Morphological and Phylogenetic Appraisal of Novel and Extant Taxa of Stictidaceae from Northern Thailand

Stictidaceae comprises taxa with diverse lifestyles. Many species in this family are drought resistant and important for studying fungal adaptation and evolution. Stictidaceae comprises 32 genera, but many of them have been neglected for decades due to the lack of field collections and molecular data. In this study, we introduce a new species Fitzroyomyces hyaloseptisporus and a new combination Fitzroyomycespandanicola. We also provide additional morphological and molecular data for Ostropomyces pruinosellus and O. thailandicus based on new collections isolated from an unidentified woody dicotyledonous host in Chiang Rai, Thailand. Taxonomic conclusions are made with the aid of morphological evidence and phylogenetic analysis of combined LSU, ITS and mtSSU sequence data. Characteristics such as the shape and septation of ascospores and conidia as well as lifestyles among genera of Stictidaceae are discussed.

Novel Antarctic yeast adapts to cold by switching energy metabolism and increasing small RNA synthesis

The novel extremophilic yeast Rhodotorula frigidialcoholis, formerly R. JG1b, was isolated from ice-cemented permafrost in University Valley (Antarctic), one of coldest and driest environments on Earth. Phenotypic and phylogenetic analyses classified R. frigidialcoholis as a novel species. To characterize its cold-adaptive strategies, we performed mRNA and sRNA transcriptomic analyses, phenotypic profiling, and assessed ethanol production at 0 and 23 °C. Downregulation of the ETC and citrate cycle genes, overexpression of fermentation and pentose phosphate pathways genes, growth without reduction of tetrazolium dye, and our discovery of ethanol production at 0 °C indicate that R. frigidialcoholis induces a metabolic switch from respiration to ethanol fermentation as adaptation in Antarctic permafrost. This is the first report of microbial ethanol fermentation utilized as the major energy pathway in response to cold and the coldest temperature reported for natural ethanol production. R. frigidialcoholis increased its diversity and abundance of sRNAs when grown at 0 versus 23 °C. This was consistent with increase in transcription of Dicer, a key protein for sRNA processing. Our results strongly imply that post-transcriptional regulation of gene expression and mRNA silencing may be a novel evolutionary fungal adaptation in the cryosphere.

Transposable elements in the genome of the lichen-forming fungus Umbilicaria pustulata, and their distribution in different climate zones along elevation

Background: Transposable elements (TEs) are an important source of genome plasticity across the tree of life. Accumulating evidence suggests that TEs may not be randomly distributed in the genome. Drift and natural selection are important forces shaping TE distribution and accumulation, acting directly on the TE element or indirectly on the host species. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the host's ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold-temperate. We trace the occurrence of the newly identified TEs in populations along three replicated elevation gradients using a Pool-Seq approach, to identify TE insertions of potential adaptive significance. Results: We found that TEs cover 21.26 % of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. Out of a total of 182 TE copies we identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function. Conclusions: This pioneering study into the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution. Particularly, it may serve as a foundation for assessing the impact of TE dynamics on fungal adaptation to the abiotic environment, and the impact of TE activity on the evolution and maintenance of a symbiotic lifestyle.

Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi

The efficiency of microorganisms to degrade lignified plants is of great importance in the Earth’s carbon cycle, but also in industrial biorefinery processes, such as for biofuel production. Here, we present a large-scale proteomics approach to investigate and compare the enzymatic response of five filamentous fungi when grown on five very different substrates: grass (sugarcane bagasse), hardwood (birch), softwood (spruce), cellulose and glucose. The five fungi included the ascomycetes Aspergillus terreus, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa and the white-rot basidiomycete Phanerochaete chrysosporium, all expressing a diverse repertoire of enzymes. In this study, we present comparable quantitative protein abundance values across five species and five diverse substrates. The results allow for direct comparison of fungal adaptation to the different substrates, give indications as to the substrate specificity of individual carbohydrate-active enzymes (CAZymes), and reveal proteins of unknown function that are co-expressed with CAZymes. Based on the results, we present a quantitative comparison of 34 lytic polysaccharide monooxygenases (LPMOs), which are crucial enzymes in biomass deconstruction.

Fun(gi)omics: Advanced and Diverse Technologies to Explore Emerging Fungal Pathogens and Define Mechanisms of Antifungal Resistance

ABSTRACT The landscape of infectious fungal agents includes previously unidentified or rare pathogens with the potential to cause unprecedented casualties in biodiversity, food security, and human health. The influences of human activity, including the crisis of climate change, along with globalized transport, are underlying factors shaping fungal adaptation to increased temperature and expanded geographical regions. Furthermore, the emergence of novel antifungal-resistant strains linked to excessive use of antifungals (in the clinic) and fungicides (in the field) offers an additional challenge to protect major crop staples and control dangerous fungal outbreaks. Hence, the alarming frequency of fungal infections in medical and agricultural settings requires effective research to understand the virulent nature of fungal pathogens and improve the outcome of infection in susceptible hosts. Mycology-driven research has benefited from a contemporary and unified approach of omics technology, deepening the biological, biochemical, and biophysical understanding of these emerging fungal pathogens. Here, we review the current state-of-the-art multi-omics technologies, explore the power of data integration strategies, and highlight discovery-based revelations of globally important and taxonomically diverse fungal pathogens. This information provides new insight for emerging pathogens through an in-depth understanding of well-characterized fungi and provides alternative therapeutic strategies defined through novel findings of virulence, adaptation, and resistance.

