed hyphae containing both GFP- and RFP-tagged proteins following cell-cell fusion, maintaining a stable dikaryotic state throughout hyphal elongation and branching, with green and red fluorescent signals overlapping or appearing in close proximity (S2B Fig). In the basidial heads, the final stages of the sexual cycle were observed, including karyogamy, where the two parental nuclei fused, followed by meiosis and subsequent inheritance of fluorescent tags from both parents in the haploid basidiospores. In total, 27 basidia from two independent crosses were observed to produce spores that contained both fluorescent markers.

Nuclear dynamics in pph22Δ crosses were notably distinct from those in wild-type crosses. As expected, pph22Δa x H99α exhibited decreased mating efficiency, but spore production was still observed after six to eight weeks of incubation (S2A Fig). Fluorescence imaging showed that both red and green nuclear markers initially coexisted within the same hyphal compartment and could be tracked along the elongating hyphal branch. However, at certain branch points, the green pph22Δ nucleus was selectively lost, allowing only the red wild-type nucleus to undergo meiosis in the terminating basidium (S2C Fig). Among 40 basidia analyzed from four independent crosses, 15 did not produce spores. Of these, 12 displayed both red and green signals in the basidium head, while the remaining three showed only the red signal. The other 25 basidia successfully produced spores, with 20 containing only the signal from the wild-type parent. This finding aligns with previous work suggesting that hyphal branching may facilitate nuclear exclusion, which can be followed by endoreplication and meiosis of the retained nucleus [38].

In contrast, there were also cases in crosses of pph22Δ x WT where the wild-type nucleus was lost following hyphal branching, after which the resulting sexual structures exhibited abnormal morphology (Fig 3). Several observations suggested that, after the loss of the red wild-type signal in a hyphal branch, the green pph22Δ nucleus could undergo endoreplication followed by nuclear division, resulting in two green nuclei within a single hyphal compartment, indicating a mechanism by which a dikaryotic-like state could be reestablished (Fig 3A). There were also instances where, following wild-type nuclear exclusion, only the pph22Δ fluorescent signal was inherited in the basidiospores, which appeared collapsed onto the basidium head (Fig 3B and 3C). Notably, five basidia that produced spores exhibited only the green signal in the progeny, suggesting that the pph22Δ mutant nucleus is capable of migrating into the basidium, undergoing diploidization, and potentially entering the meiotic process. However, despite this apparent progression, the consistent inability to recover pph22Δ mutant progeny from any dissection of pph22Δ x WT crosses, indicates a meiotic or post-meiotic defect that prevents successful germination. There was also evidence that maintenance of only the pph22Δ nucleus negatively impacted hyphal morphology, significantly altering both the shape and structure. Additionally, in another hyphal branch, there were two nuclei detected for each color to yield four nuclei in individual cells, suggesting a failure in nuclear sorting or segregation following DNA replication (Fig 3D). Together with spore genotyping analyses, these findings suggest that: 1) pseudosexual reproduction may occur through the loss of one parental nucleus during hyphal branching and 2) the pph22Δ mutation disrupts normal sexual development, including nuclear migration, hyphal morphology, and sporulation.

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Fig 3. Fluorescence microscopy depicts loss of one parental nucleus following hyphal branching during pseudosexual reproduction.

A) Loss of the wild-type nucleus at a hyphal branch point in pph22Δ Nop1-GFP x WT Nop1-RFP. B) and C) Inheritance of only the pph22Δ nucleus in basidiospores, which appear collapsed on the basidial head and have an irregular morphology, is observed after loss of the WT nucleus in a hyphal compartment (B) and following branching (C). D) Loss of the wild-type nucleus and retention of the pph22Δ nucleus results in severely abnormal hyphal development (white arrowhead). In another hyphal branch, both nuclei have undergone division in the dikaryon without segregating into separate hyphal cells (grey arrowheads). The white arrowheads in A – C indicate the point in the hypha where one parental nucleus is lost. The scale bar in each panel represents 5 μm.

https://doi.org/10.1371/journal.pgen.1011774.g003

α-a sexual reproduction is restored in pph22Δ suppressor mutants

pph22Δ strains can accumulate spontaneous suppressor mutations that restore mating, hyphal growth, basidia production, and sporulation nearly to the wild-type level (Fig 4A) [30]. To determine if the pph22Δ suppressor (sup) mutant strains could also suppress pseudosexual reproduction, F1 progeny from pph22Δ sup x WT crosses were analyzed. In one such cross, germinated spores containing the NAT drug-resistance marker were recovered during dissection, in contrast to strictly NAT-sensitive pph22Δ x WT progeny, providing evidence the suppressor mutation could restore pph22Δ spore viability following α-a sexual mating, although the resulting colonies were notably smaller in size (Fig 4B). The frequency of pph22Δ::NAT among the progeny was 45%, consistent with the expected segregation of a single selectable allele. However, in crosses with two other independently derived pph22Δ sup strains, no NAT resistant progeny were recovered, suggesting incomplete suppression of pph22Δ mutant defects following mating in those strains.

