Fungal Genomes Research Articles

Role and structure of the small subunit forming heterodimers with laccase-like enzymes.

Unlike laccases sensu stricto, which are usually monomeric enzymes, laccase-like enzymes recently re-classified as Novel Laccases (NLACs) are characterized by the formation of heterodimers with small proteins (subunits) of unknown function. Here the NLAC from Pleurotus eryngii (PeNL) and a small protein selected from the fungal genome, that is homologous to reported POXA3 from Pleurotus ostreatus, were produced in Aspergillus oryzae separately or together. The two proteins interacted regardless of whether the small subunit was co-expressed or exogenously added to the enzyme. The stability and catalytic activity of PeNL was significantly enhanced in the presence of the small subunit. SEC-MALS analysis confirmed that the complex PeNL-ss is a heterodimer of 77.4 kDa. The crystallographic structure of the small protein expressed in Escherichia coli was solved at 1.6 Å resolution. This is the first structure elucidated of a small subunit of a NLAC. The helix bundle structure of the small subunit accommodates well with the enzyme model structure, including interactions with specific regions of NLACs and some amino acid residues of the substrate-binding loops. This article is protected by copyright. All rights reserved.

Comparison of assembly platforms for the assembly of the nuclear genome of Trichoderma harzianum strain PAR3

BackgroundTrichoderma is a diverse genus of fungi that includes several species that possess biotechnological and agricultural applications, including the biocontrol of pathogenic fungi and nematodes. The mitochondrial genome of a putative strain of Trichoderma harzianum called PAR3 was analyzed after isolation from the roots of Scarlet Royal grapevine scion grafted to Freedom rootstock, located in a grapevine vineyard in Parlier, CA, USA. Here, we report the sequencing, comparative assembly, and annotation of the nuclear genome of PAR3 and confirm its identification as a strain of T. harzianum. We subsequently compared the genes found in T. harzianum PAR3 to other known T. harzianum strains. Assembly of Illumina and/or Oxford Nanopore reads by the popular long-read assemblers, Flye and Canu, and the hybrid assemblers, SPAdes and MaSuRCA, was performed and the quality of the resulting assemblies were compared to ascertain which assembler generated the highest quality draft genome assembly.ResultsMaSuRCA produced the most complete and high-fidelity assembly yielding a nuclear genome of 40.7 Mb comprised of 112 scaffolds. Subsequent annotation of this assembly produced 12,074 gene models and 210 tRNAs. This included 221 genes that did not have equivalent genes in other T. harzainum strains. Phylogenetic analysis of ITS, rpb2, and tef1a sequences from PAR3 and established Trichoderma spp. showed that all three sequences from PAR3 possessed more than 99% identity to those of Trichoderma harzianum, confirming that PAR3 is an isolate of Trichoderma harzianum. We also found that comparison of gene models between T. harzianum PAR3 and other T. harzianum strains resulted in the identification of significant differences in gene type and number, with 221 unique genes identified in the PAR3 strain.ConclusionsThis study gives insight into the efficacy of several popular assembly platforms for assembly of fungal nuclear genomes, and found that the hybrid assembler, MaSuRCA, was the most effective program for genome assembly. The annotated draft nuclear genome and the identification of genes not found in other T. harzainum strains could be used to investigate the potential applications of T. harzianum PAR3 for biocontrol of grapevine fungal canker pathogens and as source of anti-microbial compounds.

Identification of a (+)-cubenene synthase from filamentous fungi Acremonium chrysogenum

Sesquiterpene synthases convert farnesyl diphosphate into various sesquiterpenes, which find wide applications in the food, cosmetics and pharmaceutical industries. Although numerous putative sesquiterpene synthases have been identified in fungal genomes, many lack biochemical characterization. In this study, we identified a putative terpene synthase AcTPS3 from Acremonium chrysogenum. Through sequence analysis and in vitro enzyme assay, AcTPS3 was identified as a sesquiterpene synthase. To obtain sufficient product for NMR testing, a metabolic engineered Saccharomyces cerevisiae was constructed to overproduce the product of AcTPS3. The major product of AcTPS3 was identified as (+)-cubenene (55.46%) by GC-MS and NMR. Thus, AcTPS3 was confirmed as (+)-cubenene synthase, which is the first report of (+)-cubenene synthase. The optimized S. cerevisiae strain achieved a biosynthesis titer of 597.3 mg/L, the highest reported for (+)-cubenene synthesis.

