Abstract
Sclerotinia sclerotiorum is a phytopathogenic soilborne fungus that damages diverse crops worldwide. It has recently been discovered that this fungus distributes its 16 chromosomes irregularly between two nuclei within each haploid ascospore. Here, we study the meiotic process by tracking phenotypic segregation and recombination events in the progeny of a strain resulting from the fusion of two morphologically distinct mutants. We show that, despite the unusual chromosomal distribution into two nuclei, chromosome segregation and genetic recombination occur normally in S. sclerotiorum, likely due to a tight coordination between the two complementing nuclei during meiosis. Consistently, an atypical ascospore development process deviating from the conventional model was observed. Furthermore, we show that our co-segregation analyses method can be used to identify causal mutations in uncharacterized mutants, uncovering a role for deubiquitination in fungal development and virulence.
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Introduction
Sclerotinia sclerotiorum is a soilborne plant fungal pathogen with a broad host range, infecting over 600 plant species including economically significant crops such as soybean, canola, sunflower, and various vegetables1. Its persistence in soil is largely attributed to the formation of sclerotia—hardened, melanized structures that enable long-term survival. Under favorable environmental conditions, sclerotia can germinate to produce apothecia, which are small mushroom-like fruiting bodies that can release large quantities of ascospores. These airborne spores play a critical role in the pathogen’s disease cycle, initiating infection by colonizing nearby host tissues2. S. sclerotiorum is homothallic, capable of self-fertilization without requiring a genetically distinct mating partner3. However, occasional outcrossing likely occurs in nature, potentially contributing to genetic diversity4. Its reproductive strategy provides a significant advantage for the pathogen’s adaptability, survival, and dissemination5. Therefore, understanding the molecular mechanisms underlying S. sclerotiorum apothecium development and ascospore formation will be crucial for its disease control, which remains largely underexplored.
Genomic analyses, including high-throughput sequencing and chromosome assembly, have revealed that S. sclerotiorum has a haploid set of 16 chromosomes (1 N = 16)6,7. As the ascospores of S. sclerotiorum contain two nuclei, they have long been considered pseudodiploid, with each nucleus harboring a full set of 16 chromosomes, likely resulting from an additional mitotic division post mitosis and meiosis8. However, our recent careful analysis using various cell and molecular biology techniques revealed that S. sclerotiorum distributes its 16 chromosomes irregularly between the two nuclei in both ascospores and mycelial cells9. These findings raise fundamental questions about how meiosis occurs in S. sclerotiorum to generate the haploid, binucleate ascospores, as a genome partitioned between two nuclei poses significant challenges for sexual reproduction, raising the possibility of substantial deviations from conventional meiosis process. It was recently hypothesized that the compartmentalization of haploid chromosomes in S. sclerotiorum ascospores may occur either through direct nuclear fission without mitosis or via an additional mitotic division with suppressed DNA replication following meiosis II10.
To address the meiotic events in S. sclerotiorum, we applied a combination of genetic and cell biology approaches to investigate its ascospore development. We developed a method for genetic analysis using morphologically distinct mutants previously generated by ultraviolet (UV) mutagenesis11, enabling accurate tracking of both phenotypes and genotypes. Phenotypic segregation and next-generation sequencing (NGS) analysis of the progeny from ascospores post fusion of the mutants revealed regular chromosome segregation and genetic recombination during meiosis. Additionally, our method is highly efficient in identifying causal mutations in uncharacterized mutants and facilitating epistasis analysis in S. sclerotiorum. As a proof of concept, epistasis analysis indicated that the mitogen-activated protein kinase (MAPK) cascade is upstream of autophagy. We further identified a mutation in the deubiquitinating enzyme gene SsJAMM1, which results in the loss of sclerotia and virulence. Further microscopic examination using a histone 4 (SsH4)-mCherry strain supported an atypical pattern of ascospore development in S. sclerotiorum, shedding new light on its reproductive biology.
Results
A method designed to analyze mating and meiosis in S. sclerotiorum
S. sclerotiorum can form heterokaryons through hyphal fusion between genetically different strains12,13. In our previous forward genetic studies, we identified numerous UV-induced morphological mutants11,14,15. Among them, two well-characterized mutants, Sssmr1-1 that forms pink sclerotia, and Ssatg4 that cannot form sclerotia, are able to fuse through hyphae and produce normal black sclerotia (Fig. 1)11,13,15. Therefore, the two mutants were used in the initial trial to establish a method for mating and meiosis analyses (Fig. 1a). After co-cultivating Sssmr1-1 and Ssatg4 on carrot medium, large wild-type (WT)-like black sclerotia formed, indicating successful fusion and heterokaryon formation. These sclerotia were then induced to produce apothecia (Fig. 1b). This is possible as S. sclerotiorum is homothallic3. Ascospores from individual apothecia were collected and inoculated onto standard PDA (potato dextrose agar) plates. Single colonies derived from each ascospore were subsequently transferred to 96-well plates for phenotyping. As expected, phenotypic segregation was observed in the resulting colonies, suggesting that the apothecia were also heterokaryotic.