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Fig 4. The pph22Δ suppressor strains can restore α-a sexual mating in crosses with wild type.

A) Mating efficiencies in wild-type and pph22Δ sup crosses. Hyphal production and elongation, and sporulation were observed to similar extents. B) Germination of spores dissected from pph22Δ sup x WT after two days of incubation on YPD. Each row depicts spores from separate basidia. Colonies that grew on YPD + NAT (containing the pph22Δ::NAT mutant allele) are indicated by the white boxes. C) Genotyping analysis of progeny dissected from pph22Δ sup strains crossed with the KN99a strain bearing recombinant mitochondria (SSH118). Each spot is from a separate mating patch and is considered an independent experiment.

https://doi.org/10.1371/journal.pgen.1011774.g004

We next investigated mating type and mitochondrial genome segregation in pph22Δ sup x WT crosses. All pph22Δ sup isolates that were obtained in our previous study possess the MATα allele and therefore were crossed with KN99a. This led to a complication in using the same PCR genotyping strategy to determine how the mitochondria were inherited in the progeny from these crosses, as KN99a and KN99α background strains have identical mitochondria genomes. Therefore, pph22Δ sup strains were crossed with the KN99a strain SSH118, which carries a recombinant mitochondrial genome, and restriction fragment length polymorphism (RFLP) analysis was used to identify the parental source of the mitochondrial genome in the progeny (Figs 4C and S3). In each pph22Δ sup x WT cross, the basidia that were dissected each came from a different mating spot, so that the progeny analyzed were the result of independent mating events. The MAT allele was determined through PCR genotyping of the STE20 gene, as in previous experiments, and mitochondrial type was identified through PCR amplification of COX1 followed by BsrI digestion and RFLP analysis (S3B, S3C, and S3D Fig). In two independent crosses, both a and α mating types were found among the genotyped basidia and, interestingly, many progeny were heterozygous for the MAT allele (Figs 4C and S3E). However, in a third independent cross with a different pph22Δ sup strain, progeny of only one mating type were recovered, consistent with either unisexual or pseudosexual reproduction. The fact that progeny exhibiting MAT heterozygosity also came from basidia with high germination rates suggests that this cannot be explained by aneuploidy alone, as an imbalance in chromosome number in aneuploid spores would lead to reduced germination rates.

Flow cytometry analysis of progeny heterozygous for the MAT locus revealed that increased DNA content correlated with the presence of the pph22Δ::NAT deletion allele (S3F Fig). Specifically, NAT-sensitive progeny remained haploid, while all NAT-resistant progeny displayed peaks at 1N, 2N, and 4N, suggesting altered DNA replication or disrupted ploidy regulation in these cells. These results suggest that PPH22 may have a role in ploidy regulation, and its loss may disrupt cell cycle regulation and lead to altered DNA inheritance patterns. Further analysis, such as whole-genome sequencing, could help clarify which process may be maintaining MAT heterozygosity in the NAT-sensitive progeny. While mitochondria in the majority of progeny were inherited from the MATa parent, progeny from two out of seven basidia from independent mating spots in one of the pph22Δ sup x WT crosses inherited mitochondria from the MATα parent (S3E Fig). Together, with the genotyping results from pph22Δ x WT crosses, these findings suggest that deletion of PPH22 may also influence mitochondrial inheritance. Although α-a sexual reproduction could be restored in the pph22Δ sup crosses, the observed outcome was atypical, characterized by an unexpected segregation of mating-type alleles.

Overexpression of PPG1 suppresses loss of PPH22

All independently obtained pph22Δ sup mutant strains were found to be aneuploid for a segment of chromosome 6 encompassing seven genes [30]. Of these genes, PPG1, which encodes a putative PP2A catalytic subunit and shares significant sequence similarity with PPH22, was overexpressed 40-fold in the pph22Δ sup mutants compared to wild type [30]. To determine if PPG1 overexpression was the causative mutation suppressing pph22Δ phenotypes, a PPG1 allele with a constitutively active promoter (P_TEF1-PPG1-NEO) was integrated into the safe haven 1 of the PPH22/pph22Δ heterozygous mutant diploid strain and homologous recombination was confirmed through PCR genotyping. Following self-filamentation on MS media, random spores were dissected and germinated on YPD (Fig 5A). Colonies containing both NAT and NEO drug resistance markers, confirmed by growth on selective media and PCR genotyping, were faster growing and larger in size than pph22Δ mutant colonies, suggesting that overexpression of PPG1 can partially rescue growth defects of pph22Δ.