MIF-like domain containing protein orchestrates cellular differentiation and virulence in the fungal pathogen Magnaporthe oryzae

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of an MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results suggest that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

TheiaEuk: a species-agnostic bioinformatics workflow for fungal genomic characterization.

The clinical incidence of antimicrobial-resistant fungal infections has dramatically increased in recent years. Certain fungal pathogens colonize various body cavities, leading to life-threatening bloodstream infections. However, the identification and characterization of fungal isolates in laboratories remain a significant diagnostic challenge in medicine and public health. Whole-genome sequencing provides an unbiased and uniform identification pipeline for fungal pathogens but most bioinformatic analysis pipelines focus on prokaryotic species. To this end, TheiaEuk_Illumina_PE_PHB (TheiaEuk) was designed to focus on genomic analysis specialized to fungal pathogens. TheiaEuk was designed using containerized components and written in the workflow description language (WDL) to facilitate deployment on the cloud-based open bioinformatics platform Terra. This species-agnostic workflow enables the analysis of fungal genomes without requiring coding, thereby reducing the entry barrier for laboratory scientists. To demonstrate the usefulness of this pipeline, an ongoing outbreak of C. auris in southern Nevada was investigated. We performed whole-genome sequence analysis of 752 new C. auris isolates from this outbreak. Furthermore, TheiaEuk was utilized to observe the accumulation of mutations in the FKS1 gene over the course of the outbreak, highlighting the utility of TheiaEuk as a monitor of emerging public health threats when combined with whole-genome sequencing surveillance of fungal pathogens. A primary result of this work is a curated fungal database containing 5,667 unique genomes representing 245 species. TheiaEuk also incorporates taxon-specific submodules for specific species, including clade-typing for Candida auris (C. auris). In addition, for several fungal species, it performs dynamic reference genome selection and variant calling, reporting mutations found in genes currently associated with antifungal resistance (FKS1, ERG11, FUR1). Using genome assemblies from the ATCC Mycology collection, the taxonomic identification module used by TheiaEuk correctly assigned genomes to the species level in 126/135 (93.3%) instances and to the genus level in 131/135 (97%) of instances, and provided zero false calls. Application of TheiaEuk to actual specimens obtained in the course of work at a local public health laboratory resulted in 13/15 (86.7%) correct calls at the species level, with 2/15 called at the genus level. It made zero incorrect calls. TheiaEuk accurately assessed clade type of Candida auris in 297/302 (98.3%) of instances. TheiaEuk demonstrated effectiveness in identifying fungal species from whole genome sequence. It further showed accuracy in both clade-typing of C. auris and in the identification of mutations known to associate with drug resistance in that organism.

Performance evaluation of InfectID-BSI: A rapid quantitative PCR assay for detecting sepsis-associated organisms directly from whole blood

