Frontiers in Fungal Biology

Accessory genes are thought to contribute to fungal adaptation and pathogenicity by modulating host immunity, while core genes play crucial roles in maintaining fundamental biological processes. Rust fungi (order Pucciniales) are obligate biotrophic plant-pathogens and infect economically relevant crops. Here, we characterize core and accessory gene repertoires across rust fungi, with a particular focus on Phakopsora pachyrhizi, the causal agent of Asian soybean rust. Across Pucciniales genomes, accessory genes represented the largest fraction of gene content (~44.6% on average), whereas core genes accounted for a smaller proportion (~18-35%). Notably, variations in accessory gene content among rust fungi are perhaps attributed to lineage-specific gene expansions and losses. Core gene content was positively correlated with total gene number across Pucciniales genomes, suggesting retention after gene duplication events, consistent with their essential biological functions. Among P. pachyrhizi genes expressed during soybean infection, core effectors were associated with cysteine-rich proteins, pectin-degrading enzymes, and SPFH/Band 7 family, while accessory effectors included phosphatidylethanolamine-binding proteins, trehalose phosphatases, and CFEM domain-containing proteins. The in-plant induced core and accessory genes in P. pachyrhizi also comprised multiple families of CAZymes (GH5/GH7 cellulases, CE5 cutinases, CE8 pectinesterases, CE4/GH18 chitin-modifying enzymes); proteases (aspartyl proteases, serine carboxypeptidases, alpha/beta hydrolases); transporters (amino acid permeases, ferric reductase-like transmembrane proteins, and OPT oligopeptide transporter), and transcription factors (bZIP, GATA zinc finger, STE-like, and homeobox KN). Our study highlights that core and accessory gene families have shaped P. pachyrhizi-soybean interactions, identifying promising targets for functional studies aimed at elucidating host-adaptation mechanisms in rust fungi.

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.

The contribution of accessory or conditionally dispensable chromosomes to host-specific virulence was first demonstrated in members of the Fusarium solani species complex (FSSC) that are pathogens of garden pea, Pisum sativum L. The phenomenon has since been shown to exist in many fungal plant pathogens, including the closely related F. oxysporum species complex (FOSC). Genome analysis of members of the FSSC and FOSC pathogenic on pea revealed a diverse size range of the accessory genome of these fungi. Despite the ~65 million years of diverging time, regions on a chromosome known to carry host-specific virulence factors for pea, including the cytochrome P450 pisatin demethylase (PDA) and other pea pathogenicity (PEP) genes, were present in all genomes of these pea pathogens. Genes directly involved in virulence on pea - PEP2, PDA, and PEP5- were the most frequently clustered together. Transcriptome analysis of fungal mycelia treated with the pea phytoalexin pisatin, identified 1,155 differentially expressed genes where many were involved in cellular stress responses. As wilt pathogens that invade host xylem, members of the FOSC encode more putative effectors, when compared to those in the FSSC, and several FOSC effectors were identified to confer race specificity. The conservation of part of the accessory genomes across two evolutionarily diverged species complexes suggests a common origin. Horizontal transfer of accessory chromosomes containing genetic loci involved in pathogenesis for garden pea offers a parsimonious explanation of the polyphyletic origin of host specificity.

The ability to opportunistically infect humans has evolved multiple times across fungi and is a major burden to public health. Opportunistic pathogenicity requires the confluence of pre-existing traits in the fungus that facilitate host colonization (e.g., the ability to grow at 37C) and the existence of host immune filters that permit the survival of some colonizers (e.g., inborn errors of immunity). Numerous studies have previously shown that fungal pathogens can exhibit extensive strain-to-strain variation in the ability to cause disease. Moreover, it is also well established that non-pathogenic fungi can occasionally cause severe infections. Together, these observations provoke the question: what differentiates opportunistic fungal pathogens from non-pathogens? To empirically address this, we directly compared phenotypic, metabolomic, and genomic variation between Aspergillus fumigatus, an organism responsible for more than 300,000 infections per year, and Aspergillus fischeri, a close relative of A. fumigatus that is not considered clinically relevant. By examining 26 phenotypic traits across 16 representative strains of A. fumigatus and 16 of A. fischeri, we find that infection-relevant traits measured under in vitro monoculture conditions show species-specific distributions, whereas traits measured under in vitro coculture with murine macrophages overlap in their distributions. Strikingly, strains of the two species also overlap in their virulence profiles in an immunocompromised murine model of pulmonary aspergillosis; three strains of A. fischeri exhibit lethality rates of >50% while two A. fumigatus strains were among the least virulent of all 32 strains tested. Consistent with the observed overlap, we could not statistically associate variation in virulence to variation in the presence of specific genomic elements, phenotypic traits, or secondary metabolites. Our results raise the hypothesis that opportunistic pathogenicity can extend beyond the boundaries of individual species. We propose a conceptual model where the opportunistic pathogenic potential of any fungal strain is the product of complex interactions among numerous genomic, ecological, and host immunity factors.