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Fig 5. Overexpression of PPG1 suppresses the pph22Δ mutation.

A) Germination of spores dissected from a PPH22/pph22Δ heterozygous mutant diploid strain expressing TEF1-PPG1 (PPG1-OE) on YPD. The genotypes of the colonies indicated were determined from growth on YPD + NAT (pph22Δ::NAT allele) and YPD + NAT + NEO (pph22Δ::NAT and P_TEF1-PPG1-NEO alleles). B) The indicated strains were serially diluted and grown on YPD solid media at 25⁰C, 30⁰C, and 37⁰C; or YPD supplemented with 1 M sorbitol, 100 ng/mL rapamycin, 1 μg/mL FK506, or 100 μg/mL cyclosporine A, each at 30⁰C. Images were taken after 3-4 days of incubation. C) Assay of melanin production. Strains were grown in liquid YPD media overnight at 30⁰C and plated onto Niger seed agar. Images were captured after four days of incubation at 30⁰C. D) Analysis of capsule formation by India ink staining. The indicated strains were grown for 3 days in RPMI media at 30⁰C. Images are representative of two biological replicates. Scale bar is 5 μm. E) Mating of ppg1Δ and pph22Δ PPG1-OE strains crossed to wild type on MS media. Images were taken after six weeks of incubation in the dark. White arrowheads indicate bald basidia heads that did not produce spores. The scale bars in the middle and bottom panels are 200 μm and 50 μm, respectively.

https://doi.org/10.1371/journal.pgen.1011774.g005

Next, to examine the role of PPG1 overexpression in response to different nutrient and stress conditions, pph22Δ PPG1-OE, pph22Δ, pph22Δ sup, ppg1Δ, and wild-type strains were serially diluted and spotted onto YPD at 25⁰C, 30⁰C, and 37⁰C; YPD with 1 M sorbitol; YPD with rapamycin; and YPD with the immunosuppressive drugs FK506 or cyclosporine A (CsA) (Fig 5B). pph22Δ exhibited severe growth defects or no growth on YPD at 25⁰C, 30⁰C, and 37⁰C, as well as on YPD supplemented with sorbitol, FK506, or cyclosporine A, which was partially reversed in the pph22Δ sup strain, consistent with previously published results. On YPD supplemented with rapamycin, both pph22Δ sup and pph22Δ PPG1-OE strains displayed significant growth advantages. Collectively, overexpression of PPG1 in the pph22Δ mutant was able to partially or fully restore growth to wild-type levels under each condition tested, suggesting that P_TEF1-PPG1 is more effective at compensating for pph22Δ than overexpression of PPG1 due to aneuploidy in pph22Δ sup strains.

It was surprising that ppg1Δ did not phenocopy pph22Δ, and only exhibited severe growth defects on YPD at 25⁰C and on YPD supplemented with sorbitol or rapamycin, compared to growth on YPD at 30⁰C. In fact, ppg1Δ displayed similar growth to pph22Δ PPG1-OE under several conditions, including growth at 30⁰C and 37⁰C as well as in the presence of FK506 and CsA. These results suggest that while Pph22 and Ppg1 are hypothesized to have similar functions and may play partially compensatory roles, Pph22 is likely the primary catalytic subunit of PP2A, as its loss leads to more severe effects than the loss of PPG1. Next, the abilities of ppg1Δ and pph22Δ PPG1-OE strains to produce melanin pigment and polysaccharide capsule, two important virulence factors to protect cells from the host immune response during Cryptococcus infection, were assessed. To induce melanin and capsule production, cells were grown on Niger seed agar and in RPMI media, respectively, with both incubated at 30⁰C. pph22Δ sup and pph22Δ PPG1-OE produced similar amounts of melanin and were comparable to the wild type, while ppg1Δ could not produce melanin, similar to the lac1Δ negative control (Fig 5C). These results suggest that PPG1 is required for melanin production, while PPG1 overexpression in the pph22Δ background is sufficient to restore this virulence trait. India ink counterstaining of RPMI-grown cells showed that PPG1 overexpression restored normal cell morphology in the pph22Δ mutant, though capsule production was still impaired (Fig 5D). In contrast, ppg1Δ cells could produce capsule, albeit to a lesser extent than wild-type cells. Together, these findings are consistent with the idea that PPG1 can functionally compensate for the loss of PPH22 in certain contexts.

