Fungi and humans: closer than you think

The budding yeast, Saccharomyces cerevisiae, has long been used as a model system to study the functions of human genes. Now that the genome sequences from several other fungal species are nearly complete, we can characterize the genetic diversity in the fungal kingdom at the genomic level. This diversity means that the number of human genes with homologues in the fungal kingdom is double that with homologues in S. cerevisiae only. Therefore, functional studies of human genes in the fungal model systems should look beyond S. cerevisiae.     

Fungal genome sequencing: basic biology to biotechnology

The genome sequences provide a first glimpse into the genomic basis of the biological diversity of filamentous fungi and yeast. The genome sequence of the budding yeast, Saccharomyces cerevisiae, with a small genome size, unicellular growth, and rich history of genetic and molecular analyses was a milestone of early genomics in the 1990s. The subsequent completion of fission yeast, Schizosaccharomyces pombe and genetic model, Neurospora crassa initiated a revolution in the genomics of the fungal kingdom. In due course of time, a substantial number of fungal genomes have been sequenced and publicly released, representing the widest sampling of genomes from any eukaryotic kingdom. An ambitious genome-sequencing program provides a wealth of data on metabolic diversity within the fungal kingdom, thereby enhancing research into medical science, agriculture science, ecology, bioremediation, bioenergy, and the biotechnology industry. Fungal genomics have higher potential to positively affect human health, environmental health, and the planet’s stored energy. With a significant increase in sequenced fungal genomes, the known diversity of genes encoding organic acids, antibiotics, enzymes, and their pathways has increased exponentially. Currently, over a hundred fungal genome sequences are publicly available; however, no inclusive review has been published. This review is an initiative to address the significance of the fungal genome-sequencing program and provides the road map for basic and applied research.     

Genomics of the filamentous fungi - moving from the shadow of the bakers yeast

Fungi have now well and truly entered the genomic age. We currently know the complete DNA sequence for 18 fungal species and many more fungal genome sequencing projects are in progress. Whilst yeasts dominated the early genomic years, recently there has been a dramatic increase in filamentous fungal genome projects. The implications of this wealth of genetic information for mycologists worldwide is immense. In this review we summarise the background to fungal genome projects with an emphasis on the filamentous fungi. We discuss efforts to determine gene function and to compare genomes from different species. Since this is such a fast-moving field, useful web sites are listed that will enable the reader to keep up to date with developments.     

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.     

Branching out: Towards a trait-based understanding of fungal ecology

Fungal ecology lags behind in the use of traits (i.e. phenotypic characteristics) to understand ecological phenomena. We argue that this is a missed opportunity and that the selection and systematic collection of trait data throughout the fungal kingdom will reap major benefits in ecological and evolutionary understanding of fungi. To develop our argument, we first employ plant trait examples to show the power of trait-based approaches in understanding ecological phenomena such as identifying species allocation resources patterns, inferring community assembly and understanding diversity–ecosystem functioning relationships. Second, we discuss ecologically relevant traits in fungi that could be used to answer such ecological phenomena and can be measured on a large proportion of the fungal kingdom. Third, we identify major challenges and opportunities for widespread, coordinated collection and sharing of fungal trait data. The view that we propose has the potential to allow mycologists to contribute considerably more influential studies in the area of fungal ecology and evolution, as has been demonstrated by comparable earlier efforts by plant ecologists. This represents a change of paradigm, from community profiling efforts through massive sequencing tools, to a more mechanistic understanding of fungal ecology.     

Not seeing the genomes for the DNA

This Short Communication highlights the diversity of 'secondary' genome data (like mitochondrial and plastid genomes) that can be gleaned from next-generation sequencing projects, and encourages researchers to be mindful that these data are often as informative and useful as the 'primary' genome data.     

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging ‘omics’-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia.     

Influenza genome diversity and evolution

The influenza viruses contain highly variable genomes and are able to infect a wide range of host species. Large-scale sequencing projects have collected abundant influenza sequence data for assessing influenza genome diversity and evolution. This work reviews current influenza sequence databases characteristics and statistics, as well as recent studies utilizing these databases to unravel influenza virus diversity and evolution. Also discussed are the newest deep sequencing methods and their applications to influenza virus research.     

