Fungal Genetics and Biology

Leptosphaeria maculans is a plant-pathogenic fungus that infects Brassica species, including Brassica napus (oilseed rape). Breeding oilseed rape varieties with genetic resistance is an efficient way to control the disease; however, L. maculans can adapt and overcome these resistances. Understanding the mechanisms that enable L. maculans to adapt is crucial for managing the emergence of better-adapted isolates. Brassica carinata, the Ethiopian Mustard, although closely related to B. napus, is considered a nonhost species of L. maculans because this fungus cannot infect it. Despite the extreme resistance of B. carinata, one natural L. maculans isolate has been identified as unable to infect B. napus, causing moderate and atypical symptoms on this species. We performed a cross between this isolate and an isolate adapted to B. napus, followed by a QTL analysis, which identified seven QTL, each encompassing candidate genes involved in L. maculans adaptation to B. carinata or B. napus. Additionally, we observed transgression in the progeny, wherein a few strains caused significantly more or less aggressive symptoms on both species of Brassica. We found that epistasis within the L. maculans genome contributes to the observed transgression. These initial findings provide further opportunities to study the adaptive capacities of L. maculans, as well as data to initiate analysis of the extreme resistance of B. carinata to L. maculans. HighlightsO_LISeven pathogenicity QTL identified, carrying several interesting candidate genes C_LIO_LITransgression of some progeny isolates on B. napus and B. carinata was reported C_LIO_LIEpistasis plays a significant role in the adaptation of L. maculans toward host and nonhost Brassica species C_LI

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

Domestication of molds for cheese production has repeatedly shaped Penicillium fungi, most notably giving rise to the emblematic P. camemberti lineage, derived from P. biforme. Here, we identified a new P. biforme lineage, named cheesy, likely selected from P. biforme for food fermentation, including cheese and sausage production. This lineage exhibits evidence of a severe bottleneck, with little nucleotide polymorphism and a single mating type. The cheesy lineage has evolved advantageous traits for cheesemaking: compared to other P. biforme strains and its wild relative P. fuscoglaucum, it displays faster growth on cheese, galactose and lactose media, higher sporulation and germination rates on cheese, elevated lipolytic activity, enhanced inhibition capacities, and produced specific volatile organic compounds. Additionally, P. camemberti and P. biforme cheesy differ in their content of Starship mobile elements, acquired through horizontal transfers. These elements carry cargo genes potentially relevant for adaptation to cheese. Notably, the cheesy lineage has acquired a 20 kb Starship element (Rattus), nested within a much larger 160 kb Starship (Bilge), and carrying cargo genes with predicted functions involved in antagonistic interactions among micro-organisms. SignificanceO_LIWe identified a new Penicillium biforme lineage, named cheesy, which was likely selected from P. biforme for food fermentation (cheese and sausage), genetically and phenotypically different from the emblematic P. camemberti lineage. This lineage suffered from a severe bottleneck, displaying very little nucleotide polymorphism and a single mating type. C_LIO_LIThis newly identified lineage has evolved advantageous traits for cheesemaking; compared to other P. biforme strains and its wild relative P. fuscoglaucum, it displayed faster growth on cheese, galactose and lactose media, higher sporulation and germination rates on cheese, higher lipolytic activity, better inhibition capacities and specific volatile organic compounds. C_LIO_LIPenicillium camemberti and P. biforme cheesy also differed by their content in Starship mobile elements, acquired through horizontal transfers, and carrying cargo genes potentially relevant for adaptation to cheese. C_LIO_LINotably, the cheesy lineage has acquired a specific 20 kb Starship nested within a much larger 160 kb Starship, and likely involved in antagonistic interactions. C_LI