a Colony morphology of wild type (WT), Sssmr1-1 and Ssatg4 on PDA plates. The pictures were taken at 14 days post inoculation (dpi). b Flow chart of a method designed for the genetic analysis of mating and meiosis in S. sclerotiorum. Step 1: Young mycelia of the Sssmr1-1 mutant, which forms pink sclerotia, and the Ssatg4 mutant, which does not form sclerotia, are co-inoculated onto carrot medium. To ensure maximum mycelial fusion, the two mycelial discs are placed face-to-face in direct contact. Step 2: The large WT-like black sclerotia produced on the carrot medium, which indicate successful fusion of the two mutants, are collected and washed. Step 3: Following sterilization, the large black sclerotia are induced to produce apothecia. Step 4: Ascospores from individual apothecium are collected and plated onto PDA at an optimal density of 50–80 ascospores per plate. After about two days, single colonies from each ascospore are transferred to 96-well plates. Step 5: The growth and morphology of each colony in the 96-well plates are monitored and assessed for segregation analysis. Step 6: Genomic DNA is extracted from selected single colonies, followed by NGS analysis.
We then quantified the number of colonies exhibiting a WT-like phenotype with black sclerotia, an Sssmr1-1-like phenotype with pink sclerotia, and an Ssatg4-like phenotype with no sclerotia from two different apothecia. The observed ratios were 12:11:25 (WT: pink: Ssatg4-like) for Apothecium 1 and 13:11:24 for Apothecium 2. Interestingly, both segregation patterns closely matched the expected Mendelian 1:1:2 ratio (χ² = 0.125, p = 0.9394 for Apothecium 1; χ² = 0.167, p = 0.9200 for Apothecium 2), which is consistent with the expected inheritance of two traits in a haploid organism (Fig. 2a). Furthermore, the non-sclerotia phenotype of Ssatg4 appears to act upstream of sclerotia melanization, as the morphology of Sssmr1-1 Ssatg4 double mutant is similar to that of Ssatg4 (Fig. 2a).
a Genetic dissection of mating between Sssmr1-1 and Ssatg4. The genotypes of Sssmr1-1 and Ssatg4 mutants are denoted as sR and Sr, respectively. At the Ssatg4 locus, R represents the WT allele and r is the mutant Ssatg4 allele. At the Sssmr1-1 locus, S represents the WT allele and s is the mutant Sssmr1-1 allele. Following mating, the resulting diploid fusion cell goes through meiosis and gives rise to four types of gametes with equal probability. These gametes produce progeny with three phenotypic classes, segregating in a 1:1:2 ratio (WT-like: Sssmr1-1-like: Ssatg4-like) according to the Mendelian law. b The relative positions of sscle_12g089570 (for SsATG4) and sscle_12g091490 (for SsSMR1) on Chromosome 12. The two gene loci are highlighted in cyan and red, respectively. c A summary table showing the markers and their presence in the colonies derived from ascospores resulted from the mating of Sssmr1-1 and Ssatg4. The phenotypes of the colonies are indicated as WT, Sssmr1-1, or Ssatg4-like in the table. The table includes chromosome number (Chrom), gene code, and single nucleotide polymorphism (SNP) in red or black, indicating markers derived from Sssmr1-1 or Ssatg4, respectively. Two gene codes connected by a dash (-) indicate that the SNP is located in the intergenic region between them. Genes with causal mutations in Sssmr1-1 and Ssatg4 are highlighted in bold. The number of reads from NGS are presented as two numbers in each cell, color-coded for clarity. The number before the comma represents the SNP counts of the indicated marker in the reference genome (WT S. sclerotiorum 1980), while the number after the comma represents the SNP counts of the indicated marker in the mutant genome. For all sequenced progeny, cyan indicates markers from Ssatg4, red indicates markers from Sssmr1-1, and white indicates no information. d Chromosome maps of selected progeny, as translated from the marker information from c. The chromosome regions containing only Ssatg4 markers are arbitrarily colored in cyan, extending either to the end of the chromosome or to the appearance of an Sssmr1-1 marker. Similarly, regions containing only Sssmr1-1 markers are arbitrarily colored in red, extending either to the end of the chromosome or to the appearance of an Ssatg4 marker. Chromosome regions with no marker information are shown in white. The maps were drawn using MapChart (v2.32). Note that the chromosome map does not fully represent the true physical arrangement due to the limitations in the density of available markers.