Perchance to dream: solving the mystery of sleep through genetic analysis

Sleep has been identified in all mammals and nonmammalian vertebrates that have been critically evaluated. In addition, sleep-like states have also been identified and described in several invertebrates. Despite this prevalence throughout the animal kingdom, the function of sleep remains a mystery. The completion of several genome sequencing projects has led to the expectation that fundamental aspects of sleep can be elucidated through genetic dissection. Indeed, studies in both the mouse and fly have begun to reveal tantalizing suggestions about the underlying principles that regulate sleep homeostasis. In this article we will review recent studies that have used genetic techniques to evaluate sleep in the fruit fly and the mouse.     

Microbial Pathogens in the Fungal Kingdom

The fungal kingdom is vast, spanning ~1.5 to as many as 5 million species diverse as unicellular yeasts, filamentous fungi, mushrooms, lichens, and both plant and animal pathogens. The fungi are closely aligned with animals in one of the six to eight supergroups of eukaryotes, the opisthokonts. The animal and fungal kingdoms last shared a common ancestor ~1 billion years ago, more recently than other groups of eukaryotes. As a consequence of their close evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models for mammalian biology, but prove more difficult to treat in infected animals. The last common ancestor to the fungal/metazoan lineages is thought to have been unicellular, aquatic, and motile with a posterior flagellum, and certain extant species closely resemble this hypothesized ancestor. Species within the fungal kingdom were traditionally assigned to four phyla, including the basal fungi (Chytridiomycota, Zygomycota) and the more recently derived monophyletic lineage, the dikarya (Ascomycota, Basidiomycota). The fungal tree of life project has revealed that the basal lineages are polyphyletic, and thus there are as many as eight to ten fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and virulence arose multiple times independently. A sobering recent development involves the species Batrachochytrium dendrobatidis from the basal fungal phylum, the Chytridiomycota, which has emerged to cause global amphibian declines and extinctions. Genomics is revolutionizing our view of the fungal kingdom, and genome sequences for zygomycete pathogens (Rhizopus, Mucor), skin-associated fungi (dermatophytes, Malassezia), and the Candida pathogenic species clade promise to provide insights into the origins of virulence. Here we survey the diversity of fungal pathogens and illustrate key principles revealed by genomics involving sexual reproduction and sex determination, loss of conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic transitions in form. The overarching conclusion is that fungal pathogens of animals have arisen repeatedly and independently throughout the fungal tree of life, and while they share general properties, there are also unique features to the virulence strategies of each successful microbial pathogen.     

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found—a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker.     

Complete,high-quality genomes from long-read metagenomic sequencing of two wolf lichen thalli reveals enigmatic genome architecture

Fungal genomes display incredible levels of complexity and diversity, and are exceptional study systems for genome evolution. Here we used the Oxford Nanopore MinION sequencing platform to generate high-quality fungal genomes from complex metagenomic samples of lichen thalli. We sequenced two wolf lichens using one flow cell per sample, generating 17.1 Gbps for Letharia lupina and 14.3 Gbps for Letharia columbiana. The resulting L. lupina genome is one of the most contiguous lichen genomes available to date, with 49.2 Mbp contained on 31 contigs. The L. columbiana genome, while less contiguous, is still relatively high quality, with 52.3 Mbp on a total of 161 contigs. Each thallus for both species contained multiple distinct haplotypes, a phenomenon that has rarely been empirically demonstrated. The Oxford Nanopore sequencing technologies are robust and effective when applied to complex symbioses, and have the potential to fundamentally transform our understanding of fungal genetics.     

Fungal evolution: major ecological adaptations and evolutionary transitions

Fungi are a highly diverse group of heterotrophic eukaryotes characterized by the absence of phagotrophy and the presence of a chitinous cell wall. While unicellular fungi are far from rare, part of the evolutionary success of the group resides in their ability to grow indefinitely as a cylindrical multinucleated cell (hypha). Armed with these morphological traits and with an extremely high metabolical diversity, fungi have conquered numerous ecological niches and have shaped a whole world of interactions with other living organisms. Herein we survey the main evolutionary and ecological processes that have guided fungal diversity. We will first review the ecology and evolution of the zoosporic lineages and the process of terrestrialization, as one of the major evolutionary transitions in this kingdom. Several plausible scenarios have been proposed for fungal terrestralization and we here propose a new scenario, which considers icy environments as a transitory niche between water and emerged land. We then focus on exploring the main ecological relationships of Fungi with other organisms (other fungi, protozoans, animals and plants), as well as the origin of adaptations to certain specialized ecological niches within the group (lichens, black fungi and yeasts). Throughout this review we use an evolutionary and comparative‐genomics perspective to understand fungal ecological diversity. Finally, we highlight the importance of genome‐enabled inferences to envision plausible narratives and scenarios for important transitions.     