BackgroundBloodstream infections (BSIs) (presence of pathogenic organism in blood) that progress to sepsis (life-threatening organ dysfunction caused by the body's dysregulated response to an infection) is a major healthcare issue globally with close to 50 million cases annually and 11 million sepsis-related deaths, representing about 20% of all global deaths. A rapid diagnostic assay with accurate pathogen identification has the potential to improve antibiotic stewardship and clinical outcomes. MethodsThe InfectID-Bloodstream Infection (InfectID-BSI) test is a real-time quantitative PCR assay, which detects 26 of the most prevalent BSI-causing pathogens (bacteria and yeast) directly from blood (without need for pre-culture). InfectID-BSI identifies pathogens using highly discriminatory single nucleotide polymorphisms located in conserved regions of bacterial and fungal genomes. This report details the findings of a patient study which compared InfectID-BSI with conventional blood culture at two public hospitals in Queensland, Australia, using 375 whole blood samples (from multiple anatomical sites, eg. left arm, right arm, etc.) from 203 patients that have been clinically assessed to have signs and symptoms of suspected BSI, sepsis and septic shock. FindingsInfectID-BSI was a more sensitive method for microorganism detection compared with blood culture (BacT/ALERT, bioMerieux) for positivity rate (102 vs 54 detections), detection of fastidious organisms (Streptococcus pneumoniae and Aerococcus viridans) (25 vs 0), detection of low bioburden infections (measured as genome copies/0.35 mL of blood), time to result (<3 h including DNA extraction for InfectID-BSI vs 16 h–48 h for blood culture), and volume of blood required for testing (0.5 mL vs 40–60 mL). InfectID-BSI is an excellent ‘rule out’ test for BSI, with a negative predictive value of 99.7%. InfectID-BSI's ability to detect ‘difficult to culture’ microorganisms re-defines the four most prevalent BSI-associated pathogens as E. coli (28.4%), S. pneumoniae (17.6%), S. aureus (13.7%), and S. epidermidis (13.7%). InterpretationInfectID-BSI has the potential to alter the clinical treatment pathway for patients with BSIs that are at risk of progressing to sepsis.

Extreme Diversity of Mycoviruses Present in Single Strains of Rhizoctonia cerealis, the Pathogen of Wheat Sharp Eyespot.

Rhizoctonia cerealis is the pathogen of wheat sharp eyespot, which occurs throughout temperate wheat-growing regions of the world. In this project, the genomes of viruses from four strains of R. cerealis were analyzed based on Illumina high-throughput transcriptome sequencing (RNA-Seq) data. After filtering out reads that mapped to the fungal genome, viral genomes were assembled. In total, 131 virus-like sequences containing complete open reading frames (ORFs), belonging to 117 viruses, were obtained. Based on phylogenetic analysis, some of them were identified as novel members of the families Curvulaviridae, Endornaviridae, Hypoviridae, Mitoviridae, Mymonaviridae, and Phenuiviridae, while others were unclassified viruses. Most of these viruses from R. cerealis were significantly different from the viruses already reported. We propose the establishment of a new family, Rhizoctobunyaviridae, and two new genera, Rhizoctobunyavirus and Iotahypovirus. We further clarified the distribution and coinfection of these viruses in the four strains. Surprisingly, 39 viral genomes of up to 12 genera were found in strain R1084. Strain R0942, containing the fewest viruses, also contained 21 viral genomes belonging to 10 genera. Based on the RNA-Seq data, we estimated the accumulation level of some viruses in host cells and found that the mitoviruses in R. cerealis generally have very high accumulation. In conclusion, in the culturable phytopathogenic fungus R. cerealis, we discovered a considerable diversity of mycoviruses and a series of novel viruses. This study expands our understanding of the mycoviral diversity in R. cerealis and provides a rich resource for the further use of mycoviruses to control wheat sharp eyespot. IMPORTANCE Rhizoctonia cerealis is a binucleate fungus that is widely distributed worldwide and can cause sharp eyespot disease in cereal crops. In this study, 131 virus-like sequences belonging to 117 viruses were obtained based on analysis of high-throughput RNA-Seq data from four strains of R. cerealis. Many of these viruses were novel members of various virus families, while others were unclassified viruses. As a result, a new family named Rhizoctobunyaviridae and two new genera, Rhizoctobunyavirus and Iotahypovirus, were proposed. Moreover, the discovery of multiple viruses coinfecting a single host and the high accumulation levels of mitoviruses have shed light on the complex interactions between different viruses in a single host. In conclusion, a significant diversity of mycoviruses was discovered in the culturable phytopathogenic fungus R. cerealis. This study expands our understanding of mycoviral diversity, and provides a valuable resource for the further utilization of mycoviruses to control wheat diseases.

