Functional analysis of chromatin-associated proteins in Sordaria macrospora reveals similar roles for RTT109 and ASF1 in development and DNA damage response.

We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.

Comparative genomics of Cryptococcus and Kwoniella reveals pathogenesis evolution and contrasting karyotype dynamics via intercentromeric recombination or chromosome fusion.

A large-scale comparative genomic analysis was conducted for the global human fungal pathogens within the Cryptococcus genus, compared to non-pathogenic Cryptococcus species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species of both genera, resulting in a dataset encompassing virtually all of their known diversity. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at pre-adaptive pathogenic potential, our analysis found evidence in pathogenic Cryptococcus species of specific examples of gene gain (via horizontal gene transfer) and gene loss, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the two genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5 or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes, underwent chromosome reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Taken together, our findings advance our understanding of genomic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.

Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora.

IMPORTANCE: Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

The Role of Chromatin and Transcriptional Control in the Formation of Sexual Fruiting Bodies in Fungi.

Fungal fruiting bodies are complex, three-dimensional structures that arise from a less complex vegetative mycelium. Their formation requires the coordinated action of many genes and their gene products, and fruiting body formation is accompanied by major changes in the transcriptome. In recent years, numerous transcription factor genes as well as chromatin modifier genes that play a role in fruiting body morphogenesis were identified, and through research on several model organisms, the underlying regulatory networks that integrate chromatin structure, gene expression, and cell differentiation are becoming clearer. This review gives a summary of the current state of research on the role of transcriptional control and chromatin structure in fruiting body development. In the first part, insights from transcriptomics analyses are described, with a focus on comparative transcriptomics. In the second part, examples of more detailed functional characterizations of the role of chromatin modifiers and/or transcription factors in several model organisms (Neurospora crassa, Aspergillus nidulans, Sordaria macrospora, Coprinopsis cinerea, and Schizophyllum commune) that have led to a better understanding of regulatory networks at the level of chromatin structure and transcription are discussed.

Draft genome of the aardaker (Lathyrus tuberosus L.), a tuberous legume.

OBJECTIVES: Lathyrus tuberosus is a nitrogen-fixing member of the Fabaceae which forms protein-rich tubers. To aid future domestication programs for this legume plant and facilitate evolutionary studies of tuber formation, we have generated a draft genome assembly based on Pacific Biosciences sequence reads. DATA DESCRIPTION: Genomic DNA from L. tuberosus was sequenced with PacBio's HiFi sequencing chemistry generating 12.8 million sequence reads with an average read length of 14 kb (approximately 180 Gb of sequence data). The reads were assembled to give a draft genome of 6.8 Gb in 1353 contigs with an N50 contig length of 11.1 Mb. The GC content of the genome assembly was 38.3%. BUSCO analysis of the genome assembly indicated a genome completeness of at least 96%. The genome sequence will be a valuable resource, for example, in assessing genomic consequences of domestication efforts and developing marker sets for breeding programs. The L. tuberosus genome will also aid in the analysis of the evolutionary history of plants within the nitrogen-fixing Fabaceae family and in understanding the molecular basis of tuber evolution.

Obligate sexual reproduction of a homothallic fungus closely related to the Cryptococcus pathogenic species complex.