Poly(ADP-ribose) polymerases (PARPs) catalyze ADP-ribosylation, a conserved post- translational modification involved in DNA repair, transcriptional regulation, and chromatin remodeling. Although extensively studied in animals, the evolution and diversification of PARPs across the fungal kingdom remain largely unexplored. Here, we present the first kingdom-wide comparative genomic analysis of PARP proteins across 534 fungal species spanning eight phyla. We identified two primary conserved fungal PARP protein types corresponding to human PARP1 and PARP6. Both exhibit highly dynamic evolutionary histories characterized by frequent independent gain and loss events. Ancestral state reconstruction supports the presence of PARP1 in the last common ancestor of fungi, whereas the PARP6-like family has undergone repeated lineage-specific gains and losses. Fungal PARP6-like proteins retain a compact PARP catalytic domain fused to a C-terminal E2 ubiquitin-conjugating domain, whereas the PARP1 family displays extensive structural diversification through domain shuffling and lineage-specific fusions associated with DNA metabolism, chromatin remodeling, and signal transduction. Reconstruction of ancestral catalytic motifs across fungi and other eukaryotes revealed convergent evolution of a non-canonical H-Y-Y catalytic triad, with multiple motif variants co-occurring within individual proteins, suggesting functional diversification. In the Fusarium oxysporum species complex, we identified a lineage-specific expansion of the PARP family, driven exclusively by accessory chromosomes. Genomes with expanded PARP1 repertoires exhibited elevated basal PARylation, increased resistance to DNA-damaging agents that induce single strand breaks, and DNA damage- induced expression of accessory Parp genes. These findings reveal fungal PARPs as evolutionarily dynamic proteins that likely contribute to genome stability, adaptation, and pathogenicity.

Ada1 (All Development Altered-1) is a conserved but poorly characterised basic leucine zipper (bZIP) transcription factor found throughout filamentous fungi. In the wheat pathogen Parastagonospora nodorum, PnAda1 is required for full virulence and is transcriptionally associated with the virulence regulator PnPf2, but its biological functions remain unclear. Here, we combined comparative RNA sequencing with targeted phenotypic analyses to define the role of PnAda1 during vegetative growth and host infection. Deletion of PnAda1 did not abolish pathogenicity but delayed disease progression, with the PnAda1-deletion mutant transcriptome at 7 days post-inoculation resembling that of the wildtype SN15 at 3 days. This developmental delay was associated with impaired activation of early infection-associated genes, including putative carbohydrate-active enzymes, proteases, transporters and other host-colonisation factors. In contrast, expression of major necrotrophic effector genes was not reduced and instead remained elevated during later stages of infection, indicating that PnAda1 is required for the timely progression of infection-associated transcriptional regulation rather than direct activation of effector genes. Beyond virulence, transcriptomic and phenotypic analyses revealed roles for PnAda1 in nitrogen assimilation, carbon utilisation, abiotic stress responses and fungicide sensitivity. Notably, PnAda1 deletion increased sensitivity to succinate dehydrogenase inhibitor fungicides and reduced expression of succinate dehydrogenase subunit genes. Collectively, our findings identify PnAda1 as a broad regulator of developmental and infection-associated transitions in P. nodorum and expand current understanding of the transcriptional network underlying virulence, metabolism and stress adaptation in an important fungal wheat pathogen.

Colletotrichum graminicola causes significant losses of the staple crop maize worldwide. The fungus produces two distinct asexual spore types, oval and falcate conidia, which show unique processes in development and plant interaction. Based on genome resequencing of our laboratory strain (CgM2/ M1.001), we investigated the gene expression profiles of oval and falcate conidia during development and early leaf infection using RNA-seq. Our results reveal specific gene expression profiles between the two spore types, indicating fundamental differences in their developmental programs that reflect different modes of infection. We identified expression patterns discriminating both conidia types from mycelium and spore type-specific ones for genes encoding transcription factors, conserved fungal developmental genes, transporters, genes of secondary metabolite clusters, and pathogenicity-related functions, including effectors and carbohydrate-active enzymes (CAZymes). Our study shows that despite the identical genomic basis, oval and falcate conidia show unique transcriptomes across vegetative development and early plant interaction. Taking together, these results provide new insights into the molecular mechanisms determining the biology of C. graminicola and its interaction with the plant host.

