Insect-pathogenic fungi use subtilisin-like serine proteases (SLSPs) to degrade chitin-associated proteins in the insect procuticle. Most insect-pathogenic fungi in the order Hypocreales (Ascomycota) are generalist species with a broad host-range, and most species possess a high number of SLSPs. The other major clade of insect-pathogenic fungi is part of the subphylum Entomophthoromycotina (Zoopagomycota, formerly Zygomycota) which consists of high host-specificity insect-pathogenic fungi that naturally only infect a single or very few host species. The extent to which insect-pathogenic fungi in the order Entomophthorales rely on SLSPs is unknown. Here we take advantage of recently available transcriptomic and genomic datasets from four genera within Entomophthoromycotina: the saprobic or opportunistic pathogens Basidiobolus meristosporus, Conidiobolus coronatus, C. thromboides, C. incongruus, and the host-specific insect pathogens Entomophthora muscae and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants (Formica polyctena), respectively. In total 154 SLSP from six fungi in the subphylum Entomophthoromycotina were identified: E. muscae (n = 22), P. formicae (n = 6), B. meristosporus (n = 60), C. thromboides (n = 18), C. coronatus (n = 36), and C. incongruus (n = 12). A unique group of 11 SLSPs was discovered in the genomes of the obligate biotrophic fungi E. muscae, P. formicae and the saprobic human pathogen C. incongruus that loosely resembles bacillopeptidase F-like SLSPs. Phylogenetics and protein domain analysis show this class represents a unique group of SLSPs so far only observed among Bacteria, Oomycetes and early diverging fungi such as Cryptomycota, Microsporidia, and Entomophthoromycotina. This group of SLSPs is missing in the sister fungal lineages of Kickxellomycotina and the fungal phyla Mucoromyocta, Ascomycota and Basidiomycota fungi suggesting interesting gene loss patterns.

The fungus Entomophthora muscae (Entomophthoromycota, Entomophthorales, Entomophthoraceae) is a widespread insect pathogen responsible for fatal epizootic events in many dipteran fly hosts. During epizootics in 2011 and 2012 in Durham, North Carolina, we observed a transition of fungal infections from one host, the plant-feeding fly Delia radicum, to a second host, the predatory fly Coenosia tigrina. Infections first appeared on Delia in the middle of March, but by the end of May, Coenosia comprised 100% of infected hosts. Multilocus sequence typing revealed that E. muscae in Durham comprises two distinct subpopulations (clades) with several haplotypes in each. Fungi from either clade are able to infect both fly species, but vary in their infection phenologies and host-specificities. Individuals of the more phylogenetically diverse clade I predominated during the beginning of the spring epizootic, infecting mostly phytophagous Delia flies. Clade II dominated in late April and May and affected mostly predatory Coenosia flies. Analysis of population structure revealed two subpopulations within E. muscae with limited gene exchange. This study provides the first evidence of recombination and population structure within the E. muscae species complex, and illustrates the complexity of insect-fungus relationships that should be considered for development of biological control methods.

Fungi of the Conidiobolus group belong to the family Ancylistaceae (Entomophthorales, Entomophthoromycotina, Zoopagomycota) and include over 70 predominantly saprotrophic species in four similar and closely related genera, that were separated phylogenetically recently. Entomopathogenic fungi of the genus Batkoa are very close morphologically to the Conidiobolus species. Their thalli share similar morphology, and they produce ballistic conidia like closely related entomopathogenic Entomophthoraceae. Ballistic conidia are traditionally considered as an efficient tool in the pathogenic process and an important adaptation to the parasitic lifestyle. Our study aims to reconstruct the phylogeny of this fungal group using molecular and genomic data, ancestral lifestyle and morphological features of the conidiobolus-like group and the direction of their evolution. Based on phylogenetic analysis, some species previously in the family Conidiobolaceae are placed in the new families Capillidiaceae and Neoconidiobolaceae, which each include one genus, and the Conidiobolaceae now includes three genera. Intermediate between the conidiobolus-like groups and Entomophthoraceae, species in the distinct Batkoa clade now belong in the family Batkoaceae. Parasitism evolved several times in the Conidiobolus group and Ancestral State Reconstruction suggests that the evolution of ballistic conidia preceded the evolution of the parasitic lifestyle.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions.IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.IMPORTANCE Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

Phylogenomic approaches have the potential to improve confidence about the inter-relationships of species in the order Mucorales within the fungal tree of life. Rhizopus species are especially important as plant and animal pathogens and bioindustrial fermenters for food and metabolite production. A dataset of 192 orthologous genes was used to construct a phylogenetic tree of 21 Rhizopus strains, classified into four species isolated from habitats of industrial, medical and environmental importance. The phylogeny indicates that the genus Rhizopus consists of three major clades, with R. microsporus as the basal species and the sister lineage to R. stolonifer and two closely related species R. arrhizus and R. delemar A comparative analysis of the mating type locus across Rhizopus reveals that its structure is flexible even between different species in the same genus, but shows similarities between Rhizopus and other mucoralean fungi. The topology of single-gene phylogenies built for two genes involved in mating is similar to the phylogenomic tree. Comparison of the total length of the genome assemblies showed that genome size varies by as much as threefold within a species and is driven by changes in transposable element copy numbers and genome duplications.

Zygomycete fungi were classified as a single phylum, Zygomycota, based on sexual reproduction by zygospores, frequent asexual reproduction by sporangia, absence of multicellular sporocarps, and production of coenocytic hyphae, all with some exceptions. Molecular phylogenies based on one or a few genes did not support the monophyly of the phylum, however, and the phylum was subsequently abandoned. Here we present phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins, and we demonstrate that zygomycetes comprise two major clades that form a paraphyletic grade. A formal phylogenetic classification is proposed herein and includes two phyla, six subphyla, four classes and 16 orders. On the basis of these results, the phyla Mucoromycota and Zoopagomycota are circumscribed. Zoopagomycota comprises Entomophtoromycotina, Kickxellomycotina and Zoopagomycotina; it constitutes the earliest diverging lineage of zygomycetes and contains species that are primarily parasites and pathogens of small animals (e.g. amoeba, insects, etc.) and other fungi, i.e. mycoparasites. Mucoromycota comprises Glomeromycotina, Mortierellomycotina, and Mucoromycotina and is sister to Dikarya. It is the more derived clade of zygomycetes and mainly consists of mycorrhizal fungi, root endophytes, and decomposers of plant material. Evolution of trophic modes, morphology, and analysis of genome-scale data are discussed.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade natures polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.