.Fungi are enigmatic organisms that flourish in soil, on decaying plants, or during infection of animals or plants. Growing in myriad forms, from single-celled yeast to multicellular molds and mushrooms, fungi have also evolved a variety of strategies to reproduce. Normally, fungi reproduce in one of two ways: either they reproduce asexually, with one individual producing a new individual identical to itself, or they reproduce sexually, with two individuals of different 'mating types' contributing to produce a new individual. However, individuals of some species exhibit 'homothallism' or self-fertility: these individuals can produce reproductive cells that are universally compatible, and therefore can reproduce sexually with themselves or with any other cell in the population.Homothallism has evolved multiple times throughout the fungal kingdom, suggesting it confers advantage when population numbers are low or mates are hard to find. Yet some homothallic fungi been overlooked compared to heterothallic species, whose mating types have been well characterised. Understanding the genetic basis of homothallism and how it evolved in different species can provide insights into pathogenic species that cause fungal disease.With that in mind, Passer, Clancey et al. explored the genetic basis of homothallism in Cryptococcus depauperatus, a close relative of C. neoformans, a species that causes fungal infections in humans. A combination of genetic sequencing techniques and experiments were applied to analyse, compare, and manipulate C. depauperatus' genome to see how this species evolved self-fertility.Passer, Clancey et al. showed that C. depauperatus evolved the ability to reproduce sexually by itself via a unique evolutionary pathway. The result is a form of homothallism never reported in fungi before. C. depauperatus lost some of the genes that control mating in other species of fungi, and acquired genes from the opposing mating types of a heterothallic ancestor to become self-fertile.Passer, Clancey et al. also found that, unlike other Cryptococcus species that switch between asexual and sexual reproduction, C. depauperatus grows only as long, branching filaments called hyphae, a sexual form. The species reproduces sexually with itself throughout its life cycle and is unable to produce a yeast (asexual) form, in contrast to other closely related species.This work offers new insights into how different modes of sexual reproduction have evolved in fungi. It also provides another interesting case of how genome plasticity and evolutionary pressures can produce similar outcomes, homothallism, via different evolutionary paths. Lastly, assembling the complete genome of C. depauperatus will foster comparative studies between pathogenic and non-pathogenic Cryptococcus species.

Fungi are enigmatic organisms that flourish in soil, on decaying plants, or during infection of animals or plants. Growing in myriad forms, from single-celled yeast to multicellular molds and mushrooms, fungi have also evolved a variety of strategies to reproduce. Normally, fungi reproduce in one of two ways: either they reproduce asexually, with one individual producing a new individual identical to itself, or they reproduce sexually, with two individuals of different 'mating types' contributing to produce a new individual. However, individuals of some species exhibit 'homothallism' or self-fertility: these individuals can produce reproductive cells that are universally compatible, and therefore can reproduce sexually with themselves or with any other cell in the population. Homothallism has evolved multiple times throughout the fungal kingdom, suggesting it confers advantage when population numbers are low or mates are hard to find. Yet some homothallic fungi been overlooked compared to heterothallic species, whose mating types have been well characterised. Understanding the genetic basis of homothallism and how it evolved in different species can provide insights into pathogenic species that cause fungal disease. With that in mind, Passer, Clancey et al. explored the genetic basis of homothallism in Cryptococcus depauperatus, a close relative of C. neoformans, a species that causes fungal infections in humans. A combination of genetic sequencing techniques and experiments were applied to analyse, compare, and manipulate C. depauperatus' genome to see how this species evolved self-fertility. Passer, Clancey et al. showed that C. depauperatus evolved the ability to reproduce sexually by itself via a unique evolutionary pathway. The result is a form of homothallism never reported in fungi before. C. depauperatus lost some of the genes that control mating in other species of fungi, and acquired genes from the opposing mating types of a heterothallic ancestor to become self-fertile. Passer, Clancey et al. also found that, unlike other Cryptococcus species that switch between asexual and sexual reproduction, C. depauperatus grows only as long, branching filaments called hyphae, a sexual form. The species reproduces sexually with itself throughout its life cycle and is unable to produce a yeast (asexual) form, in contrast to other closely related species. This work offers new insights into how different modes of sexual reproduction have evolved in fungi. It also provides another interesting case of how genome plasticity and evolutionary pressures can produce similar outcomes, homothallism, via different evolutionary paths. Lastly, assembling the complete genome of C. depauperatus will foster comparative studies between pathogenic and non-pathogenic Cryptococcus species.

Draft genome sequences of strains CBS6241 and CBS6242 of the basidiomycetous yeast Filobasidium floriforme.