Recent work has established septation as a critical process for virulence in A. fumigatus, as strains lacking septa are avirulent. While our work has focused on the well-studied Septation Initiation Network (SIN), understanding of septation machinery upstream and downstream of SIN remains limited in filamentous fungi. Proximity labeling techniques are powerful tools to study pathway interactions, especially those that are transient. Recent advances have seen TurboID used to study pathways and processes important for fungal pathogenesis. In this study, we adapt TurboID for use in Aspergillus fumigatus and use overlapping datasets of two SIN components, the terminal NDR kinase SidB and its binding partner MobA, to identify putative septation effectors. Many known septation effectors, including septins and cell wall synthesis proteins, were enriched in our datasets, validating the approach. Phenotypic characterization of hits identified by LC-MS/MS revealed several previously uncharacterized gene products involved in septum formation and cell wall stress tolerance. These included the PP2A regulatory subunit PabA and IQGAP SepG, which have been shown to regulate septation in Aspergillus nidulans. Thus, TurboID is a useful tool to study fungal signaling and physiology in A. fumigatus and may be used to improve understanding of pathogenic processes.

Fungal mycoparasitism is commonly viewed as a broadly conserved lifestyle relying on the secretion of cell wall-degrading enzymes and specialized metabolites. However, whether Trichoderma deploys a universal antagonistic program or dynamically adapts its molecular arsenal according to prey identity remains largely unknown. Here, we investigated the phenotypic and transcriptomic responses of four highly antagonistic Trichoderma atroviride strains confronted with damping-off pathogens from three phylogenetically distinct lineages: the ascomycete Alternaria brassicicola, the basidiomycete Rhizoctonia solani, and the oomycete Globisporangium ultimum. In vitro confrontation assays were performed between the four T. atroviride strains and nine pathogen strains (three strains per species). Despite notable intraspecific variation in pathogen sensitivity, all four T. atroviride strains remained highly effective against all tested pathogens. Comparative RNA-seq analyses during direct confrontation with one representative strain per pathogen species revealed strong prey-dependent transcriptional plasticity. Interactions with the two fungal pathogens elicited a robust induction of classical mycoparasitic machinery, including glycoside hydrolases, secreted peptidases, and effector-like proteins, whereas these responses were markedly attenuated in interaction with G. ultimum. Specialized metabolite biosynthetic genes were broadly induced across all interactions, but with largely distinct gene sets depending on the pathogen, with the strongest divergence observed for confrontation with G. ultimum. Beyond this shared response, each fungal pathogen also triggered a distinct host-specific program. In response to A. brassicicola and R. solani, T. atroviride induced distinct sets of genes associated with cell wall organization, while the response to R. solani specifically involved the activation of peroxisomal functions and aromatic amino acid biosynthesis pathways. Receptors potentially involved in pathogen perception also showed pathogen-dependent expression patterns, with distinct GPCRs (G protein-coupled receptors) overexpressed depending on the pathogen encountered. NLRs (NOD-like receptors) displayed a similar pattern, although overexpression was detected only in response to A. brassicicola and G. ultimum. Together, these results demonstrate that T. atroviride does not rely on a fixed mycoparasitic program but instead dynamically remodels its transcriptome according to prey identity, revealing a high degree of transcriptional plasticity underlying broad-spectrum antagonism.

Fungal diseases cause devastating crop infestations and millions of deaths annually. To improve disease treatment development, we must understand the biological processes these treatments target and the fungal cellular responses they elicit. Current methods to identify genetic responses of fungi to antifungal drugs and fungicides typically involve laborious molecular genetics experiments or transcriptomics. We utilized high-throughput screens in the basidiomycete yeast Rhodotorula (Rhodosporidium) toruloides to rapidly identify genes associated with the fungal response to a copper-zinc fungicide developed by VM Agritech and compared identified genes to those transcriptionally activated in response to the fungicide in R. toruloides and the filamentous ascomycete Neurospora crassa. These 'omics technologies combined with molecular genetics and biochemical analyses showed copper and zinc synergize to inhibit fungal growth due, at least in part, to an increase in cellular zinc concentrations when copper is present. Intriguingly, although we identified genes involved in the copper-zinc fungicide response using both high-throughput screens and transcriptomics, there was little overlap in genes identified through the two methods. Our findings demonstrate the power of combining functional genomics and transcriptomics approaches in identifying a more comprehensive map of the genetic mechanisms necessary for responding to fungicides.