That's no moon, it's a Starship: Giant transposons driving fungal horizontal gene transfer.

To date, most reports of horizontal gene transfer (HGT) in fungi rely on genome sequence data and are therefore an indirect measure of HGT after the event has occurred. However, a novel group of class II-like transposons known as Starships may soon alter this status quo. Starships are giant transposable elements that carry dozens of genes, some of which are host-beneficial, and are linked to many recent HGT events in the fungal kingdom. These transposons remain active and mobile in many fungal genomes and their transposition has recently been shown to be driven by a conserved tyrosine-recombinase called 'Captain'. This perspective explores some of the remaining unanswered questions about how these Starship transposons move, both within a genome and between different species. We seek to outline several experimental approaches that can be used to identify the genes essential for Starship-mediated HGT and draw links to other recently discovered giant transposons outside of the fungal kingdom.

Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

GtAI: an improved species-specific tRNA adaptation index using the genetic algorithm.

The tRNA adaptation index (tAI) is a translation efficiency metric that considers weighted values (S ij values) for codon-tRNA wobble interaction efficiencies. The initial implementation of the tAI had significant flaws. For instance, generated S ij weights were optimized based on gene expression in Saccharomyces cerevisiae, which is expected to vary among different species. Consequently, a species-specific approach (stAI) was developed to overcome those limitations. However, the stAI method employed a hill climbing algorithm to optimize the S ij weights, which is not ideal for obtaining the best set of S ij weights because it could struggle to find the global maximum given a complex search space, even after using different starting positions. In addition, it did not perform well in computing the tAI of fungal genomes in comparison with the original implementation. We developed a novel approach named genetic tAI (gtAI) implemented as a Python package (https://github.com/AliYoussef96/gtAI), which employs a genetic algorithm to obtain the best set of S ij weights and follows a new codon usage-based workflow that better computes the tAI of genomes from the three domains of life. The gtAI has significantly improved the correlation with the codon adaptation index (CAI) and the prediction of protein abundance (empirical data) compared to the stAI.

Qualitative metabolomics-based characterization of a phenolic UDP-xylosyltransferase with a broad substrate spectrum from Lentinus brumalis

Wood-decaying fungi are the major decomposers of plant litter. Heavy sequencing efforts on genomes of wood-decaying fungi have recently been made due to the interest in their lignocellulolytic enzymes; however, most parts of their proteomes remain uncharted. We hypothesized that wood-decaying fungi would possess promiscuous enzymes for detoxifying antifungal phytochemicals remaining in the dead plant bodies, which can be useful biocatalysts. We designed a computational mass spectrometry-based untargeted metabolomics pipeline for the phenotyping of biotransformation and applied it to 264 fungal cultures supplemented with antifungal plant phenolics. The analysis identified the occurrence of diverse reactivities by the tested fungal species. Among those, we focused on O-xylosylation of multiple phenolics by one of the species tested, Lentinus brumalis. By integrating the metabolic phenotyping results with publicly available genome sequences and transcriptome analysis, a UDP-glycosyltransferase designated UGT66A1 was identified and validated as an enzyme catalyzing O-xylosylation with broad substrate specificity. We anticipate that our analytical workflow will accelerate the further characterization of fungal enzymes as promising biocatalysts.

First Report of Stem Rot Caused by Botryosphaeria dothidea and Phomopsis velata on Forsythia suspensa in China.