To investigate the genetic basis of meiosis in S. sclerotiorum, we conducted next-generation sequencing (NGS) on nine colonies derived from single ascospores from Apothecium 1, exhibiting the three different phenotypes. Random mutations induced by UV in S. sclerotiorum are primarily single nucleotide polymorphisms (SNPs)16. Since the Sssmr1-1 and Ssatg4 mutants were isolated from the same genetic background, these SNPs serve as precise and reliable genetic markers to examine recombination events. Based on our previous data11,15, Sssmr1-1 and Ssatg4 harbor causal mutations in SsSMR1 (Sclerotial Melanogenesis Regulatory gene 1; sscle_12g091490) and SsATG4 (Autophagy-related gene 4; sscle_12g089570), respectively. These two genes are located quite far from each other on Chromosome 12 (Fig. 2b), which explains the observed 1:1:2 segregation ratio, as they become genetically unlinked and segregate independently.
After analyzing the NGS data of Sssmr1-1 and Ssatg4, we identified 29 markers covering all chromosomes, except Chromosome 8 and 13 (Fig. 2c and Supplementary Fig. 1). The presence of each marker was assessed in each progeny showing WT-like, Sssmr1-1-like, or Ssatg4-like phenotypes. As shown in Fig. 2c, d, none of the nine progeny shared the exact same marker composition pattern, supporting that each ascus within an apothecium undergoes its own meiosis process17. Additionally, the causal mutations of Sssmr1-1 and Ssatg4 co-segregated with their corresponding phenotypes in all progeny (Fig. 2c). Moreover, despite the limited availability of genetic markers (Supplementary Fig. 1), plenty of crossovers were detected on different chromosomes, indicating normal recombination events during meiosis (Fig. 2d). Taken together, although S. sclerotiorum separates its haploid chromosomes into two nuclei9, chromosome segregation and genetic recombination appear to occur regularly during meiosis.
Genetic analysis of mating and meiosis using the S. sclerotiorum Ssste50-1 and Ssatg4 mutants
To confirm our observation from the fusion of Ssatg4 and Sssmr1-1, we paired Ssatg4 with another non-sclerotia UV mutant Ssste50-1 (Fig. 3a), carrying a causal mutation in SsSte50 (sscle_07g058440), which locates on a different chromosome than Ssatg4. SsSte50 encodes an adaptor protein in a MAPK pathway13. Although both mutants cannot form sclerotia, their colony morphologies are visibly distinct (Fig. 3a). Similarly, co-cultivation of Ssatg4 and Ssste50-1 led to sclerotia production; however, the sclerotia amount was markedly reduced due to the fusion defects of Ssste50-113, and sclerotia formation was observed only when the two mycelial discs were forcibly brought into direct contact to promote fusion. The progeny from a single apothecium exhibited a WT-like phenotype with black sclerotia, an Ssatg4-like non-sclerotia phenotype, and an Ssste50-1-like non-sclerotia phenotype, resulting in a ratio of 14:13:21 (WT: Ssatg4-like: Ssste50-1-like). This segregation ratio closely matched the expected Mendelian 1:1:2 ratio (χ² = 0.792, p = 0.6731), suggesting that the MAPK pathway functions upstream of autophagy, as approximately half of the progeny exhibited the Ssste50-1-like phenotype (Fig. 3b). Moreover, NGS data from the apothecium confirmed its heterokaryotic nature (Fig. 3c). As with the Sssmr1-1 and Ssatg4 mating, recombination events were inferred from the NGS data analysis of four WT-like progeny from the mating between Ssste50-1 and Ssatg4 (Fig. 3c, d and Supplementary Fig. 2). Agreeably, neither of the causal mutations of Ssste50-1 nor Ssatg4 was present in the WT-like progeny (Fig. 3c). These results validate independent chromosome segregation and normal genetic recombination during meiosis in S. sclerotiorum.
a Colony morphology of Ssste50-1 and Ssatg4 on PDA plates. The pictures were taken at 14 dpi. b Genetic dissection of mating between Ssste50-1 and Ssatg4. The genotypes of Ssste50-1 and Ssatg4 mutants are denoted as zR and Zr, respectively. At the Ssatg4 locus, R represents the WT allele and r is the mutant Ssatg4 allele. At the Ssste50-1 locus, Z represents the WT allele and z is the mutant Ssste50-1 allele. Following mating, the resulting diploid fusion cell goes through meiosis and gives rise to four types of gametes with equal probability. These gametes produce progeny with three phenotypic classes, segregating in a 1:1:2 ratio (WT-like: Ssatg4-like: Ssste50-1-like) according to the Mendelian law. c A summary table showing the SNP/INDEL polymorphisms detected by NGS and their presence in the genomes of an apothecium and four WT-like colonies derived from ascospores originated from the mating of Ssste50-1 and Ssatg4. The table includes chromosome number (Chrom), gene code, and single nucleotide polymorphism/short insertion or deletion (SNP/INDEL) in red or black, indicating markers derived from Ssste50-1 or Ssatg4, respectively. Two gene codes connected by a dash (-) indicate that the SNP is located in the intergenic region between them. Genes with causal mutations in Ssste50-1 and Ssatg4 are highlighted in bold. The results from NGS analysis are presented as two numbers in each cell, color-coded for clarity. The number before the comma represents the SNP counts of the indicated marker in the reference genome (WT S. sclerotiorum 1980), while the number after the comma represents the SNP counts of the indicated marker in the mutant genome. For all sequenced progeny, cyan indicates markers from Ssatg4, and green indicates markers from Ssste50-1. d Chromosome maps of the selected progeny based on the marker information from c. The chromosome regions containing only Ssatg4 markers are arbitrarily colored in cyan, extending either to the end of the chromosome or to the appearance of an Ssste50-1 marker. Similarly, regions containing only Ssste50-1 markers are arbitrarily colored in green, extending either to the end of the chromosome or to the appearance of an Ssatg4 marker. Chromosome regions with no marker information are shown in white. The maps were drawn using MapChart (v2.32). Note that the chromosome map does not fully represent the true genetic arrangement due to the limitations in the number of available markers.