To face the fungal infections that affect millions of people every year, a better understanding of resistance-conferring mutations is necessary. One obstacle is the considerable time and effort necessary to construct point mutations in pathogenic species, which is required to validate their role in resistance. To overcome this problem, we have constructed a series of heterologous expression plasmids that allow expression of the ERG3, ERG6 and ERG11 orthologs from Candida albicans, Candidozyma auris, Nakaseomyces glabratus, Candida parapsilosis, Candida tropicalis, and the CYP51A and CYP51B genes of Aspergillus fumigatus. All orthologs are codon-optimized and synthesized for expression in the budding yeast Saccharomyces cerevisiae. We also constructed host strains in which the native ERG genes are either deleted or under the control of a repressible promoter. We showed that all orthologs complement the function of the native genes when expressed from the S. cerevisiae native promoters. We recreated previously reported resistance-conferring mutations, and showed that expression in S. cerevisiae replicates known resistance phenotypes. We then used the system to test corresponding mutations across orthologs, and showed that most resistance phenotypes are conserved. The heterologous expression system allows rapid construction of mutants, by taking advantage of the abundant genetic tools available for S. cerevisiae. Screening of mutants can be done within a few experimental steps, greatly increasing the throughput compared to the introduction of mutations in pathogenic fungi. As a proof-of-concept, we used the system to discover new resistance-conferring mutations, using random mutagenesis. All plasmids are made publicly available.

The coprophilous agaricomycete Coprinopsis cinerea is a model organism for antagonistic fungal-bacterial interactions. Previous studies showed that C. cinerea responds to antagonistic bacteria with strong induction of a set of genes encoding secreted antibacterial molecules. However, little is known about the elicitors of this response. Key open questions in this respect include whether individual antibacterial defence genes are induced by different bacteria and/or by specific bacterial soluble molecules. Here, we present a new C. cinerea reporter system to monitor antibacterial defence induction and address related outstanding issues with minimal hands-on time. In this system, the promoter of the endogenous bacterial-induced gene cclys1 drives the expression of cnluc, which encodes a secreted variant of the deep-sea shrimp luciferase Nluc. We show that cNluc allows to detect and quantify cclys1 induction by measuring luminescence directly in the culture medium of reporter strain colonies. Building on these features, we successfully leveraged the inducible cNluc reporter strain for the development of a novel 96-well plate assay that allows the high-throughput screening of antibacterial defence elicitors. As cNluc can be subject to degradation by secreted proteases of fungal or bacterial origin in the culture medium, we coupled this assay to confirmatory qRT-PCR. Testing this set-up by confronting the reporter strain with several different bacteria revealed that cclys1 induction occurs independently of the bacterial ecological niche. Based on these results, we also recommend qRT-PCR exclusively for validation of negative results. We conclude that cNluc offers significant advantages over cytoplasmic reporter proteins, especially for preliminary rapid screening of multiple conditions.