Forsythia suspensa (Thunb.) Vahl (Oleaceae) is a well-known traditional Chinese medicine. It exhibits antioxidant activity and exerts antibacterial, antiviral, and antiemetic effects (Li and Chen 2005). From May 2020 to October 2021, a disease was observed on field-grown forsythia plants in Lingbao City, Henan Province, China (110°33'25.74″E, 34°30'19.34″W). The diseased plants were characterized by stem rot, retarded growth, a declined fruit quality, and in extreme cases, death of F. suspensa. Approximately 3.0% to 5.0% individuals exhibited stem rotten in the main branches. On average, 60% of the branches of infected individual trees were affected by this disease. During the initial infection stage, the bark of the plants was raised and curled, and the xylem and phloem of the stems turned brown color, whereas in the late stage of the infection, the outer bark had dried and become detached, and the inner xylem and phloem had blackened. Upon infection, the growth of plants was reduced, and the main branches became desiccated as the disease progressed. We randomly selected five diseased branches from five plant fields, the bark tissues (about 25 mm²) of which were surface-sterilized in 75% ethanol for 30 s, treated with 1% NaClO for 5 min, rinsed five times with sterile water, and placed on potato dextrose agar (PDA). After incubating 3 days, 20 clones were observed, and two representative strains (FSJF11 and FSJF13, three replicates for each) was selected for intensive study. Samples of these strains have been deposited in Institutes of Traditional Chinese Medicine, Henan University. On PDA, the colonies of FSJF11 were initially white and fluffy in appearance, later turning gray, and finally black. The vigorously growing hyphae were branched and septate. However, no spores was observed during culture. FSJF13 colonies were rapidly growing, initially white in color and later turning gray. After culturing for 20 days, black conidia appeared and yellow conidial horns were released. The alpha conidia were elliptical, slightly pointed at both ends, and each end possessed an oil ball (6.40±0.60 × 1.86±0.25 μm). The beta conidia were slender, linear, and hook shaped with a slightly curved end (28.92±2.81 × 0.96±0.14 μm). DNA of the isolates was extracted using a Fungal Genome DNA Extraction Kit (Sangon Biotech, Shanghai), and selected genes were amplified using the primer pairs ITS1/ITS4 (Tian et al. 2018), LROR/ LR5, and NS1/NS4 (Aiello et al. 2020). Sequences have been deposited in GenBank (ITS: MW834579 and MW834580; LSU: MW829566 and MW829567; SSU: MW834582 and MW834583). The lengths of the amplified ITS, LSU and SSU sequences were 491, 759, and 1013 bp for FSJF11, respectively, and these in FSJF13 were 543, 927, and 901 bp, respectively. The ITS, LSU, and SSU sequences of FSJF11 were found to have sequence identities of 99.19%, 100%, and 100% with those of Botryosphaeria dothidea stains AY259092, EU673243, and Eu673174, respectively, and a phylogenetic tree was constructed based on the concatenated sequences (ITS, LSU, and SSU) revealed that FSJF11 and B. dothidea formed a clade with 96% support. A BLAST search of the Genbank database revealed that the ITS sequence of FSJF13 showed 99.81% identity with that of Phomopsis velata (MN183778). Given that no LSU or SSU sequences of this species are currently available, we constructed a phylogenetic tree based solely on ITS sequences, which revealed that FSJF13 and P. velata formed a clade with 99% support. Based on the morphological and molecular characteristics(Qi et al. 2007), the isolates of FSJF11 and FSJF13 were identified as B. dothidea and P. velata, respectively. Healthy branches of F. suspensa were wounded in vitro after inoculating active fungal cake of B. dothidea or P. velata (diameter = 5 mm) on the bark, and control branches were treated with PDA. In total, each branch was inoculated via four holes were inoculated on each branch, and three branches were used for each treatment. The inoculation sites were covered with a piece of wet absorbent cotton and then wrapped with plastic film, and the branches were incubated at 26 °C. The branches continued to grow after removal of the cotton and the film on the fourth day. All inoculated points on the branches showed lesions similar to those observed in the field, whereas the control branches were asymptomatic. The pathogenicity rates of FSJF11 and FSJF13 (three replicates for each) were 66.67% and 83.33%, respectively. Both species were re-isolated from the symptomatic branches respectively, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of B. dothidea and P. velata causing branches rot in F. suspensa. The findings of this study will contribute to developing effective strategies for the control of this newly emerging plant disease.

The Sordariomycetes: an expanding resource with Big Data for mining in evolutionary genomics and transcriptomics.