In fungi, hyphal fusion is beneficial within an individual, but fusion between individuals comes with the risks of infection or exploitation. To manage this risk, fungi have developed mechanisms to restrict sustained fusion to be within a genetic individual, called allorecognition. In Ascomycete fungi, this recognition is based on allelic identity at several polymorphic allorecognition genes, often triggering cell death. However, the genetic basis of allorecognition is unknown in basidiomycetes, the clade that includes mushroom-forming fungi. Here, we map the first locus for this trait, which we call somA, in the mushroom-forming fungus Coprinopsis cinerea. We combined F1 offspring phenotypes with independent backcross lines to identify a region on chromosome 5 linked with the production of a barrage zone, a classic allorecognition phenotype. Fine-mapping of this region resulted in a region with a set of kinases and NACHT domain proteins, flanked by a leucine-rich repeat (LRR) protein. While the NACHT and kinase proteins are diverse between the parents, the LRR-encoding protein shows signs of purifying selection. Additional C. cinerea genomes show that this region contains several highly divergent alleles, consistent with long-term balancing selection. These polymorphic alleles all contain a single monomorphic LRR, which may indicate a novel mechanism for fungal nonself recognition. Based on a phylogenetic survey of related Basidiomycetes, this specific locus architecture appears to be restricted to closely related species. This finding of a multiallelic locus may explain the general trend of few nonself recognition loci in basidiomycetes. These results provide a first understanding of how individuality is maintained in basidiomycetes.

Though fungi have largely been studied in the context of terrestrial niches, aquatic species that originated from terrestrial ancestors can be found across the fungal kingdom. To date, the mechanisms of these transitions from land to sea have remained poorly understood, and it is unclear what traits are associated with the specialization of a fungal to marine environments. Here we develop Kluyveromyces budding yeasts, sampled from terrestrial, estuarine, and marine niches, as a model for the evolution of fungi into the ocean. Comparative analyses of genomes from the genus revealed a contraction in genome size and gene number in aquatic Kluyveromyces compared to their terrestrial relatives, including at genes annotated in alcoholic fermentation. In laboratory culture, we uncovered evidence for phenotypic losses in aquatic Kluyveromyces species, namely compromised desiccation and cold resistance relative to the terrestrial clade. Aquatic Kluyveromyces also exhibited better salt tolerance than terrestrial species, reflecting an evolutionary gain consonant with their provenance from seawater. Furthermore, in molecular-evolution analyses, we found robust signal for positive selection in the aquatic Kluyveromyces lineage, most notably at genes annotated in respiration. We interpret these results under a model in which the release of ethanol, which allows yeasts in terrestrial niches to kill off bacterial competitors at close range, has little use in the water; aquatic Kluyveromyces thus evolved to lose fermentation but gained other metabolic and stress-tolerance innovations essential for fitness in the marine niche. We propose that the syndrome of genomic features and phenotypes in aquatic Kluyveromyces reflects broadly relevant mechanisms of evolutionary transitions by fungi into ocean environments.

Fusarium oxysporum MRL8996 is a fungal strain isolated from a severe case of contact lens-associated keratitis. Here we report a near-complete genome assembly of this isolate using a hybrid Nanopore and Hi-C scaffolding approach. The assembly resolves the genome into 16 distinct chromosomes, including 11 core and 5 lineage-specific chromosomes. This high-quality reference genome provides an unprecedented tool for investigating the large-scale structural variations and evolutionary mechanisms driving adaptation in this highly versatile fungal lineage.