Genetic analysis of mating between Ssste50-1 and an uncharacterized mutant M135 reveals its likely causal mutation
To further corroborate our observations, we next fused Ssste50-1 with another non-sclerotia mutant M135 (Fig. 4a), which was also isolated from our previous forward genetic screen11. However, its causal mutation has not yet been identified due to the presence of five significant mutations, making it difficult to guess (Fig. 4b). Given that the causal mutations perfectly co-segregated with the corresponding phenotypes in the two previous mating experiments (Fig. 2c and Fig. 3c), we hypothesized that genetic analysis using our established method could help identify the causal mutation for M135. Consistent with the former two mating experiments, the phenotypic segregation ratio for the Ssste50-1 and M135 mating aligned with the expected Mendelian 1:1:2 ratio (WT-like: M135-like: Ssste50-1-like = 12:13:23; χ² = 0.125, p = 0.9394), suggesting that the M135 mutant phenotype is most likely caused by a mutation in a single gene that functions downstream of the MAPK pathway (Fig. 4c). After evaluating the five candidates in the genome of four WT-like progeny derived from ascospores from fusing Ssste50-1 and M135, only the T1436G mutation in the sscle_12g086990 gene was absent, strongly suggesting that this is the causal mutation of M135 (Fig. 4d). Overall, genetic recombination was still observed despite the lack of marker information on four chromosomes (Fig. 4e and Supplementary Fig. 3).
a Colony morphology of Ssste50-1 and M135 on PDA plates. The pictures were taken at 14 dpi. b Candidate mutations in M135 as identified from NGS data analysis. The most likely gene from M135 mutant is highlighted in bold, as deduced from data in (d). Chrom: chromosome number, del: deletion, fs: frameshift, dup: duplication. c Genetic dissection of mating between Ssste50-1 and M135. The genotypes of Ssste50-1 and M135 mutants are denoted as zM and Zm, respectively. At the Ssste50-1 locus, Z represents the WT allele and z is the mutant Ssste50-1 allele. At the M135 locus, M represents the WT allele and m is the mutant M135 allele. Following mating, the resulting diploid fusion cell goes through meiosis and gives rise to four types of gametes with equal probability. These gametes produce progeny with three phenotypic classes, segregating in a 1:1:2 ratio (WT-like: M135-like: Ssste50-1-like) according to the Mendelian law. d A summary table showing the markers and their presence in the genomes of four WT-like colonies derived from ascospores originated from the mating of Ssste50-1 and M135. The table includes chromosome number (Chrom), gene code, and single nucleotide polymorphism/short insertion or deletion (SNP/INDEL) in red or black, indicating markers derived from Ssste50-1 or M135, respectively. Two gene codes connected by a dash (-) indicate that the SNP is located in the intergenic region between them. The results from NGS analysis are presented as two numbers in each cell, color-coded for clarity. The number before the comma represents the SNP counts of the indicated marker in the reference genome (WT S. sclerotiorum 1980), while the number after the comma represents the SNP counts of the indicated marker in the mutant genome. For all sequenced progeny, magenta indicates markers from M135, and green indicates markers from Ssste50-1. e Chromosome maps of the selected progeny based on the marker information from (d). The chromosome regions containing only M135 markers are arbitrarily colored in magenta, extending either to the end of the chromosome or to the appearance of a Ssste50-1 marker. Similarly, regions containing only Ssste50-1 markers are arbitrarily colored in green, extending either to the end of the chromosome or to the appearance of an M135 marker. Chromosome regions with no marker information are shown in white. The maps were drawn using MapChart (v2.32). Note that the chromosome map does not fully represent the true genetic arrangement due to the limitations in the number of available markers.