Pseudogymnoascus destructans is a psychrophilic ascomycete fungus that causes White-Nose Syndrome in North American hibernating bats. P. destructans invades the tissue present in bats' noses and wing membranes, causing the characteristic white nose and cupping erosion in the wings. P. destructans exhibits characteristics of a hemibiotrophic fungal pathogen, in which P. destructans can invade the host cells without inducing damage. During this stage, P. destructans hyphae assume a morphology similar to fungal plant pathogens during the biotrophic stage of infection. However, the mechanism by which P. destructans regulates its morphology and virulence is not fully understood. The phosphatase calcineurin is a key regulator of fungal stress response and virulence and could play a role in P. destructans virulence. Calcineurin can be inhibited using FK506 or Cyclosporin A (CSA). FK506 interacts with FKBP12, while CSA interacts with Cyclosporin A, and the protein-drug complex then inhibits calcineurin activity. Here, we show that P. destructans is intrinsically resistant to one calcineurin inhibitor (FK506), possibly because P. destructans FKBP12 has a lysine instead of the conserved arginine at position 89, which has been shown to reduce the affinity of the FKBP12-FK506 complex for calcineurin. Using cyclosporin A, we found that calcineurin is important for fungal growth. Additionally, calcineurin inhibition increases voriconazole inhibition. In contrast, calcineurin inhibition was antagonistic to the anti-cell wall drug micafungin. Taken together, calcineurin's role in fungal growth and response to voriconazole is conserved in P. destructans but may play a unique role in P. destructans response to micafungin.

Currently, the fungal class Paraglomeromycetes (phylum Glomeromycota) consists of one order, Paraglomerales, with two families: Paraglomeraceae, comprising the genera Paraglomus with ten species and Innospora with one species, and the monogeneric and monospecific Pervetustaceae represented by Pervetustus with Pe. simplex. However, our morphological analyses of these species and an undescribed specimen morphologically resembling other Paraglomeromycetes species, as well as analyses of sequences of these fungi and environmental sequences, indicated that the current taxonomic composition of this class should be updated. We did so here by creating in Paraglomeromycetes Pervetustales ord. nov. with Pervetustus and Opikia catenata gen. nov. and sp. nov., as well as transferring Innospora with In. majewskii to Innosporaceae fam. nov. These novelties were introduced primarily based on strong genetic divergences of the analyzed taxa, as many morphological and histochemical features of their spores and mycorrhizal structures overlap. Only Op. catenata possesses the unique trait of occasionally producing spores in chains. This mode of sporulation has also not been recognized in any other Glomeromycota species. Unlike species of Pervetustales and Innosporaceae, which produce single-walled spores, the subcellular spore structure of Paraglomus species contains one or two walls.

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

Abstract Tricyclic coumarins (TCs) exhibit defensive properties against herbivorous insects and possess valuable pharmacological properties for humans. They are mainly distributed in the Apiaceae, Rutaceae, Moraceae, and Fabaceae families. The chemical structural diversity of TCs in these families primarily arises from differences in skeletal variety and attachment position of the third ring, as observed in unsaturated furanocoumarins (FCs) and saturated dihydrofuranocoumarins (DHFCs). In TC biosynthesis, the FC and DHFC pathways diverge through distinct oxidation patterns. The product specificity of the reported cytochrome P450 monooxygenase at the branching point suggests that these TC groups are produced in nearly identical ratios; however, some species of the genus Angelica (Apiaceae) accumulate DHFCs at higher levels than FCs. In this study, we investigated the enzymatic mechanism responsible for forming these two TC groups using A. keiskei, a Japanese native species used as a medicinal and edible herb. Organ-specific transcriptome analysis highlighted a P450 member CYP71AJ62 that exhibited a close correlation with TC accumulation across organs. Enzymatic characterization demonstrated that CYP71AJ62 predominantly forms a hydroxylated DHFC, vaginidiol, which may promote DHFC over FC skeletal formation in TC biosynthesis. Phylogenetic analysis suggested that CYP71AJ62 has been derived from a gene responsible for FC formation by functional shift without gene duplication. Subsequent three-dimensional structural modeling and site-directed mutagenesis revealed that two amino acid residues on substrate recognition sites cooperatively determine the product specificity of this P450 enzyme. This study provides new insights into the molecular evolutionary process underlying ring-structure diversification and, more broadly, the adaptation of plants to their environments.