To further explore the functional differences between Pph22 and Ppg1, 3D structural models of the STRIPAK complex with either Pph22 or Ppg1 as the catalytic subunit were generated with AlphaFold3 [50]. The resulting Pph22-containing structure was comparable to the previously published model generated with AlphaFold2, though slight differences in folding were observed [30,51]. The two models appeared largely similar, indicating that Ppg1 can potentially assemble into a complex with the other STRIPAK subunits (S4A and S4B Fig). Cryo-EM studies of the human STRIPAK complex have demonstrated that Far8 forms a homotetramer, serving as a scaffold at the base of the complex [12]. To assess whether Cryptococcus Far8 could similarly assemble into a tetramer, a structural model was generated using four copies of the Far8 protein (S4C Fig). This model was then combined with either the Pph22-Tpd3 or Ppg1-Tpd3 heterodimers to examine PP2A assembly. Both models appeared highly similar overall, though the four Far8 proteins adopted slightly different conformations in each structure (S4D and S4E Fig). These findings suggest that both Pph22 and Ppg1 can potentially integrate into STRIPAK in a comparable manner, perhaps filling analogous roles within the complex.

Finally, the involvement of Ppg1 during mating was examined by crossing ppg1Δ and pph22Δ PPG1-OE strains with wild type. Similar to what has been observed in pph22Δ crosses, ppg1Δ x WT exhibited delayed mating and hyphae were produced only in isolated spots on the mating patch after six weeks of incubation (Fig 5E compared to Fig 1A). With further incubation, more extended hyphal branches were produced but mostly bald basidia heads with almost no spore formation. In contrast, pph22Δ PPG1-OE x WT crosses displayed signs of mating at a similar rate to wild type and produced abundant basidia with spore chains (Fig 5E compared to Fig 4A). These findings indicate that Ppg1 plays a crucial role during mating and sexual development, similar to Pph22. Taken together, this further supports the conclusion that PPG1 overexpression is the key mutation suppressing pph22Δ, as targeted overexpression of PPG1 in the pph22Δ background similarly restored both mating and growth deficiencies.

Pseudosexual reproduction also occurs at a high rate in far8Δ mutant crosses

It has been reported that pseudosexual reproduction occurs at a rate of ~1% between wild-type mating partners with compatible parental genomes, as well as in partners with incompatible parental genomes due to genetic divergence or genome structure variation [38,52]. The analyses presented here demonstrated that deletion of PPH22 results in exclusively pseudosexual reproduction following α-a sexual mating. To further investigate whether other mutations in the STRIPAK complex influence pseudosexual reproduction, far8Δ mutant crosses were analyzed. Far8 has also been shown to be critical during Cryptococcus sexual development, with far8Δ strains exhibiting severe defects in hyphal production and sporulation. To inspect the sexual structures of far8Δ x WT crosses in higher resolution, hyphae, basidia, and spore chains were imaged with SEM (Fig 6A). Spores were observed budding from the basidia in irregular numbers, often exceeding the typical set of four. In some cases, seven or more spores emerged independently from separate sites on a single basidium. It was previously demonstrated that far8Δ mutant strains underwent whole-genome endoreplication to become predominantly diploid [30], thus, when crossed with wild type, mating could result in triploid meiosis. This raises the possibility that the irregular spore structures observed may be influenced by the unique genomic composition in the basidium.

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Fig 6. far8Δ mutants display a high rate of pseudosexual reproduction.

A) SEM of mated wild-type and far8Δ shows basidia heads producing spores with abnormal morphology. In one of the two basidia (right), spores are seen budding from seven independent spots on the basidial head. The scale bar represents 5 μm. B) Dissection of spores from far8Δ x WT yields colonies mostly homogenous in size on YPD. far8Δ mutants were identified based on growth on YPD + NEO and form smaller, slow-growing colonies compared to wild type. Each row of spores is from an independent basidium. C) Genotyping analysis of progeny from individual basidia in far8Δ x WT crosses. Each spot analyzed is from an independent mating experiment.