Advances in genomics and transcriptomics accompanying the rapid accumulation of omics data have provided new tools that have transformed and expanded the traditional concepts of model fungi. Evolutionary genomics and transcriptomics have flourished with the use of classical and newer fungal models that facilitate the study of diverse topics encompassing fungal biology and development. Technological advances have also created the opportunity to obtain and mine large datasets. One such continuously growing dataset is that of the Sordariomycetes, which exhibit a richness of species, ecological diversity, economic importance, and a profound research history on amenable models. Currently, 3,574 species of this class have been sequenced, comprising nearly one-third of the available ascomycete genomes. Among these genomes, multiple representatives of the model genera Fusarium, Neurospora, and Trichoderma are present. In this review, we examine recently published studies and data on the Sordariomycetes that have contributed novel insights to the field of fungal evolution via integrative analyses of the genetic, pathogenic, and other biological characteristics of the fungi. Some of these studies applied ancestral state analysis of gene expression among divergent lineages to infer regulatory network models, identify key genetic elements in fungal sexual development, and investigate the regulation of conidial germination and secondary metabolism. Such multispecies investigations address challenges in the study of fungal evolutionary genomics derived from studies that are often based on limited model genomes and that primarily focus on the aspects of biology driven by knowledge drawn from a few model species. Rapidly accumulating information and expanding capabilities for systems biological analysis of Big Data are setting the stage for the expansion of the concept of model systems from unitary taxonomic species/genera to inclusive clusters of well-studied models that can facilitate both the in-depth study of specific lineages and also investigation of trait diversity across lineages. The Sordariomycetes class, in particular, offers abundant omics data and a large and active global research community. As such, the Sordariomycetes can form a core omics clade, providing a blueprint for the expansion of our knowledge of evolution at the genomic scale in the exciting era of Big Data and artificial intelligence, and serving as a reference for the future analysis of different taxonomic levels within the fungal kingdom.

Genomes of fungi and relatives reveal delayed loss of ancestral gene families and evolution of key fungal traits

Fungi are ecologically important heterotrophs that have radiated into most niches on Earth and fulfil key ecological services. Despite intense interest in their origins, major genomic trends of their evolutionary route from a unicellular opisthokont ancestor to derived multicellular fungi remain poorly known. Here we provide a highly resolved genome-wide catalogue of gene family changes across fungal evolution inferred from the genomes of 123 fungi and relatives. We show that a dominant trend in early fungal evolution has been the gradual shedding of protist genes and the punctuated emergence of innovation by two main gene duplication events. We find that the gene content of non-Dikarya fungi resembles that of unicellular opisthokonts in many respects, owing to the conservation of protist genes in their genomes. The most rapidly duplicating gene groups included extracellular proteins and transcription factors, as well as ones linked to the coordination of nutrient uptake with growth, highlighting the transition to a sessile osmotrophic feeding strategy and subsequent lifestyle evolution as important elements of early fungal history. These results suggest that the genomes of pre-fungal ancestors evolved into the typical filamentous fungal genome by a combination of gradual gene loss, turnover and several large duplication events rather than by abrupt changes. Consequently, the taxonomically defined Fungi represents a genomically non-uniform assemblage of species.

Comparative genomics of smut fungi suggest the ability of meiosis and mating in asexual species of the genus Pseudozyma (Ustilaginales)

BackgroundThe Ustilaginales comprise hundreds of plant-parasitic fungi with a characteristic life cycle that directly links sexual reproduction and parasitism: One of the two mating-type loci codes for a transcription factor that not only facilitates mating, but also initiates the infection process. However, several species within the Ustilaginales have no described parasitic stage and were historically assigned to the genus Pseudozyma. Molecular studies have shown that the group is polyphyletic, with members being scattered in various lineages of the Ustilaginales. Together with recent findings of conserved fungal effectors in these non-parasitic species, this raises the question if parasitism has been lost recently and in multiple independent events or if there are hitherto undescribed parasitic stages of these fungi.ResultsIn this study, we sequenced genomes of five Pseudozyma species together with six parasitic species from the Ustilaginales to compare their genomic capability to perform two central functions in sexual reproduction: mating and meiosis. While the loss of sexual capability is assumed in certain lineages and asexual species are common in Asco- and Basidiomycota, we were able to successfully annotate potentially functional mating and meiosis genes that are conserved throughout the whole group.ConclusionOur data suggest that at least the key functions of a sexual lifestyle are maintained in the analyzed genomes, challenging the current understanding of the so-called asexual species with respect to their evolution and ecological role.