Fungal rhodopsins and opsin-related proteins: eukaryotic homologues of bacteriorhodopsin with unknown functions.

In the last decade, several genome sequencing projects revealed the existence of previously unknown photoreceptors. Among those are eukaryotic rhodopsins of haloarchaeal type, mostly represented by fungal sequences. We have classified and analyzed seventy-seven of these fungal proteins, which show a high similarity of their putative transmembrane regions to those of bacteriorhodopsin. Those sequences can be divided into the two subgroups, fungal rhodopsins (RDs) and opsin-related proteins (ORPs), the latter lacking the lysine residue necessary for retinal binding. We have analyzed the conservation pattern of the residues known to have functional or structural importance in bacteriorhodopsin and discussed dramatic differences in the conservation between RDs and ORPs. We found many cases of multiple forms of RDs and/or ORPs and examined possible reasons for such multiplicity. For some species the reason may lie in functional photobiological diversification, while for the others it follows the pattern of evolutionary recent genome duplication and possible functional redundancy.     

Analyses of soil fungal communities in adjacent natural forest and hoop pine plantation ecosystems of subtropical Australia using molecular approaches based on 18S rRNA genes

Soil fungal communities were studied using 18S rDNA-based molecular techniques. Soil DNA was analyzed using temperature gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP), cloning and sequencing methods, following community DNA extraction and polymerase chain reaction (PCR). The extracted community DNA was successfully amplified using the primer pair of EF4f-Fung5r which produced ca. 550bp 18S rDNA fragments. TGGE screening of the PCR products showed some differences in band position and intensity between two soil samples in adjacent natural forest (YNF) and hoop pine plantation (YHP) ecosystems at Yarraman in subtropical Australia. TGGE and SSCP could be used for screening PCR products. However, care must be exercised when interpreting the TGGE and SSCP results with respect to microbial diversity, because one band may not necessarily represent one species. It is recommended that the PCR products should be purified before TGGE or SSCP screening. SSCP screening of the clone sequences revealed differences among the clones. Sequence and phylogenetic analyses revealed that all obtained clones were affiliated to the kingdom Fungi, including three phyla, i.e., Zygomycota, Ascomycota and Basidiomycota. Our results suggested that community DNA extraction, PCR, cloning, SSCP screening of clones, sequencing of selected clones and phylogentic analyses could be a good strategy in investigation of soil fungal community and diversity.     

Biodiversity, genomes, and DNA sequence databases

There are ∼1.4 million organisms on this planet that have been described morphologically but there is no comparable coverage of biodiversity at the molecular level. Little more than 1% of the known species have been subject to any molecular scrutiny and eukaryotic genome projects have focused on a group of closely related model organisms. The past year, however, has seen an ∼80% increase in the number of species represented in sequence databases and the completion of the sequencing of three prokaryotic genomes. Large-scale sequencing projects seem set to begin coverage of a wider range of the eukaryotic diversity, including green plants, microsporidians and diplomonads.     

The Ypt/Rab family and the evolution of trafficking in fungi

The evolution of the eukaryotic endomembrane system and the transport pathways of their vesicular intermediates are poorly understood. A common set of organelles and pathways seems to be present in all free-living eukaryotes, but different branches of the tree of life have a variety of diverse, specialized organelles. Rab/Ypt proteins are small guanosine triphosphatases with tissue-specific and organelle-specific localization that emerged as markers for organelle diversity. Here, I characterize the Rab/Ypt family in the kingdom Fungi, a sister kingdom of Animals. I identify and annotate these proteins in 26 genomes representing near one billion years of evolution, multiple lifestyles and cellular types. Surprisingly, the minimal set of Rab/Ypt present in fungi is similar to, perhaps smaller than, the predicted eukaryotic ancestral set. This suggests that the saprophytic fungal lifestyle, multicellularity as well as the highly polarized secretion associated with hyphal growth did not require any major innovation in the molecular machinery that regulates protein trafficking. The Rab/Ypt and other protein traffic-related families are kept small, not paralleling increases in genome size, in contrast to the expansion of such components observed in other branches of the tree of life, such as the animal and plant kingdoms. This analysis suggests that multicellularity and cellular diversity in fungi followed different routes from those followed by plants and metazoa.