https://doi.org/10.1371/journal.pgen.1011774.g006

Next, spores were dissected from individual basidia from both far8Δ a x WTα and far8Δ α x WTa crosses. Spores that germinated formed mostly homogenously sized colonies on YPD and did not contain the drug resistance marker, but occasionally small, slow-growing far8Δ mutant colonies were obtained (Fig 6B). In total, spores from 16 basidia obtained from nine independent mating spots were dissected and genotyped (Fig 6C). Given the genomic instability of triploid meiosis, reduced spore viability was expected. However, surprisingly, 10 out of 16 basidia exhibited germination rates above 70%, with an overall average of 79%, which is comparable to the germination rate observed in progeny from wild-type crosses. PCR genotyping of the MAT locus and mitochondrial DNA in progeny from 11 out of 16 dissected basidia were consistent with pseudosexual reproduction, characterized by uniparental nuclear inheritance from only the wild-type parent, a pattern also observed in pph22Δ mutant crosses, and mitochondrial inheritance from the MATa parent. Heterozygosity at the MAT locus was also detected in some progeny from the remaining five basidia of the far8Δ x WT crosses, resembling the findings in the pph22Δ sup x WT crosses. Notably, 11 out of 16 progeny possessing both MATa and MATα also had the far8Δ mutant allele, which may be attributed to the altered genomic structure of the far8Δ parental strains. These findings suggest that the far8Δ mutation also leads to haploinsufficiency during sexual development, although to a lesser extent than in pph22Δ strains. Taken together, these results suggest that loss of STRIPAK components PPH22 or FAR8 drives pseudosexual reproduction, with the mutant nuclei at a selective disadvantage to the wild-type nucleus, as shown by uniparental nuclear inheritance predominantly from the wild-type parent across multiple independent crosses (S5 Fig).

Transcriptome profiling links STRIPAK to regulation of genome integrity and development

To investigate the molecular mechanisms underlying the defects in both asexual and sexual development observed in STRIPAK complex mutants, we performed RNA-sequencing analysis of pph22Δ and far8Δ strains compared to wild type under standard growth conditions (YPD, 30°C). Differentially expressed genes (DEGs) previously identified from these datasets [30] were examined for annotations or predicted functions, based on FungiDB entries and homology to characterized fungal genes, particularly those associated with cell cycle progression and developmental processes. These genes were organized into six functional categories: (1) DNA replication and genome integrity, (2) nuclear organization, (3) chromatin structure and transcription regulation, (4) cell wall and morphogenesis, (5) cell cycle and division machinery, and (6) RNA processing and ribosome biogenesis (Fig 7). To focus on the most robust transcriptional changes, genes from the pph22Δ dataset were filtered to include only those with log₂ fold-change values greater than 0.7 or less than –0.7. The same filtered gene list was then applied to the far8Δ dataset, and heatmaps were generated for each functional group using average linkage clustering and Euclidean distance metrics (Fig 7 and S3 Table). In general, both mutants exhibited similar expression patterns within each category, though the pph22Δ strain consistently showed stronger transcriptional responses than far8Δ.

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Fig 7. Transcriptome analysis of differential gene expression changes related to growth and development in pph22Δ and far8Δ mutants.

Heatmaps show log₂ fold-changes in gene expression for selected genes in six functional categories: DNA replication and genome integrity, nuclear organization, chromatin structure and transcription, cell wall and morphogenesis, cell cycle and division machinery, and RNA processing and biogenesis. Genes in each category were selected based on functional annotations from FungiDB (https://fungidb.org/fungidb). Differentially expressed genes were filtered in pph22Δ mutants (|log₂FC| ≥ 0.7), and the same gene set was then analyzed for far8Δ. Heatmaps were generated using average linkage with Euclidean distance measurements. Gene labels are based on annotations from FungiDB or were assigned based on sequence homology to orthologous genes in other fungal species. “FC” stands for fold-change.

https://doi.org/10.1371/journal.pgen.1011774.g007

Both mutants displayed strong upregulation of genes associated with DNA replication and genome stability, including DNA repair factors RAD5, RAD9, RAD18, RAD51, RAD54, RAD57, and PIF1, the meiotic recombinase DMC1, and GINS complex components PSF1 and PSF3. This coordinated response suggests an attempt to compensate for genome instability induced by loss of PPH22 or FAR8. There was also an overall decrease in expression of genes involved in nuclear organization, such as nuclear pore components and nuclear envelope transport proteins, along with reduced expression of core histone genes, histone-modifying enzymes, and chromatin remodeling factors, suggesting disrupted nuclear dynamics and altered chromatin structure in the mutants.

Genes involved in cell wall remodeling and morphogenesis were differentially expressed in both pph22Δ and far8Δ mutants, further implicating STRIPAK in regulating cellular architecture. Multiple chitin synthase genes, including CHS1, CHS2, CHS4, CHS5, CHS7, and CHS8, were strongly upregulated, as were regulators of polarized growth and morphogenesis such as RHO3, KIN1, CPK1, and FUS3. These transcriptional changes suggest activation of compensatory pathways to reinforce or restructure the cell wall. However, this response stands in contrast to the severe morphological defects and cell wall abnormalities previously observed in these mutants, implying that transcriptional upregulation alone is insufficient to restore proper cell wall structure or morphogenesis. Moreover, upregulation of subsets of pathway components may lead to imbalances that disrupt pathway function. Conversely, key genes involved in cell wall metabolism and stress resistance, such as HOG1, TPS1, TPS2, KRE63, and GLC1, were downregulated, which may further compromise the ability to maintain cell wall integrity under stress.