Terabase-Scale Coassembly of a Tropical Soil Microbiome.

Petabases of environmental metagenomic data are publicly available, presenting an opportunity to characterize complex environments and discover novel lineages of life. Metagenome coassembly, in which many metagenomic samples from an environment are simultaneously analyzed to infer the underlying genomes' sequences, is an essential tool for achieving this goal. We applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 terabases (Tbp) of metagenome data from a tropical soil in the Luquillo Experimental Forest (LEF), Puerto Rico. The resulting coassembly yielded 39 high-quality (>90% complete, <5% contaminated, with predicted 23S, 16S, and 5S rRNA genes and ≥18 tRNAs) metagenome-assembled genomes (MAGs), including two from the candidate phylum Eremiobacterota. Another 268 medium-quality (≥50% complete, <10% contaminated) MAGs were extracted, including the candidate phyla Dependentiae, Dormibacterota, and Methylomirabilota. In total, 307 medium- or higher-quality MAGs were assigned to 23 phyla, compared to 294 MAGs assigned to nine phyla in the same samples individually assembled. The low-quality (<50% complete, <10% contaminated) MAGs from the coassembly revealed a 49% complete rare biosphere microbe from the candidate phylum FCPU426 among other low-abundance microbes, an 81% complete fungal genome from the phylum Ascomycota, and 30 partial eukaryotic MAGs with ≥10% completeness, possibly representing protist lineages. A total of 22,254 viruses, many of them low abundance, were identified. Estimation of metagenome coverage and diversity indicates that we may have characterized ≥87.5% of the sequence diversity in this humid tropical soil and indicates the value of future terabase-scale sequencing and coassembly of complex environments. IMPORTANCE Petabases of reads are being produced by environmental metagenome sequencing. An essential step in analyzing these data is metagenome assembly, the computational reconstruction of genome sequences from microbial communities. "Coassembly" of metagenomic sequence data, in which multiple samples are assembled together, enables more complete detection of microbial genomes in an environment than "multiassembly," in which samples are assembled individually. To demonstrate the potential for coassembling terabases of metagenome data to drive biological discovery, we applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 Tbp of reads from a humid tropical soil environment. The resulting coassembly, its functional annotation, and analysis are presented here. The coassembly yielded more, and phylogenetically more diverse, microbial, eukaryotic, and viral genomes than the multiassembly of the same data. Our resource may facilitate the discovery of novel microbial biology in tropical soils and demonstrates the value of terabase-scale metagenome sequencing.

The Dynamic Fungal Genome: Polyploidy, Aneuploidy and Copy Number Variation in Response to Stress.

Fungal species have dynamic genomes and often exhibit genomic plasticity in response to stress. This genome plasticity often comes with phenotypic consequences that affect fitness and resistance to stress. Fungal pathogens exhibit genome plasticity in both clinical and agricultural settings and often during adaptation to antifungal drugs, posing significant challenges to human health. Therefore, it is important to understand the rates, mechanisms, and impact of large genomic changes. This review addresses the prevalence of polyploidy, aneuploidy, and copy number variation across diverse fungal species, with special attention to prominent fungal pathogens and model species. We also explore the relationship between environmental stress and rates of genomic changes and highlight the mechanisms underlying genotypic and phenotypic changes. A comprehensive understanding of these dynamic fungal genomes is needed to identify novel solutions for the increase in antifungal drug resistance.