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

ABSTRACT Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii. We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii. The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus. Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents. IMPORTANCE Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii. In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii. Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

Comprehensive analysis of the dermatophyte Trichophyton rubrum transcriptional profile reveals dynamic metabolic modulation

The environmental challenges imposed onto fungal pathogens require a dynamic metabolic modulation, which relies on activation or repression of critical factors and is essential for the establishment and perpetuation of host infection. Wherefore, to overcome the different host microenvironments, pathogens not only depend on virulence factors but also on metabolic flexibility, which ensures their dynamic response to stress conditions in the host. Here, we evaluate Trichophyton rubrum interaction with keratin from a metabolic perspective. We present information about gene modulation of the dermatophyte during early infection stage after shifting from glucose- to keratin-containing culture media, in relation to its use of glucose as the carbon source. Analyzing T. rubrum transcriptome using high-throughput RNA-sequencing technology, we identified the modulation of essential genes related to nitrogen, fatty acid, ergosterol, and carbohydrate metabolisms, among a myriad of other genes necessary for the growth of T. rubrum in keratinized tissues. Our results provide reliable and critical strategies for adaptation to keratin and confirm that the urea-degrading activity associated with the reduction in disulfide bonds and proteolytic activity facilitated keratin degradation. The global modulation orchestrates the responses that support virulence and the proper adaptation to keratin compared with glucose as the carbon source. The gene expression profiling of the host-pathogen interaction highlights candidate genes involved in fungal adaptation and survival and elucidates the machinery required for the establishment of the initial stages of infection.

Fungal Adaptation to the Advanced Stages of Wood Decomposition: Insights from the Steccherinum ochraceum

Steccherinum ochraceum is a white rot basidiomycete with wide ecological amplitude. It occurs in different regions of Russia and throughout the world, occupying different climatic zones. S. ochraceum colonizes stumps, trunks, and branches of various deciduous (seldom coniferous) trees. As a secondary colonizing fungus, S. ochraceum is mainly observed at the late decay stages. Here, we present the de novo assembly and annotation of the genome of S. ochraceum, LE-BIN 3174. This is the 8th published genome of fungus from the residual polyporoid clade and the first from the Steccherinaceae family. The obtained genome provides a first glimpse into the genetic and enzymatic mechanisms governing adaptation of S. ochraceum to an ecological niche of pre-degraded wood. It is proposed that increased number of carbohydrate-active enzymes (CAZymes) belonging to the AA superfamily and decreased number of CAZymes belonging to the GH superfamily reflects substrate preferences of S. ochraceum. This proposition is further substantiated by the results of the biochemical plate tests and exoproteomic study, which demonstrates that S. ochraceum assumes the intermediate position between typical primary colonizing fungi and litter decomposers or humus saprotrophs. Phylogenetic analysis of S. ochraceum laccase and class II peroxidase genes revealed the distinct evolutional origin of these genes in the Steccherinaceae family.

A Double-Edged Sword: Aneuploidy is a Prevalent Strategy in Fungal Adaptation

Aneuploidy, a deviation from a balanced genome by either gain or loss of chromosomes, is generally associated with impaired fitness and developmental defects in eukaryotic organisms. While the general physiological impact of aneuploidy remains largely elusive, many phenotypes associated with aneuploidy link to a common theme of stress adaptation. Here, we review previously identified mechanisms and observations related to aneuploidy, focusing on the highly diverse eukaryotes, fungi. Fungi, which have conquered virtually all environments, including several hostile ecological niches, exhibit widespread aneuploidy and employ it as an adaptive strategy under severe stress. Gambling with the balance between genome plasticity and stability has its cost and in fact, most aneuploidies have fitness defects. How can this fitness defect be reconciled with the prevalence of aneuploidy in fungi? It is likely that the fitness cost of the extra chromosomes is outweighed by the advantage they confer under life-threatening stresses. In fact, once the selective pressures are withdrawn, aneuploidy is often lost and replaced by less drastic mutations that possibly incur a lower fitness cost. We discuss representative examples across hostile environments, including medically and industrially relevant cases, to highlight potential adaptive mechanisms in aneuploid yeast.

Candida albicans Interactions with Mucosal Surfaces during Health and Disease

Flexible adaptation to the host environment is a critical trait that underpins the success of numerous microbes. The polymorphic fungus Candida albicans has evolved to persist in the numerous challenging niches of the human body. The interaction of C. albicans with a mucosal surface is an essential prerequisite for fungal colonisation and epitomises the complex interface between microbe and host. C. albicans exhibits numerous adaptations to a healthy host that permit commensal colonisation of mucosal surfaces without provoking an overt immune response that may lead to clearance. Conversely, fungal adaptation to impaired immune fitness at mucosal surfaces enables pathogenic infiltration into underlying tissues, often with devastating consequences. This review will summarise our current understanding of the complex interactions that occur between C. albicans and the mucosal surfaces of the human body.