The transcriptome data also revealed altered expression of genes involved in cell cycle control and RNA biogenesis, two interconnected processes affecting cellular growth and proliferation. In both pph22Δ and far8Δ mutants, genes encoding many key regulators of the cell cycle and chromosome segregation were upregulated, including the checkpoint kinase SWE1, cohesin loader SCC2, spindle assembly checkpoint component MAD2, transcriptional regulator CDK8, cohesin-associated factor PDS5, and kinetochore or spindle pole body proteins such as SPC19, SPC24, KIN4, and DAD1/3. These changes likely reflect an attempt to compensate for cell cycle stress or chromosomal instability. However, genes encoding other core cell cycle regulators were downregulated, including PHO81 and PHO85, tubulin gene TUB1, and the mitotic checkpoint proteins BUB3, CDC37, and PAT1, suggesting dysregulation throughout the cell cycle. In parallel, genes involved in RNA processing and biogenesis were predominantly downregulated, particularly those encoding translation initiation and elongation factors, ribosome assembly proteins, RNA polymerase II subunits, and pre-mRNA splicing components. In contrast, expression of several nucleolar proteins, small nucleolar RNPs (snoRNPs), and subunits of RNA polymerase I and III were increased, potentially reflecting a shift in transcriptional activity toward rRNA and tRNA biogenesis. Together, these transcriptional shifts across core cellular processes underscore the central role of the STRIPAK complex in coordinating the developmental programs in C. neoformans.

Strains, media, and growth conditions

Strains characterized in this study are listed in S1 Table. Strains were prepared for long-term storage as 20% glycerol stocks at -80°C. Fresh cultures were revived and maintained on YPD (1% yeast extract, 2% Bacto Peptone, 2% dextrose) agar medium, incubated at 30°C. Cryptococcus transformants harboring dominant drug-resistance markers were selected on YPD medium supplemented with 100 μg/mL nourseothricin (NAT), 200 μg/mL neomycin (G418), or 100 μg/mL hygromycin B (HYG). Strains were grown in YPD liquid cultures at 30°C as indicated. For plate growth assays, strains were cultivated on YPD, Murashige and Skoog (MS) (Sigma-Aldrich M5519), or Niger seed (7% Niger seed, 0.1% dextrose). Sorbitol was added to YPD medium at a 1 M concentration. To analyze cell growth in response to immunosuppressive agents, rapamycin (100 ng/mL), FK506 (1 μg/mL), and cyclosporine A (100 μg/mL) were added to YPD medium. For serial dilution assays, fresh cells were diluted to a starting OD₆₀₀ of 0.1, serially diluted 5-fold, and spotted onto plates for the indicated media and temperature conditions. Plates were incubated for two to seven days and photographed daily. For capsule analysis, strains were incubated for 3 days in RPMI media at 30˚C, followed by negative staining with India ink. For mating assays, strains were incubated on MS plates, face up, in the dark at room temperature for up to eight weeks. Spores dissected from MS plates were germinated on YPD at 30°C for five days.

Strain construction

Mutant strains analyzed in this study were generated in the C. neoformans H99α, YL99a, or KN99a/KN99α backgrounds. All genetic manipulations were done using CRISPR-Cas9-mediated mutagenesis and the TRACE system, according to previously published protocols [61,62]. In brief, Cas9 was amplified from the pBHM2403 plasmid and gRNAs were generated with sequence specific-primers and amplification of the gRNA scaffold from the pBHM2329 plasmid [62]. Donor DNA templates for transformations were generated by PCR or plasmid digestion and purified with the QIAquick gel extraction kit (Qiagen #28704). Cells and DNA were prepared for transformation as described in the TRACE system, and electroporated utilizing a BioRad gene pulser (0.45 kV, 125 μF, 600 Ω). Cells were recovered in YPD media for 2–3 hours at 30⁰C following electroporation.

