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Aspergillus fumigatus is a deadly agent of human fungal disease where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics with the potential to address the full genetic repertoire of a species. Here, we evaluate 260 genome sequences of A . fumigatus including 62 newly sequenced strains, using a combination of population genomics, phylogenomics, and pan-genomics. Our results offer a high-resolution assessment of population structure and recombination frequency, phylogenetically structured gene presence–absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes. Although A . fumigatus disperses primarily via asexual conidia, we identified extraordinarily high levels of recombination with the lowest linkage disequilibrium decay value reported for any fungal species to date. We provide evidence for 3 primary populations of A . fumigatus , with recombination occurring only rarely between populations and often within them. These 3 populations are structured by both gene variation and distinct patterns of gene presence–absence with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism suggesting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. Altogether, the pan-genome of A . fumigatus represents one of the largest fungal pan-genomes reported to date including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome-based approach for evaluating intraspecific variation and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

Transposable elements (TEs) shape genome evolution through periodic bursts of amplification. In this study prior knowledge of the mPing/Ping/Pong TE family is exploited to track their copy numbers and distribution in genome sequences from 3,000 accessions of domesticated Oryza sativa (rice) and the wild progenitor Oryza rufipogon . We find that mPing bursts are restricted to recent domestication and is likely due to the accumulation of two TE components, Ping16A and Ping16A_Stow , that appear to be critical for mPing hyperactivity. Ping16A is a variant of the autonomous element with reduced activity as shown in a yeast transposition assay. Transposition of Ping16A into a Stowaway element generated Ping16A_Stow , the only Ping locus shared by all bursting accessions, and shown here to correlate with high mPing copies. Finally, we show that sustained activity of the mPing/Ping family in domesticated rice produced the components necessary for mPing bursts, not the loss of epigenetic regulation. Transposable element (TE) bursts shape genome evolution but their origin remains unclear. Here, the authors show that a burst is restricted to only a few domesticated rice accessions and is associated with the acquisition of two TE variants, Ping16A and Ping16A_Stow, not the loss of TE silencing.

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.

Nutritional deprivation triggers a switch from a saprotrophic to predatory lifestyle in soil-dwelling nematode-trapping fungi (NTF). In particular, the NTF Arthrobotrys oligospora secretes food and sex cues to lure nematodes to its mycelium and is triggered to develop specialized trapping devices. Captured nematodes are then invaded and digested by the fungus, thus serving as a food source. In this study, we examined the transcriptomic response of A . oligospora across the stages of sensing, trap development, and digestion upon exposure to the model nematode Caenorhabditis elegans . A . oligospora enacts a dynamic transcriptomic response, especially of protein secretion–related genes, in the presence of prey. Two-thirds of the predicted secretome of A . oligospora was up-regulated in the presence of C . elegans at all time points examined, and among these secreted proteins, 38.5% are predicted to be effector proteins. Furthermore, functional studies disrupting the t-SNARE protein Sso2 resulted in impaired ability to capture nematodes. Additionally, genes of the DUF3129 family, which are expanded in the genomes of several NTF, were highly up-regulated upon nematode exposure. We observed the accumulation of highly expressed DUF3129 proteins in trap cells, leading us to name members of this gene family as T rap E nriched P roteins (TEPs). Gene deletion of the most highly expressed TEP gene, TEP1 , impairs the function of traps and prevents the fungus from capturing prey efficiently. In late stages of predation, we observed up-regulation of a variety of proteases, including metalloproteases. Following penetration of nematodes, these metalloproteases facilitate hyphal growth required for colonization of prey. These findings provide insights into the biology of the predatory lifestyle switch in a carnivorous fungus and provide frameworks for other fungal–nematode predator–prey systems.

MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

This work was supported by Spanish National Research Plan (Projects AGL2011-30495 and AGL2014-55971-R) and FEDER funds; Public University of Navarre; U.S. Department of Energy Joint Genome Institute; and Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. T

Snake fungal disease (SFD; ophidiomycosis), caused by the pathogen Ophidiomyces ophiodiicola ( Oo ), has been documented in wild snakes in North America and Eurasia, and is considered an emerging disease in the eastern United States of America. However, a lack of historical disease data has made it challenging to determine whether Oo is a recent arrival to the USA or whether SFD emergence is due to other factors. Here, we examined the genomes of 82 Oo strains to determine the pathogen’s history in the eastern USA. Oo strains from the USA formed a clade (Clade II) distinct from European strains (Clade I), and molecular dating indicated that these clades diverged too recently (approximately 2,000 years ago) for transcontinental dispersal of Oo to have occurred via natural snake movements across Beringia. A lack of nonrecombinant intermediates between clonal lineages in Clade II indicates that Oo has actually been introduced multiple times to North America from an unsampled source population, and molecular dating indicates that several of these introductions occurred within the last few hundred years. Molecular dating also indicated that the most common Clade II clonal lineages have expanded recently in the USA, with time of most recent common ancestor mean estimates ranging from 1985 to 2007 CE. The presence of Clade II in captive snakes worldwide demonstrates a potential mechanism of introduction and highlights that additional incursions are likely unless action is taken to reduce the risk of pathogen translocation and spillover into wild snake populations.

• While there has been significant progress characterizing the ‘symbiotic toolkit’ of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. • We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs) and G-protein coupled receptors (GPCRs). • Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. • These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity.

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis. worms, and mammals, but is absent from yeasts that have been examined. We identified and analyzed H3K27me3 in the filamentous fungus Neurospora crassa and in other Neurospora species. H3K27me3 covers 6.8% of the N. crassa genome, encompassing 223 domains, including 774 genes, all of which are transcriptionally silent N. crassa H3K27me3-marked genes are less conserved than unmarked genes and only ~35% of genes marked by H3K27me3 in N. crassa are also H3K27me3-marked in Neurospora discreta and Neurospora tetrasperma. We found that three components of the Neurospora Polycomb repressive complex 2 (PRC2)—[Su-(var) 3-9; E(z); Trrthorax] (SET)-7, embryonic ectoderm development (EED), and SU(Z)12 (suppressor of zeste12)—are required for H3K27me3, whereas the fourth component Neurospora protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, caused by deletion of the gene encoding the catalytic PRC2 subunit, set-7, resulted in up-regulation of 130 genes, including genes in both H3K27me3-marked and unmarked regions.

About the Authors: Brian Lovett Affiliation: Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia, United States of America Angie Macias Affiliation: Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia, United States of America ORCID logo http://orcid.org/0000-0002-2975-686X Jason E. Stajich Affiliation: Department of Microbiology and Plant Pathology and Institute for Integrative Genome Biology, University of California, Riverside, California, United States of America ORCID logo http://orcid.org/0000-0002-7591-0020 John Cooley Affiliation: Department of Ecology and Evolutionary Biology, University of Connecticut, Hartford, Connecticut, United States of America ORCID logo http://orcid.org/0000-0002-3691-2592 Jørgen Eilenberg Affiliation: Department of Plant and Environmental Science, University of Copenhagen, Frederiksberg, Denmark ORCID logo http://orcid.org/0000-0002-9273-5252 Henrik H. de Fine Licht Affiliation: Department of Plant and Environmental Science, University of Copenhagen, Frederiksberg, Denmark ORCID logo http://orcid.org/0000-0003-3326-5729 Matt T. Kasson * E-mail: mtkasson@mail.wvu.edu Affiliation: Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia, United States of America ORCID logo http://orcid.org/0000-0001-5602-7278 Citation: Lovett B, Macias A, Stajich JE, Cooley J, Eilenberg J, de Fine Licht HH, et al. Though the effects of entomopathogenic fungi on their hosts are a far cry from behavior-modifying viruses such as rabies or the phantasmic world of brain-eating zombies that drag their way through our popular culture, both rabies and select entomopathogenic fungi are nevertheless archetypal examples of pathogens that actively enlist their living hosts for successful transmission, a phenomenon referred to hereafter as active host transmission (AHT) [4]. Many fungi from the subphylum Entomophthoromycotina are so highly specialized for life on their preferred insect hosts that they can be cultivated beyond a vegetative stage outside of their host only with difficulty (or even not at all), making laboratory studies on transmission, host behavior, etc. based on fungi produced in vitro difficult. The infrequent and ephemeral occurrence of other AHT hosts in nature limits our ability to find adequate specimens for formal investigation. Because of these challenges, few actual examples of entomopathogens relying on AHT have been documented.