Targeted gene knockout confirms that sscle_12g086990 is the responsible gene for the M135 mutant phenotype
The sscle_12g086990 gene in S. sclerotiorum encodes a JAB1/MPN/MOV34 (JAMM)-type deubiquitinating enzyme (DUB), with an N-terminal Ubiquitin-Specific Peptidase 8 (USP8) dimerization domain and a JAMM metalloenzyme domain that functions as an endosome-associated ubiquitin isopeptidase (Fig. 5a)18,19. However, the function of DUBs from the JAMM subfamily has not been reported in any plant pathogenic fungi. Therefore, we designated this gene SsJAMM1. The T1436G point mutation in M135 leads to an early stop codon shortly after the JAMM domain (Fig. 5a), and M135 was thus renamed as Ssjamm1-1.
a Protein structure diagram of SsJAMM1 encoded by sscle_12g086990. Two domains predicted by InterPro (https://www.ebi.ac.uk/interpro/) are indicated by green and orange rectangle, respectively. The position of the premature stop codon is marked by a blue triangle. The diagram was drawn using Illustrator for Biological Sequencing (IBS)27. b PCR verification of the sscle_12g086990 (SsJAMM1) gene deletion. Genomic DNA isolated from WT and mutant alleles Ssjamm1-2 and Ssjamm1-3 were used as PCR templates. Three pairs of primers were designed to verify the presence of the upstream and downstream fragments of the hygromycin resistance gene (7 F + H855R and H855F + 8 R) and the SsJAMM1 deletion (5 F + 6 R), with expected amplicon sizes indicated in brackets. M shows the lane for DNA marker. The experiment was repeated twice independently with similar results. Source data are provided as a Source Data file. c Colony morphology of WT, Ssjamm1-1, Ssjamm1-2 and Ssjamm1-3. All genotypes were cultured on PDA plates. Photos were taken at 14 dpi. d The Ssjamm1 deletion alleles failed to complement the sclerotia formation defect of the original UV-induced mutant Ssjamm1-1 by hyphal fusion. All mutants were inoculated on the same plate for genetic complementation test. Ssatg4 was used as a positive control. Representative photos were taken at 14 dpi, with red triangles highlighting sclerotia formed in the hyphal fusion interface. e Mycelial growth of WT, Ssjamm1-1, Ssjamm1-2 and Ssjamm1-3 on PDA plates. The colony diameter was measured every 12 hours. Data represent means of three replicates (n = 3). Error bars show standard deviation (SD). Source data are provided as a Source Data file. f Pathogenicity assay for WT, Ssjamm1-1, Ssjamm1-2 and Ssjamm1-3 on detached Nicotiana benthamiana leaves. A representative photo was taken at 36 hours post inoculation (hpi). Quantification of lesion sizes was acquired from six replicates. Dots represent lesion areas measured by ImageJ (https://imagej.net/ij/). The statistics analysis was carried out by One-way ANOVA. Different letters represent statistical significance (p = 3.7962 × 10−12). Error bars represent means ± standard deviation (SD, n = 6). Scale bar = 1 cm. Source data are provided as a Source Data file. g Compound appressoria observation. PDA plugs from WT, Ssjamm1-1, Ssjamm1-2 and Ssjamm1-3 were transferred onto glass slides and photographed at 24 hpi. Scale bar = 20 μm.
Using a previously established targeted gene knockout (KO) method via homologous recombination11, we generated two independent deletion alleles Ssjamm1-2 and Ssjamm1-3. The presence of a PCR amplicon within the SsJAMM1 gene in the WT strain, but not in the two KO mutants, along with the detection of the hygromycin resistance gene (HYG) amplicons exclusively in the KO mutants, confirmed successful gene replacement (Fig. 5b). Ssjamm1-2 and Ssjamm1-3 exhibited identical phenotypic defects as Ssjamm1-1, including the absence of sclerotia and slightly reduced mycelial growth (Fig. 5c, e). Our further hyphal fusion test among Ssjamm1-1 and the two KO mutants showed failed complementation, as evidenced by the absence of sclerotia formation at the fusion interface. In contrast, robust sclerotia formation was observed in the positive control (Ssjamm1-1 × Ssatg4), supporting the conclusion that the mutants are allelic (Fig. 5d). Moreover, these Ssjamm1 mutants failed to cause any necrotic lesions when inoculated onto detached Nicotiana benthamiana leaves (Fig. 5f). This loss of pathogenicity is likely due to the impaired compound appressoria formation, which is essential for host penetration (Fig. 5g). Altogether, these results demonstrate that the deubiquitination process mediated by the JAMM-type DUB SsJAMM1 regulates both sclerotia formation and pathogenicity in S. sclerotiorum.