To generate the PPG1 overexpression allele, the TEF1 promoter (1.2 kb) and PPG1 coding regions were amplified from H99α genomic DNA and assembled into plasmid pSDMA57, digested with XhoI and SpeI, via Gibson assembly (New England Biolabs E5510S) for targeting to the safe haven (SH) locus [49]. GFP tagging of Nop1 was also performed through integration at the safe haven. To facilitate homologous recombination at the target locus, the safe haven plasmid pYSCE5 (pSH-P_H3-GFP-HYG) was designed to include a safe haven genomic sequence containing AscI and PacI restriction sites for linearization, along with the histone H3 promoter, GFP, and the hygromycin B resistance gene (HYG). The NOP1 gene, amplified from H99α genomic DNA without its stop codon, was incorporated into plasmid pYSCE5 between the H3 promoter and GFP, generating pYSCE8 (pSH-H3-NOP1-GFP-HYG). Plasmids for targeted integration at the safe haven were linearized via overnight digestion with AscI and PacI and gel-purified prior to transformation. The ppg1::NEO deletion allele was made via PCR amplification of approximately 1 kb homologous 5’ and 3’ flanking regions to the PPG1 open reading frame, which were then assembled with the neomycin resistance gene expression cassette (NEO) through overlap PCR [63,64]. Transformants were plated onto selective media (YPD + NEO or YPD + HYG) and allowed to grow up to 7 days at 30⁰C. Stable genomic integrants were verified by diagnostic PCR amplifying the 5′ and 3′ flanking junctions of the insertion site.

Primers, PCR genotyping, and RFLP analysis

Primers employed in this study are listed in S2 Table. Genomic DNA from strains generated in this study was extracted from cells with the MasterPure Yeast DNA Purification Kit (LGC Biosearch Technologies). Dissected progeny from mated crosses were genotyped by colony PCR. Single colonies were picked into 0.25% SDS and 1 mM EDTA, briefly frozen at -20 °C, and then heated at 100⁰C for 10 minutes in a thermocycler. The resulting supernatants were used as DNA templates for PCR. To determine the mating type of C. neoformans strains, the STE20 gene in the MAT locus was amplified using primers specific to the STE20α and STE20a alleles. The mitochondrial COX1 gene, which differs in size in KN99 background strains compared to H99 due to the presence of an intron, was probed to determine mitochondrial inheritance. For KN99α pph22Δ sup crosses, the KN99a SSH118 strain, which has a recombinant mitochondrial genome, was used as the wild-type parent to differentiate between mitochondrial genotypes in the KN99 background progeny. After PCR amplification of COX1, the resulting PCR product was digested with restriction enzyme BsrI for restriction fragment length polymorphism (RFLP) analysis. Digestion of the COX1 PCR product amplified from KN99a, which contains three BsrI recognition sites, and SSH118, which has two BsrI sites, were used as controls to determine the mitochondrial inheritance of the progeny from pph22Δ sup x SSH118 crosses.

Microscopy

For sample preparation for scanning electron microscopy from mated strains of Cryptococcus, an agar slice of the plated cells was fixed in a solution of 4% formaldehyde and 4% glutaraldehyde for 2 hours at 4°C. The fixed cells were then gradually dehydrated in a graded ethanol series (30%, 50%, 70%, and 95%), with a 15-minute incubation at 4°C for each concentration. This was followed by three washes with 100% ethanol, each for 15 minutes at 4°C. The samples were further dehydrated using a Ladd CPD3 Critical Point Dryer and coated with a layer of gold using a Denton Desk V Sputter Coater (Denton Vacuum, USA). Hyphae, basidia, and basidiospores were observed with a scanning electron microscope with an EDS detector (Apreo S, ThermoFisher, USA). Brightfield, differential interference contrast (DIC), and fluorescence microscopy images were visualized with an AxioScop 2 fluorescence microscope and captured with an AxioCam MRm digital camera (Zeiss, Germany). Consistent exposure times were used for all images captured from the same experiment. Image processing and merging channels of fluorescent images was performed with ImageJ/Fiji software.

Cell-cell fusion and mating assays

To induce mating, the indicated strains were grown overnight in YPD medium, diluted to an OD₆₀₀ of 1.0, and 4 μL of each cell suspension was spotted onto MS agar plates. The plates were incubated at room temperature in the dark and monitored for signs of filamentation and sporulation for up to eight weeks. For wild-type mating crosses, MATa cells (KN99a) were mixed with MATα cells (H99α) in equal amounts and spotted onto MS plates. For crosses involving pph22Δ and far8Δ strains, the following modifications were made to compensate for the impaired growth of these mutants on MS medium. Fresh pph22Δ and far8Δ mutant cells were first spotted onto MS agar and pre-incubated in the dark at room temperature for two days, without the wild-type mating partner. After pre-growth, wild-type cells of the opposite mating type were added directly on top of the mutant cells at a ten-fold lower cell amount. Twelve mating spots were prepared per plate, with each spotted treated as an independent replicate. A wild-type H99α x KN99a cross was included on each plate as a control. Basidiospores were dissected from individual basidia, or randomly dissected, and incubated on YPD medium at 30⁰C for 3–4 days to determine spore viability and germination rates.