Compared with bacterial and archaeal extremophiles, single-celled eukaryotes living in extreme habitats are understudied and underrepresented in genomic databases. An exception is the obligately halophilic stramenopile Halocafeteria seosinensis strain EHF34. A transcriptome-focused analysis of this extremophilic protists revealed the importance of organic osmolyte regulation and transport in its adaptation to hypersaline environments. However, genomic resources for H. seosinensis are currently limited to a highly fragmented assembly generated by short-read sequencing, which has hindered further investigation of the genome biology and evolution of this fascinating organism. Here, we used long-read Oxford Nanopore sequencing to generate a highly contiguous, chromosome-scale genome assembly for H. seosinensis. The assembly is 38.8 megabase pairs (Mbp) in size and contains 60 nuclear contigs, making it the most contiguous genome for a member of the order Bicosoecida. Approximately 19% of the genome is comprised of transposable elements. Of the 11,684 predicted protein-coding genes, many appear to be associated with DNA mobility-related functions, and several may be linked to adaptation to a hypersaline environment. Analysis of the H. seosinensis long-read genome assembly presented herein will facilitate our understanding of the ways in which protists have adapted to extreme environments. SignificanceHalocafeteria seosinensis is an extremophilic protist adapted to hypersaline environments. Previous analyses of a transcriptome and short-read draft genome assembly for this organism provided insights into the molecular mechanisms underlying osmotic regulation, which facilitate its adaptation to high-salt conditions. However, the lack of contiguity and quality of the draft assembly prevented the characterization of complex genomic regions, including transposable elements and viral insertions, as well as genomic comparisons with related species. Here we present a highly contiguous, chromosome-scale genome assembly for H. seosinensis that enables accurate gene prediction, detailed analysis of repeat content, and comparative genomic analysis. This long-read genome assembly will serve as a valuable resource for studying one of the few tractable halophilic protists sequenced to date.

Green algae must continuously balance resource availability to maintain photosynthetic performance. The O2:CO2 ratio is a key determinant of their metabolic mode. Under hyperoxia or low CO2, many algae induce a carbon concentrating mechanism (CCM). In the model green alga Chlamydomonas reinhardtii, the CCM relies on a pyrenoid, a specialized microcompartment that elevates CO2 around rubisco. While ambient CO2 acclimation is well-studied, responses to hyperoxia remain poorly understood, despite its frequent occurrence in nature under high light. Using controlled bioreactors, we exposed two diverse Chlamydomonas ecotypes, CC1009 and CC2343, to 95% oxygen to analyze time-dependent, genome-wide transcriptomic and phenotypic changes. Both ecotypes induced CCM genes, but they exhibited distinct molecular and physiological phenotypes. The tolerant ecotype (CC1009) successfully adapted, developing a functional CCM with a structured starch sheath. Conversely, the sensitive ecotype (CC2343) suffered growth arrest and formed malformed pyrenoids. Transcriptomics revealed that CC1009 initiated a rapid initial response, upregulating chloroplast proteostasis and downregulating nucleotide metabolism. CC2343 showed a massive, delayed transcriptional response, downregulating genes coding for photosystems and tetrapyrrole biosynthesis. This unbiased transcriptomic approach identifies key candidate genes driving algal acclimation to hyperoxic stress in natural, high-light environments.

Entamoeba histolytica is a clinically important pathogenic eukaryote and the causative agent of amoebic dysentery. Entamoeba dispar, a nonpathogenic commensal species that resides in the human colon, is the closest sibling species, and serves as an appropriate comparator for genome-wide analysis. Although the genome of E. histolytica is approximately 26.9 Mb, and the largest known genome within the genus, that of E. invadens, is approximately 40.9 Mb, obtaining high-quality assemblies in this genus has remained challenging due to extensive repetitive regions, tRNA gene arrays, and aneuploidy. Here, we used PacBio HiFi sequencing to assemble the genomes of the pathogenic E. histolytica and the nonpathogenic E. dispar. We reconstructed all 36 chromosomes of E. histolytica and 35 chromosomes of E. dispar, assembling each as a single continuous DNA sequence (contig). The two species exhibited high genome-wide nucleotide similarity and conserved synteny at the amino acid level. At one end of each chromosome, we identified tRNA arrays, whereas the opposite end lacked such arrays, resulting in an asymmetric chromosomal architecture. Analysis of unique-read depth revealed widespread aneuploidy in both species: E. histolytica is predominantly tetraploid, whereas E. dispar is diploid, a conclusion further supported by SNP allele-frequency distributions. These assemblies provide a robust foundation for comparative genomics in Entamoeba and offer detailed insights into chromosome-end structure and ploidy.