The Tremellomycetes are a species-rich group within the basidiomycete fungi; however, most analyses of this group to date have focused on pathogenic Cryptococcus species within the order Tremellales. Recent genome-assisted studies of other Tremellomycetes have identified interesting features with respect to biotechnological applications as well as the evolution of genes involved in mating and sexual development. Here, we report genome sequences of two strains of Filobasidium floriforme, a species from the order Filobasidiales, which branches basally to the Tremellales, Trichosporonales, and Holtermanniales. The assembled genomes of strains CBS6241 and CBS6242 are 27.4 Mb and 26.4 Mb in size, respectively, with 8314 and 7695 predicted protein-coding genes. Overall sequence identity at nucleic acid level between the strains is 97%. Among the predicted genes are pheromone precursor and pheromone receptor genes as well as two genes encoding homedomain (HD) transcription factors, which are predicted to be part of the mating type (MAT) locus. Sequence analysis indicates that CBS6241 and CBS6242 carry different alleles for both the pheromone/receptor genes as well as the HD transcription factors. Orthology inference identified 1482 orthogroups exclusively found in F. floriforme, some of which were involved in carbohydrate transport and metabolism. Subsequent CAZyme repertoire characterization identified 267 and 247 enzymes for CBS6241 and CBS6242, respectively, the second highest number of CAZymes among the analyzed Tremellomycete species. In addition, F. floriforme contains five CAZymes absent in other species and several plant-cell-wall degrading CAZymes with the highest copy number in Tremellomycota, indicating the biotechnological potential of this species.

Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora.

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Endoplasmic reticulum membrane receptors of the GET pathway are conserved throughout eukaryotes.

Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plant Arabidopsis thaliana, most components of the pathway were identified through in silico sequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation-mass spectrometry analysis to detect in vivo interactors of AtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses with AtGET1, and binds to Arabidopsis GET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of other Atget lines, its heterologous expression together with the coreceptor AtGET1 rescues growth defects of Δget1get2 yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 in Arabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype.

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context.

Sordaria macrospora: 25 years as a model organism for studying the molecular mechanisms of fruiting body development.

Fruiting bodies are among the most complex multicellular structures formed by fungi, and the molecular mechanisms that regulate their development are far from understood. However, studies with a number of fungal model organisms have started to shed light on this developmental process. One of these model organisms is Sordaria macrospora, a filamentous ascomycete from the order Sordariales. This fungus has been a genetic model organism since the 1950s, but its career as a model organism for molecular genetics really took off in the 1990s, when the establishment of a transformation protocol, a mutant collection, and an indexed cosmid library provided the methods and resources to start revealing the molecular mechanisms of fruiting body development. In the 2000s, "omics" methods were added to the S. macrospora tool box, and by 2020, 58 developmental genes have been identified in this fungus. This review gives a brief overview of major method developments for S. macrospora, and then focuses on recent results characterizing different processes involved in regulating development including several regulatory protein complexes, autophagy, transcriptional and chromatin regulation, and RNA editing. KEY POINTS: •Sordaria macrospora is a model system for analyzing fungal fruiting body development. •More than 100 developmental mutants are available for S. macrospora. •More than 50 developmental genes have been characterized in S. macrospora.

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages.

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly.

Comparative Genomics and Transcriptomics To Analyze Fruiting Body Development in Filamentous Ascomycetes.

Many filamentous ascomycetes develop three-dimensional fruiting bodies for production and dispersal of sexual spores. Fruiting bodies are among the most complex structures differentiated by ascomycetes; however, the molecular mechanisms underlying this process are insufficiently understood. Previous comparative transcriptomics analyses of fruiting body development in different ascomycetes suggested that there might be a core set of genes that are transcriptionally regulated in a similar manner across species. Conserved patterns of gene expression can be indicative of functional relevance, and therefore such a set of genes might constitute promising candidates for functional analyses. In this study, we have sequenced the genome of the Pezizomycete Ascodesmis nigricans, and performed comparative transcriptomics of developing fruiting bodies of this fungus, the Pezizomycete Pyronema confluens, and the Sordariomycete Sordaria macrospora With only 27 Mb, the A. nigricans genome is the smallest Pezizomycete genome sequenced to date. Comparative transcriptomics indicated that gene expression patterns in developing fruiting bodies of the three species are more similar to each other than to nonsexual hyphae of the same species. An analysis of 83 genes that are upregulated only during fruiting body development in all three species revealed 23 genes encoding proteins with predicted roles in vesicle transport, the endomembrane system, or transport across membranes, and 13 genes encoding proteins with predicted roles in chromatin organization or the regulation of gene expression. Among four genes chosen for functional analysis by deletion in S. macrospora, three were shown to be involved in fruiting body formation, including two predicted chromatin modifier genes.