To assess cell-cell fusion frequency in wild-type x pph22Δ crosses, KN99a or KN99α strains carrying a NEO resistance marker (JOHE10493, JOHE18842) were mixed in 1:10 proportions with pph22Δ:NAT a or α strains, spotted onto MS agar, and incubated in the dark at room temperature for 3 days. A KN99a::NAT (JOHE18853) x KN99α::NEO (JOHE18842) wild-type cross was used as a control. The entire patch of cells was harvested and serially diluted in PBS before plating onto YPD + NAT, YPD + NEO, and YPD + NAT + NEO selective media. Colony-forming units (CFUs) were counted after incubation at 30°C for two days (for wild-type selection) or four days (for pph22Δ and dual-resistant fusion products). Fusion frequency was calculated as the number of CFUs on YPD + NAT + NEO divided by the total number of colonies on YPD + NAT and YPD + NEO and expressed as a percentage relative to the wild-type control. Each assay was performed in triplicate.

Whole-genome sequencing and SNP analysis

Genomic DNA for whole-genome sequencing was extracted from saturated 4 mL YPD cultures with the MasterPure Yeast DNA Purification Kit. The precipitated DNA was dissolved in 35 μL of 1x TE buffer (100 mM Tris-HCl, 10 mM EDTA, pH = 8.0), and the concentration was estimated using Qubit. Illumina sequencing was performed at the Duke Sequencing and Genomic Technologies core facility (https://genome.duke.edu) with Novaseq X Plus, providing 250 bp paired-end reads. The Illumina sequences were trimmed using trim-galore (v 0.6.7) and mapped to the H99α genome assembly (RefSeq: GCF_000149245.1) using Geneious software. The resulting BAM files were converted to TDF format, and read coverage was visualized in IGV to verify uniform read coverage across the genome. For SNP calling, the Illumina sequences were mapped to the H99α genome assembly using the Geneious default mapper with five iterations. Variant calling was performed with BAM mapped read files, with parameters set to a 0.9 variant frequency and a minimum of 100x coverage per variant. For the KN99a/KN99α diploid strain (CnLC6683), variant calling was performed with a 0.45 frequency and 100x coverage per variant. Illumina sequences from H99α and KN99a/KN99α served as controls for SNP calling analysis. Variants were exported from Geneious as VCF files and imported into IGV for visualization.

Flow cytometry

For ploidy determination of Cryptococcus samples, fluorescence activated cell sorting (FACS) was performed according to a previously published protocol, with slight modifications [65]. Wild-type and mutant strains were grown on YPD plate medium at 30⁰C overnight, harvested, and washed with PBS. The cells were then fixed in 70% ethanol for 16 hours at 4⁰C. Fixed cells were pelleted and washed with 1 mL NS buffer (10 mM Tris-HCl, 0.25 M sucrose, 1 mM EDTA, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.4 mM phenylmethylsulfonyl fluoride, and 7 mM β-mercaptoethanol). After centrifugation, the cells were treated with RNase (0.5 mg/mL) and stained with propidium iodide (10 μg/mL) in a 200 μL suspension of NS buffer for 2 hours in the dark. Then, 50 μL of the stained cells were diluted into 200 μL of 50 mM Tris-HCl, pH = 8.0, and submitted to the Duke Cancer Institute Flow Cytometry Shared Resource for analysis. Fluorescence was measured using a BD FACSCanto flow cytometer and analyzed with BD FACSDiva software. Approximately 15,000–20,000 events were analyzed for each sample.

RNA sequencing and differential expression analysis

The RNA-seq dataset was previously published in Peterson et al., 2024 [30]. Briefly, cultures of wild-type strains H99 and KN99a, along with three independent strains of pph22Δ and far8Δ were grown in 30 mL YPD at 30⁰C to an OD₆₀₀ of 0.8-1.0. Total RNA was harvested from cells and subjected to stranded mRNA-sequencing with poly(A) enrichment with Illumina NovaSeq X Plus. Processing of read files and differential expression analysis were performed as previously described [30]. Each set of mutant samples was compared to an independent set of wild-type controls, with all comparisons conducted as separate experiments. Heatmaps of log₂ fold-changes in the mutant groups compared to wild type were created in Morpheus using average linkage clustering with Euclidean distance measurements [66].

Quantification and statistical analysis

All statistical analyses and data visualizations were performed with GraphPad Prism v10 software. Details regarding replication, quantification, and statistical tests used for each experiment are provided in the methods and corresponding figure legends.