Atypical ascospore development in S. sclerotiorum observed using confocal microscopy
To investigate how haploid binucleate ascospores are formed in S. sclerotiorum following mating and meiosis, we used a previously generated histone 4 (SsH4)-mCherry strain to track ascospore development. As shown in Fig. 6, emerging, premature, and mature apothecia were dissected to examine potential crozier cells, early-stage asci, and mature asci containing eight binucleate ascospores, respectively. Notably, in all our attempts to track the early stages of ascospore development, we never observed cells with a single nucleus or odd number of nuclei within the developing apothecia. Instead, we detected a potential tetrakaryotic precursor cell, which may represent the zygote, with each nucleus potentially retaining approximately half of the genome (Fig. 6a). Strikingly, the direct precursor of the mature ascus appeared as a long, thread-like structure composed of four cells, each containing two nuclei (Fig. 6b). As the mature ascospores are haploid based on the recent finding9, these precursor cells likely represent four haploid gametes post meiosis. This organization differs significantly from the conventional model of the tetranucleate stage during ascospore development (Fig. 7a)8. Through this atypical process, eight haploid binucleate ascospores were ultimately formed in S. sclerotiorum after another round of mitosis (Fig. 6c and Fig. 7b).
Representative images showing the ascospore formation process, scale bar = 10 μm. a A potential tetrakaryotic cell (or zygote) is pointed out with a white rectangle. b A thin thread (young ascus) containing four gametes. White labels n1 and n2 denote the two nuclei in each cell, while white arrows point to septa stained with Calcofluor-White. c Formation of the mature ascus with eight binucleate haploid ascospores. The apothecial stages used to visualize the different cells are shown below, scale bar = 5 mm. The experiment was independently repeated at least three times with similar results.
a The widely recognized “one whole genome one nucleus” textbook model assuming each nucleus contains a full complement of 16 chromosomes. After mating, the dikaryotic cell (2 N) undergoes nuclear fusion, DNA replication, and regular meiosis, resulting in the formation of a tetranucleate cell. After two rounds of mitosis post meiosis, the assumed pseudodiploid binucleate ascospores (2 N) are formed. Note that nuclei with different DNA contents are distinguished by different colors. b The new model based on our current genetic study and microscopic data observed in Fig. 6. After mating (selfing in this case), the zygote (2 N) undergoes DNA replication and regular meiosis, resulting in the formation of a thin thread with four binucleate haploid cells (N), each nucleus containing approximately half of the full chromosome complement. After mitosis, the final eight haploid binucleate ascospores (N) are formed within the swollen ascus. Note that nuclei with different DNA contents are distinguished by different colors. As the nuclei collisions during mitosis likely lead to chromosome redistribution9, all nuclei within an ascus may exhibit distinct DNA compositions.
Discussion
Meiosis is a key process in eukaryotic sexual reproduction, leading to the formation of genetically diverse gametes. However, it remains poorly studied in most homothallic fungi, partly due to their self-fertilization mating pattern, which makes detecting recombination events challenging5. In addition to homothallism, S. sclerotiorum produces binucleate ascospores, with each nucleus carrying half of its haploid chromosome set and breaking the “one nucleus one genome” rule9, further complicating our understanding of meiosis. Here, by generating S. sclerotiorum heterokaryons through fusing two different UV-induced morphological mutants, we successfully conducted genetic analyses of mating and meiosis in this fungus. Random mutations introduced by UV irradiation served as ideal genetic markers for detecting recombination events. Although the limited number of markers restricted the inference of crossover events on certain chromosomes, clear crossover signals were detected on most chromosomes that contained more than two well-spaced markers. Based on our observation of canonical Mendelian segregation ratios and regular genetic recombination events, we conclude that meiosis in S. sclerotiorum seems to proceed normally, consistent with established Mendelian chromosome segregation and recombination principles.
Furthermore, microscopic dissection using the S. sclerotiorum SsH4-mCherry strain revealed an unusual ascospore formation process, where all observed cell types during early ascospore development contain even-numbered nuclei (Fig. 7). These cells likely also have their haploid genome separated into paired nuclei, similar to mature ascospores and mycelial cells9. Therefore, the recently proposed possibility of an additional mitotic division following meiosis II may be unnecessary10. Although not yet detected, there must be a stage when the two sets of chromosomes, originating from the mating partners in the tetrakaryotic cell, come together to enable normal genetic recombination during meiosis. However, the mechanisms underlying chromosome segregation into different nuclei while preserving genome integrity during meiosis remain unknown. It also remains mysterious how these atypical ascus precursors with four binucleate gametes are formed (Fig. 6b). We speculate that the two nuclei carrying complementary chromosome subsets likely stay in close association to ensure genomic integrity during cell divisions. Future research employing advanced cell biology techniques and comprehensive cellular and molecular genetic analyses of contributing genes will be essential to further elucidate the regulation of these atypical developmental processes during S. sclerotiorum reproduction.