Convergent evolution of linked mating-type loci in basidiomycete fungi.

Sexual development is a key evolutionary innovation of eukaryotes. In many species, mating involves interaction between compatible mating partners that can undergo cell and nuclear fusion and subsequent steps of development including meiosis. Mating compatibility in fungi is governed by the mating type (MAT) loci. In basidiomycetes, the ancestral state is hypothesized to be tetrapolar, with two genetically unlinked MAT loci containing homeodomain transcription factor genes (HD locus) and pheromone and pheromone receptor genes (P/R locus), respectively. Alleles at both loci must differ between mating partners for completion of sexual development. However, there are also basidiomycetes with bipolar mating systems, which can arise through genomic linkage of the HD and P/R loci. In the order Tremellales, bipolarity is found only in the pathogenic Cryptococcus species. Here, we describe the analysis of MAT loci from 24 species of the Trichosporonales, a sister order to the Tremellales. In all of the species analyzed, the MAT loci are fused and a single HD gene is present in each mating type, similar to the organization in the pathogenic Cryptococci. However, the HD and P/R allele combinations in the Trichosporonales are different from those in the pathogenic Cryptococci. This and the existence of tetrapolar species in the Tremellales suggest that fusion of the HD and P/R loci occurred independently in the Trichosporonales and pathogenic Cryptococci, supporting the hypothesis of convergent evolution towards fused MAT regions, similar to previous findings in other fungal groups. Unlike the fused MAT loci in several other basidiomycete lineages though, the gene content and gene order within the fused MAT loci are highly conserved in the Trichosporonales, and there is no apparent suppression of recombination extending from the MAT loci to adjacent chromosomal regions, suggesting different mechanisms for the evolution of physically linked MAT loci in these groups.

Genetic and Genomic Analyses Reveal Boundaries between Species Closely Related to Cryptococcus Pathogens.

Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes >200,000 new human infections annually. Elucidation of how these species evolved into important human-pathogenic species remains challenging and can be advanced by studying the most closely related nonpathogenic species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii However, these species have only four known isolates, and available data were insufficient to determine species boundaries within this group. By analyzing full-length chromosome assemblies, we reappraised the phylogenetic relationships of the four available strains, confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. The genomes of the three species are ∼6% divergent and exhibit significant chromosomal rearrangements, including inversions and a reciprocal translocation that involved intercentromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can still undergo sexual reproduction with C. amylolentus However, most of the resulting spores were inviable or sterile or showed reduced recombination during meiosis, indicating that intrinsic postzygotic barriers had been established. Our study and genomic data will foster additional studies addressing fungal speciation and transitions between nonpathogenic and pathogenic Cryptococcus lineages.IMPORTANCE The evolutionary drivers of speciation are critical to our understanding of how new pathogens arise from nonpathogenic lineages and adapt to new environments. Here we focus on the Cryptococcus amylolentus species complex, a nonpathogenic fungal lineage closely related to the human-pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analyses, we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. Their genomes are ∼6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) were still able to interbreed, the resulting hybrid progeny were usually inviable or sterile, indicating that barriers to reproduction had already been established. These results advance our understanding of speciation in fungi and highlight the power of genomics in assisting our ability to correctly identify and discriminate fungal species.

The transcription factor PRO44 and the histone chaperone ASF1 regulate distinct aspects of multicellular development in the filamentous fungus Sordaria macrospora.