Cryptococcus neoformans is the predominant causative agent of cryptococcal meningitis in immunocompromised individuals. Conversely in immunocompetent individuals, C. neoformans establishes a latent pulmonary infection characterized by a paucity of clinical symptoms. Using a mouse inhalation model of latent C. neoformans infection, we previously showed that CD4 T-cells are necessary for preventing fungal proliferation in the lungs. In the current study, we performed single cell RNA sequencing (scRNAseq) and found that the CD4 T-cell response was both highly heterogenous and dichotomous during pulmonary C. neoformans infection, with concomitant expression of genes related to Th1 polarization (Tbx21, Ifng) and immune regulation (Ctla4). First, we demonstrated that cells with Th1-like phenotypes are necessary and sufficient to control latent infection via adoptive transfer of T-bet positive cells into infection-matched CD4-depleted recipient mice. Second, scRNAseq analysis revealed the subpopulation of effector CD4 T-cells that co-expressed Ctla4 and Gata3 was significantly higher than a subpopulation that co-expressed Ctla4 and Tbx21. Furthermore, our data suggested that CTLA-4 upregulation is beneficial against C. neoformans infection, as CTLA-4 blockade promoted fungal proliferation. Thus, we propose a model wherein Th1 control of latent C. neoformans infection is supported by CTLA-4 suppression of detrimental Th2 activation.

Metribuzin is a versatile herbicide effective against various annual grasses and broadleaf weeds found in wheat fields. However, it can cause foliar damage to wheat, impacting plant health and yield. A clearer understanding of the genetic architecture associated with metribuzin tolerance is necessary to guide marker-based breeding strategies. This study evaluated 351 historic Gulf Atlantic Wheat Nursery (GAWN) wheat breeding lines representative of southern US soft red winter wheat (SRWW) germplasm. Field trials were conducted at Winnsboro (WN) and Baton Rouge (BR), Louisiana, in 2016 and 2017. Metribuzin was applied at specific growth stages[DN1.1], and tolerance was assessed based on visual foliar damage. Genomic data from 6,252 filtered single nucleotide polymorphism (SNP) markers were used to estimate narrow-sense heritability, conduct genome-wide association (GWAS), and assess genomic prediction accuracy using genomic best linear unbiased prediction (GBLUP). Broad-sense heritability ranged from 0.54 to 0.69 within environments and reached 0.77 across environments, while narrow-sense heritability ranged from 0.35 to 0.47, indicating moderate additive genetic control. No SNP surpassed the significance threshold, but genomic prediction (GP) showed moderate to strong predictive ability (PA) across environments, with the highest accuracy (r = 0.62) observed between BR17 and WN17. These results indicate that metribuzin tolerance in SRWW is primarily controlled by multiple small-effect loci and that GS provides a more effective breeding strategy than marker-assisted selection for improving tolerance in southern wheat germplasm.

Vacuolar-type ATPases (V-ATPases) are evolutionarily conserved rotary proton pumps that play essential roles in the eukaryotic cell. By coupling ATP hydrolysis in their cytosolic V1 region to proton translocation through their membrane-embedded VO region, V-ATPases establish and maintain an acidic pH in the lumen of several different organelles. Functional diversity in the pump is enabled by multiple paralogous genes for the subunits of the complex, which are expressed in a tissue- and organelle-specific manner. Interactions between V-ATPase and TLDc domain-containing proteins have been shown to regulate the enzyme in yeast and mammals but their relevance in plants has remained unclear. We isolated the endogenous V-ATPase from Arabidopsis thaliana leaves and determined its structure by electron cryomicroscopy. Mass spectrometry showed that most of the enzyme originated from the tonoplast. The structural analysis revealed the full rotary catalytic cycle of the plant V-ATPase, and a combination of structural and biochemical experiments showed S-acylation of subunits AP1 and the tonoplast-specific subunit a3 isoform. A subpopulation of complexes derived from the trans-Golgi network/early endosome was identified and found to bind the TLDc protein OXR5. Together, these findings reveal plant-specific features in V-ATPase and suggest organelle-specific interactions with TLDc proteins, pointing to conserved but context-dependent V-ATPase regulation in eukaryotes.

Serotonin and dopamine make dissociable contributions to reinforcement learning (RL) sub-components, yet we lack neural biomarkers capable of detecting their differential effects. Here, we report the development of a five-task EEG battery designed to probe these dissociable RL mechanisms. Using a model-free analytical approach in healthy volunteers, we identify distinct neural markers across probabilistic instrumental learning, motivational vigour, Pavlovian-instrumental transfer, reversal learning, and a working memory-RL task. A centro-parietal P300 tracked incremental learning across three paradigms and showed cross-task convergent validity. Readiness potentials and beta suppression indexed value-based motor preparation, while frontal theta captured Pavlovian-instrumental conflict. The largely independent pattern across markers supports the battery's capacity to detect selective pharmacological effects on distinct neural systems.