The distribution of a haploid chromosome set across multiple nuclei was also observed in Botrytis cinerea, another phytopathogenic fungus in the Sclerotiniaceae family, closely related to S. sclerotiorum7,9. In contrast, B. cinerea is heterothallic, and its sexual reproduction requires two distinct mating types20. Interestingly, crosses between different B. cinerea strains, characterized by known RAPD (Random Amplified Polymorphic DNA) markers, showed that most markers in the progeny segregated in a normal Mendelian ratio21, consistent with our findings in S. sclerotiorum. Therefore, the process of meiosis seems normal in fungal species that partition chromosomes in different nuclei, ensuring genome integrity and promoting genetic diversity. Whether sexual spore development follows a similar atypical process in B. cinerea, and even other fungal species as observed in S. sclerotiorum will be of interest for future investigation.
Forward genetic screens are powerful tools for uncovering novel components in biological processes through mutant analysis and causal gene cloning11. However, identifying the responsible gene associated with the causal mutation in each mutant requires significant time and effort, especially when many candidate mutations are present. In this study, the genetic analysis method we developed for investigating S. sclerotiorum mating and meiosis is highly efficient in mapping causal mutations in uncharacterized mutants. Since mutant phenotypes co-segregate with causal mutation genotypes, the NGS step can be further simplified by merely sequencing a mixed pool of a large number of WT-like progeny from mating, as the causal mutation are not expected to be present. Bulk sequencing of mutant-like progeny can also be done as in Arabidopsis thaliana mapping-by-sequencing to help identify the causal gene candidate. As a proof of concept, we successfully identified the causal mutation in a JAMM-type deubiquitinating enzyme gene in the M135 mutant from our previous forward genetic screen11. Our data reveal that the deubiquitination process is essential for sclerotia formation in S. sclerotiorum, which may be potentially linked to autophagy22. Furthermore, deubiquitination by SsJAMM1 contributes to pathogenicity as with other types of DUBs in various pathogenic fungi19,23, although the underlying mechanism warrants further investigation.
Our method can also facilitate the efficient generation of double and even higher-order mutants for epistasis analysis, which remains challenging in S. sclerotiorum and many non-model fungal species. Among all Ssatg4-like progeny from the mating between Sssmr1-1 and Ssatg4, the R2 strain carries both the causal mutation of Ssatg4 (G to A in sscle_12g089570) and that of Sssmr1-1 (C to T in sscle_12g091490) (Fig. 2c), indicating that it is a double mutant. The Ssatg4-like phenotype of the R2 double mutant confirms that autophagy functions upstream of sclerotial melanization. Likewise, our further epistasis analysis of Ssatg4, Ssste50-1, and Ssjamm1-1 mutants uncovered a previously unknown epistatic relationship among autophagy, the MAPK pathway, and deubiquitination in S. sclerotiorum, where the MAPK pathway functions upstream of both autophagy and deubiquitination. Collectively, our study advances the current understanding of fungal sexual reproduction, and the methods we developed will significantly accelerate molecular genetic analyses in S. sclerotiorum, with potential applications to other fungal species.
Methods
Fungal strains and culture conditions
The wild-type (WT) S. sclerotiorum 1980 and its derived mutant strains were maintained on potato dextrose agar (PDA) (Shanghai Bio-way Technology Co., Ltd., China) plates at room temperature. Gene knockout mutants were screened and purified using PDA supplemented with hygromycin B (Sigma-Aldrich, Cat# H3274) at a final concentration of 50 μg/mL. To grow large sclerotia for apothecium production, fresh mycelia of two mutant strains were co-inoculated from PDA onto carrot medium, which was prepared by slicing fresh carrots into 3–5 mm thick pieces, placing them in a sterile glass container, and autoclaving at 121 °C for 20 min. The inoculated cultures were incubated for 2–3 weeks until mature sclerotia are fully developed.
Apothecium induction and ascospore collection
To induce apothecia, mature sclerotia harvested from carrot medium were carefully cleaned using a test tube brush to remove residual mycelia and carrot debris. The sclerotia were then air-dried at room temperature for approximately two weeks. The dried sclerotia were surface sterilized with 33% (v/v) bleach solution (CloroxTM Disinfecting Bleach) for 10 min, followed by two washes with sterile water to remove any remaining bleach. These sclerotia were placed onto autoclaved sand in glass bottles and incubated in a cold room at 4 °C for about 4 weeks. Subsequently, they were transferred to a plant growth chamber set at 16 °C with a 16-hour light/8-hour dark cycle. Apothecium development was normally observed within 3–5 weeks. For ascospore collection, fully mature apothecia were transferred to a sterile Eppendorf tube containing 1 mL of sterile water and vortexed vigorously to release ascospores. The resulting suspension was then diluted to approximately 60 ascospores per mL of water before being spread onto PDA plates.