BACKGROUND: Fungal fruiting bodies are complex three-dimensional structures that are formed to protect and disperse the sexual spores. Their morphogenesis requires the concerted action of numerous genes; however, at the molecular level, the spatio-temporal sequence of events leading to the mature fruiting body is largely unknown. In previous studies, the transcription factor gene pro44 and the histone chaperone gene asf1 were shown to be essential for fruiting body formation in the ascomycete Sordaria macrospora. Both PRO44 and ASF1 are predicted to act on the regulation of gene expression in the nucleus, and mutants in both genes are blocked at the same stage of development. Thus, we hypothesized that PRO44 and ASF1 might be involved in similar aspects of transcriptional regulation. In this study, we characterized their roles in fruiting body development in more detail. RESULTS: The PRO44 protein forms homodimers, localizes to the nucleus, and is strongly expressed in the outer layers of the developing young fruiting body. Analysis of single and double mutants of asf1 and three other chromatin modifier genes, cac2, crc1, and rtt106, showed that only asf1 is essential for fruiting body formation whereas cac2 and rtt106 might have redundant functions in this process. RNA-seq analysis revealed distinct roles for asf1 and pro44 in sexual development, with asf1 acting as a suppressor of weakly expressed genes during morphogenesis. This is most likely not due to global mislocalization of nucleosomes as micrococcal nuclease-sequencing did not reveal differences in nucleosome spacing and positioning around transcriptional start sites between Δasf1 and the wild type. However, bisulfite sequencing revealed a decrease in DNA methylation in Δasf1, which might be a reason for the observed changes in gene expression. Transcriptome analysis of gene expression in young fruiting bodies showed that pro44 is required for correct expression of genes involved in extracellular metabolism. Deletion of the putative transcription factor gene asm2, which is downregulated in young fruiting bodies of Δpro44, results in defects during ascospore maturation. CONCLUSIONS: In summary, the results indicate distinct roles for the transcription factor PRO44 and the histone chaperone ASF1 in the regulation of sexual development in fungi.

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle.

Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.

Mating-type factor-specific regulation of the fumagillin/pseurotin secondary metabolite supercluster in Aspergillus fumigatus.

In the human pathogenic mold Aspergillus fumigatus, sexual identity is determined by the mating-type idiomorphs MAT1-1 and MAT1-2 residing at the MAT locus. Upon crossing of compatible partners, a heterothallic mating is executed to eventually form cleistothecia that contain recombinant ascospores. Given that the MAT1 gene products are DNA binding master regulators that govern this complex developmental process, we monitored the MAT1-driven transcriptomes of A. fumigatus by conditional overexpression of either MAT1 gene followed by RNA-seq analyses. Numerous genes related to the process of mating were found to be under transcriptional control, such as pheromone production and recognition. Substantial differences between the MAT1-1- and MAT1-2-driven transcriptomes could be detected by functional categorization of differentially expressed genes. Moreover, a significant and distinct impact on expression of genetic clusters of secondary metabolism became apparent, which could be verified on the product level. Unexpectedly, specific cross-regulation of the fumagillin/pseurotin supercluster was evident, thereby uncoupling its co-regulatory characteristic. These insights imply a tight interconnection of sexual development accompanied by ascosporogenesis with secondary metabolite production of a pathogenic fungus and impose evolutionary constraints that link these two fundamental aspects of the fungal lifestyle.

Visual tuning in the flashlight fish Anomalops katoptron to detect blue, bioluminescent light.

Bioluminescence is a fascinating phenomenon and can be found in many different organisms including fish. It has been suggested that bioluminescence is used for example for defense, prey attraction, and for intraspecific communication to attract for example sexual partners. The flashlight fish, Anomalops katoptron (A. katoptron), is a nocturnal fish that produces bioluminescence and lives in shallow waters, which makes it ideal for laboratory studies. In order to understand A. katoptron's ability to detect bioluminescent light (480 to 490 nm) at night, we characterized the visual system adaptation of A. katoptron using phylogenetic, electrophysiological and behavioral studies. We found that the retinae of A. katoptron contain rods and sparse cones. A. katoptron retinae express two main visual pigments, rhodopsin (RH1), and to a lesser extent, rhodopsin-like opsin (RH2). Interestingly, recombinant RH1 and RH2 are maximally sensitive to a wavelength of approximately 490 nm light (λmax), which correspond to the spectral peak of in vivo electroretinogram (ERG) measurements. In addition, behavioral assays revealed that A. katoptron is attracted by low intensity blue but not red light. Collectively, our results suggest that the A. katoptron visual system is optimized to detect blue light in the frequency range of its own bioluminescence and residual starlight.