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

Beech leaf disease (BLD), caused by the migratory plant pathogenic nematode, Litylenchus crenatae (LC, Anguinidae) is presently one of the most concerning forest diseases in the United States. BLD has spread rapidly across the landscape and is currently found in 15 U.S. states and Ontario, Canadian, making monitoring a top priority. Here, we report on the development and testing of a probe-based qPCR detection assay that integrates previously reported LC-specific primers with a newly designed probe, enabling sensitive and reproducible quantification of nematodes in plant and non-plant matrices. This assay exhibited strong in silico and in planta specificity and high quantitative performance (R2 = 0.988-0.996), with amplification efficiencies of 90.0-96.8%. The assay also performed consistently on LC-spiked spore trap membranes used to monitor the fungal aerobiome, despite matrix-associated shifts in amplification efficiency. These results support the utility of the assay for monitoring and surveillance. The reliability was further evaluated by two independent diagnostic laboratories, demonstrating high inter-laboratory concordance in detection and strong agreement in relative quantification. The analytical sensitivity and reproducible performance of the assay align with key validation principles outlined by the APS Diagnostic Assay Validation Network (DAVN) and support its application as a molecular tool in experimental, diagnostic, and surveillance contexts, with important implications for national and international LC monitoring and disease management.

Abstract: Picrotoxane-type sesquiterpene alkaloids, including dendrobine, are characteristic constituents of Dendrobium nobile, but the enzymes that produce their proposed skeletal precursors remain in completely characterized. Here, we combined tissue metabolite profiling with genome-guided screening and biochemical characterization of terpene synthases (TPSs). GC-MS analysis revealed variation in sesquiterpenoid profiles and dendrobine signals among tissues and developmental stages. Manual curation of the chromosome-level genome yielded 16 full-length TPS-a candidates. Functional screening in an FPP-supplying Escherichia coli system showed that three closely related genes on chromosome 1, DnTPS1~DnTPS3, produced copaborneol as their dominant product. The principal product of DnTPS1 was purified and identified by NMR spectroscopy, and a purified-enzyme assay confirmed its direct conversion of FPP to copaborneol. Fluorescence imaging in rice protoplasts and Nicotiana benthamiana leaves indicated predominantly cytosolic localization of DnTPS1. Guided by sequence comparisons and structural modeling, we tested 12 DnTPS1 variants and found that substitutions at W377 and I295 altered the relative distribution of sesquiterpene products. Phylogenetic and regional gene-order analyses placed DnTPS1~DnTPS3 in the same clade and identified related TPS candidates within corresponding genomic regions of six Dendrobium species. These findings establish that three D. nobile TPSs can form copaborneol and identify DnTPS1 residues that influence product selectivity, providing a basis for testing the proposed connection to dendrobine biosynthesis.

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

Filamentation, a central virulence trait of the fungal pathogen Candida albicans, is required for host cell damage, tissue invasion, and biofilm formation. The transcription factor Bcr1 was first found to be required for biofilm formation but not filamentation. Subsequent studies revealed a negative role for Bcr1 in filamentation in opaque cells, a cell type that is required for mating, under hypoxic conditions, and in a wor2del/del mutant background. Here we characterize a new context in which Bcr1 negatively regulates filamentation and present its associated gene expression impact. We compared the wild-type and bcr1del/del mutant cells in the SC5314 reference strain background at 30 degrees C in glucose media, conditions that do not induce filamentation. The bcr1del/del mutant displayed increased invasive growth in a solid medium, and some filamentation ability in a liquid medium. The bcr1del/del effect on filamentation was augmented by overexpression of hyphal cyclin gene HGC1, which caused pseudohyphal growth in wild-type, efg1del/del, or brg1del/del strains, yet caused hyphal growth in the bcr1del/del strain. RNA-sequencing (RNA-seq) analysis of 30 degrees C cells shows that the bcr1del/del mutant has elevated expression of two genes that drive filamentation: HGC1 and UME6. In published data these same two genes have decreased expression in the bcr1del/del mutant at 37 degrees C, the temperature at which biofilm formation is typically assayed. A growing cadre of biofilm/hyphal regulators can have both positive and negative effects on hypha-associated gene expression, including Bcr1, Efg1, Ndt80, and Nrg1.