Memorizing a new phone number or address is hard at first, but becomes easier with repetition, as information shifts from working memory to long-term memory. Here we investigated how repetition affects the storage and transition of different aspects of mnemonic information by comparing univariate neural markers of active object storage with multivariate decoding of memory content. Thirty participants encoded lateralized stimuli from a continuous shape space into memory. Memory items were repeated six times in a row to induce learning. In line with earlier work, EEG recordings revealed that repetition led to a reduction in contralateral delay activity (CDA), a measure of active storage that has been taken to reflect a pointer-like representation of the individual object or its original source. In contrast, shape decoding during the retention and also after an impulse perturbation remained constant across repetitions. These results suggest that learning over repetitions reflects the abolishment of active and individuated object memory representations while passive, source-independent memory representations are retained.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

Cortisol, our stress hormone, exerts widespread influence on neural activity. However, its influence on the aperiodic component of the electroencephalography power spectrum remains to be investigated. Given individual differences in the capacity to cope with stress and adversity, it also remains unclear whether trait resilience moderates this relationship. Hence, the present study examined whether individual differences in trait resilience moderates the association between resting cortisol and aperiodic activity. Participants (N=145) completed various self-report questionnaires (e.g., trait resilience). Electroencephalography was recorded over a 20-minute baseline period, followed by salivary cortisol collection. The results revealed a significant moderating effect of trait resilience in the occipital scalp region. Specifically, higher cortisol concentration was associated with flatter 1/f slopes amongst individuals with low trait resilience, whereas this association was reversed amongst those with high trait resilience. Overall, our findings highlight the role of individual differences in trait resilience in shaping hypothalamic-pituitary-adrenal axis-related neural dynamics.

Large-scale brain imaging has relied heavily on fluorescent reporters; however, photobleaching and signal variability limit quantitative analysis in intact tissues. Here, we intro-duce MelaCAST (melanin-based scattering CAST imaging), a genetically encoded scattering strategy for whole-brain imaging. AAV-mediated delivery of tyrosinase enables cell-type-specific melanin production, generating stable intracellular scattering contrast throughout the mouse brain. By integrating tissue clearing with scattering tomography, MelaCAST enables non-photobleaching, high-throughput volumetric imaging of genetically defined cell populations in in-tact brains. This approach establishes melanin as a genetically encoded scattering reporter and expands whole-organ imaging beyond fluorescence-based modalities.

Extremely elongate necks have convergently evolved in several amniote lineages, including both aquatic and terrestrial forms (Fig. 1). The development of such a feature brings with it advantages in obtaining food items, but also biomechanical challenges, such as flexibility, stability, lift, and inertia. In Tanystropheus, a particularly long-necked Triassic archosauromorph, the neck is composed of only 13, mostly extraordinarily elongated and slender cervical vertebrae and accompanying rod-like, overlapping ribs, making it arguably the most extreme example of neck elongation in tetrapod evolution (Fig. 1;1-6). Understanding the function of this remarkable neck provides insights into the limits of neck elongation in amniotes and the evolution of morphological novelties in Triassic reptiles. Here we present the first quantitative biomechanical analysis of the Tanystropheus neck using a digital model based on three-dimensionally preserved bones. We assessed its range of motion (ROM) and performed finite element analysis (FEA) on the individual cervical ribs and the neck model in different configurations. Our results indicate that the neck of Tanystropheus was not extremely stiff, as previously postulated, and the ribs likely did not impair its movements. They transferred tensile forces towards the base of the neck, similar to what hypothesized for sauropods7. This study elucidates the bauplan of an extremely specialized animal and brings us closer to understanding the patterns of achieving neck elongation in vertebrates.

Oxytocin and vasopressin are endogenous bioactive peptides with conserved roles in reproduction and, more recently recognized, in peripheral lipid metabolism. Whether this signaling system also shapes how reproduction declines with age has not been tested in any animal. Here we show that in C. elegans, the oxytocin/vasopressin-like neuropeptide nematocin restrains reproductive output as animals reach mid-life. Nematocin and its two receptors are produced throughout adult life and peak as reproduction begins to wane. Animals lacking receptor signaling produce more offspring in mid-life, an improvement that reflects better egg quality and fertilization rather than improved embryo survival. This benefit is accompanied by changes in intestinal fat metabolism, the worm's equivalent of liver and adipose tissue: nematocin normally limits the activity of a fatty-acid desaturase that is otherwise induced by mating, and it shapes how much yolk reaches developing eggs. The two receptors act through separate routes, one tuning intestinal fat metabolism and the other controlling yolk delivery to the egg. Together, these findings reveal nematocin as a regulator of the intestinal metabolic environment across reproductive age, mirroring the recently described oxytocin-hepatocyte-adipocyte lipid axis in mammals and implicate this conserved signaling system in the coordination of maternal investment during reproductive aging.