Genomic DNA extraction and NGS analysis
Genomic DNA extraction and subsequent NGS analysis of S. sclerotiorum were performed following previously established protocols11. In detail, a 5 cm × 5 cm section of young mycelia was collected from PDA plates and ground into a fine powder using liquid nitrogen. For a mature apothecium, vigorous vortexing was performed multiple times until no ascospores were observed under the microscope, after which the apothecium was ground into a fine powder using liquid nitrogen. The resulting mycelial or apothecial powders were subjected to genomic DNA extraction using a cetyltrimethylammonium bromide (CTAB; Sigma-Aldrich, Cat# 219374) buffer. The crude DNA extracts were further purified using the DNeasy Plant Maxi kit (Qiagen, Cat# 68163) before sequencing.
For NGS, purified genomic DNA samples were sent to Novogene Co. Ltd. for sequencing. Library preparation, whole-genome sequencing, and quality control were conducted by Novogene using the Illumina NovaSeq 6000 platform, generating 14 to 17 million high-quality paired-end reads per sample. To analyze NGS data, clean reads were aligned to the reference genome of S. sclerotiorum strain 1980 (ASM185786v1) using the Burrows-Wheeler Aligner (BWA-MEM) algorithm24. Mutation sites were identified using SAMtools25, and variants, including single nucleotide polymorphisms (SNPs) and insertions/ deletions (INDELs), were annotated following the GATK best practices for germline short variant discovery26.
Target gene knockout (KO)
The split-marker approach was used to construct the sscle_12g086990 gene replacement cassette and generate KO mutants based on a previous protocol11. Firstly, the replacement cassette was designed to contain the upstream region of the target gene, the hygromycin resistance gene (HYG), and the downstream region of the gene. This cassette was then introduced into WT S. sclerotiorum protoplasts via polyethylene glycol (PEG; Sigma-Aldrich, Cat# 81240)-mediated transformation. Positive transformants were selected on potato dextrose agar (PDA) supplemented with 50 μg/mL hygromycin B. To obtain pure KO mutants, protoplast purification was performed by making protoplasts from transformants that contain most HYG insertion11, until PCR analysis with the gene-specific primer pair (5 F/6 R) confirmed the absence of the WT band. All the primers used are listed in Supplementary Table 1.
Plant infection assay
To assess pathogenicity, fresh mycelial plugs (5 mm in diameter) were excised from 2-day-old PDA cultures and inoculated onto detached leaves of Nicotiana benthamiana. The infected leaves were placed on moistened paper towels inside trays and covered with lids to maintain humidity. The trays were then incubated in a growth chamber at 23 °C with a 16-hour light/8-hour dark cycle. Disease progression was quantified by measuring lesion areas using ImageJ software (https://imagej.net/ij/).
Observation of compound appressoria
Fresh mycelial plugs (5 mm in diameter) were excised from the colony margin and placed on glass slides positioned over moistened paper towels inside a clean Petri dish. The slides were incubated at room temperature for 2 days to allow appressoria development. Formation of compound appressoria was then observed using a ZEISS light microscope.
Fluorescence microscopy
Emerging, premature, and mature apothecia were dissected to examine possible crozier cells, premature thin asci, and mature asci. Apothecia were thinly sectioned longitudinally using a scalpel under an Olympus SZH stereomicroscope and placed onto a glass slide with 25 µL ddH2O. The premature apothecia sections were immersed in 0.01% Calcofluor White solution (Sigma-Aldrich, Cat# 18909) in ddH2O to visualize the cell wall and septa. The specimens were then gently squished with a coverslip and imaged using a ZEISS AXIO Imager M2 fluorescence microscope equipped with an X-Cite Series 120Q illumination system for mCherry fluorescence detection.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All data are included in the main text or the supplementary materials. The raw data of next-generation sequencing (NGS) generated in this study have been deposited in the Genome Sequence Archive (GSA) in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences under accession code CRA027406. Source data are provided with this paper.
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Acknowledgements
The authors cordially thank Dr. Jeffrey Rollins from University of Florida for S. sclerotiorum strain 1980. Mr. Ilias Kontos and Dr. Junxing Lu are thanked for preparing large sclerotia for apothecium production. This work was supported by funds to X.L. from the Canadian Natural Sciences and Engineering Research Council (NSERC) Discovery program, NSERC-CREATE-PRoTECT, Canada Research Chair (CRC), and the Canadian Foundation for Innovation (CFI) funds. L.T. and Y.X. were partly supported by China Scholarship Council (CSC) scholarships. J.T. is partly supported by a University of British Columbia Four-year Fellowship.
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X.L. conceived the idea; L.T. and X.L. designed the experiments and wrote the paper; L.T., Y.X., J. L., and J.T. conducted the experiments. Y.Z. revised the original manuscript. All authors analyzed the data, edited the manuscript and approved the final version.
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Tian, L., Xu, Y., Li, J. et al. Normal meiosis in the fungus Sclerotinia sclerotiorum despite the irregular distribution of haploid chromosomes between two nuclei. Nat Commun 16, 7492 (2025). https://doi.org/10.1038/s41467-025-62932-2
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DOI: https://doi.org/10.1038/s41467-025-62932-2