Characterization of metagenome-assembled genomes from the International Space Station

BackgroundSeveral investigations on the microbial diversity and functional properties of the International Space Station (ISS) environment were carried out to understand the influence of spaceflight conditions on the microbial population. However, metagenome-assembled genomes (MAGs) of ISS samples are yet to be generated and subjected to various genomic analyses, including phylogenetic affiliation, predicted functional pathways, antimicrobial resistance, and virulence characteristics.ResultsIn total, 46 MAGs were assembled from 21 ISS environmental metagenomes, in which metaSPAdes yielded 20 MAGs and metaWRAP generated 26 MAGs. Among 46 MAGs retrieved, 18 bacterial species were identified, including one novel genus/species combination (Kalamiella piersonii) and one novel bacterial species (Methylobacterium ajmalii). In addition, four bins exhibited fungal genomes; this is the first-time fungal genomes were assembled from ISS metagenomes. Phylogenetic analyses of five bacterial species showed ISS-specific evolution. The genes pertaining to cell membranes, such as transmembrane transport, cell wall organization, and regulation of cell shape, were enriched. Variations in the antimicrobial-resistant (AMR) and virulence genes of the selected 20 MAGs were characterized to predict the ecology and evolution of biosafety level (BSL) 2 microorganisms in space. Since microbial virulence increases in microgravity, AMR gene sequences of MAGs were compared with genomes of respective ISS isolates and corresponding type strains. Among these 20 MAGs characterized, AMR genes were more prevalent in the Enterobacter bugandensis MAG, which has been predominantly isolated from clinical samples. MAGs were further used to analyze if genes involved in AMR and biofilm formation of viable microbes in ISS have variation due to generational evolution in microgravity and radiation pressure.ConclusionsComparative analyses of MAGs and whole-genome sequences of related ISS isolates and their type strains were characterized to understand the variation related to the microbial evolution under microgravity. The Pantoea/Kalamiella strains have the maximum single-nucleotide polymorphisms found within the ISS strains examined. This may suggest that Pantoea/Kalamiella strains are much more subjective to microgravity changes. The reconstructed genomes will enable researchers to study the evolution of genomes under microgravity and low-dose irradiation compared to the evolution of microbes here on Earth.EUxsQ-aH7RTgxVLTkvgiT5Video

Strategies for Natural Product Discovery by Unlocking Cryptic Biosynthetic Gene Clusters in Fungi

Recent advancements in genome sequencing and bioinformatic analysis of fungal genomes have revealed that fungi possess cryptic or silent biosynthetic gene clusters (BGCs). This discovery suggests that our understanding of the universe of fungal secondary metabolomes is limited. In this review, we summarize recent strategies for activating cryptic BGCs in fungi, identify fungal secondary metabolites, and highlight their biological activities. We also briefly discuss the isolation and purification methods for these compounds. Our focus is on genetics-dependent and genetics-independent approaches for activating cryptic BGCs in fungi. Using the strategies discussed here, cryptic fungal natural products hold significant potential as a source for the discovery of new drug candidates.

Novel Pneumocystis Antigens for Seroprevalence Studies.

Pneumocystis jirovecii is the most common cause of fungal pneumonia in children under the age of 2 years. However, the inability to culture and propagate this organism has hampered the acquisition of a fungal genome as well as the development of recombinant antigens to conduct seroprevalence studies. In this study, we performed proteomics on Pneumocystis-infected mice and used the recent P. murina and P. jirovecii genomes to prioritize antigens for recombinant protein expression. We focused on a fungal glucanase due to its conservation among fungal species. We found evidence of maternal IgG to this antigen, followed by a nadir in pediatric samples between 1 and 3 months of age, followed by an increase in prevalence over time consistent with the known epidemiology of Pneumocystis exposure. Moreover, there was a strong concordance of anti-glucanase responses and IgG against another Pneumocystis antigen, PNEG_01454. Taken together, these antigens may be useful tools for Pneumocystis seroprevalence and seroconversion studies.