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Sunlight is an almost unavoidable environmental cue and plays a fundamental role in the biology of pro- and eukaryotic organisms. To cope with sunlight-associated stresses e.g., high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses, it is important for organisms to accurately sense and respond to changes in light. The benefits of light are obvious for green organisms such as cyanobacteria, algae and plants which use light as an energy source (photosynthesis). Less apparent are other light-dependent processes such as light-driven DNA repair by photolyases (photoreactivation) or ion pumping by microbial opsins. Fungi that can share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess many proteins for absorbing UV/blue, green, red and far-red light, produce the black 1,8 dihydroxynaphthalene (DHN) melanin and orange-red carotenoids, and may live in multispecies biofilms. Here, we are addressing the question to which extent constitutive pigment formation (melanin and carotenoids) and responses mediated by the stress-activated mitogen-activated protein (MAP) kinase contribute to the observed light (UV-B) tolerance of K. petricola.
Sunlight is an almost unavoidable environmental cue and plays a fundamental role in the biology of pro- and eukaryotic organisms. To cope with sunlight-associated stresses e.g., high temperatures, UV radiation, accumulation of reactive oxygen species, desiccation, and osmotic stress, it is important for organisms to accurately sense and respond to changes in light. The benefits of light are obvious for green organisms such as cyanobacteria, algae and plants which use light as an energy source (photosynthesis). Fungi that can share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Examples are the plant pathogen Botrytis cinerea, the gray mold fungus, and the rock inhabitant Knufia petricola, a microcolonial black fungus which forms multispecies biofilms with bacteria and algae.
The exponential rise in the number of fungal genomes sequenced by next-generation sequencing techniques makes it necessary to increase efforts to correctly annotate and assign gene functions. There are two possibilities to explore a genome and its gene functions. The hypothesis-based method proves the function of already existing gene/allele candidates by targeted mutagenesis - so called reverse genetics. The basis of forward genetics approaches is the random mutagenesis of the genome, followed by screening of obtained mutants for the phenotype of interest, and identification of the mutated genes in the respective mutants. This strategy is hypothesis-generating, means it is necessary to verify the relationship between the detected mutations and the observed phenotype by targeted mutagenesis of the identified gene. We developed a toolbox for editing the genome of the rock inhabitant Knufia petricola [Eurotiomycetes, Chaetothyriales] that allows the study of the phenotypic characteristics of black fungi such as the regulation of pigment synthesis, general stress responses, oligotrophy, and the unusual modes of cell division by advanced reverse and forward genetics approaches. The toolbox includes the annotated genome sequence of strain A95, efficient strategies for CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering. A forward genetics approach using transposon mutagenesis is currently developed for identifying essential genes. The established protocols and knowledge gained from K. petricola form a starting point for making other fungi from extreme environments accessible to genetic manipulation.
Dihydroxynaphthalene (DHN) melanin is produced by diverse Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g., in melanized reproduction and survival structures of the foliar plant pathogen Botrytis cinerea (Schumacher 2016, Mol Microbiol). In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeasts) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, DHN melanin builds a protective layer around all vegetative cells thus contributing to the survival of diverse environmental stresses even without specialized reproduction structures. For studying the relevance of constitutive DHN melanogenesis for tolerance of abiotic and biotic stresses, adhesion to substrates and subsequent damage of colonized surfaces, the rock-inhabiting fungus Knufia petricola was chosen as gene functions in this fungus can be studied by CRISPR/Cas9-based genome editing. The putative melanogenic genes were identified in the genome of K. petricola, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Phenotypes of DHN-deficient mutants are studied. Here, we will discuss the role of the DHN melanin layer on the outer cell wall in tolerating UV irradiation.
The roles of DHN melanin and the stress-activated MAP kinase in the rock inhabitant Knufia petricola
(2023)
Black fungi/yeasts exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. Due to their slow growth, robust cell walls and the lack of sexual cycles and genetic tools, the underlying mechanisms of their phenotypic traits have remained largely unexplored. Using recently developed genetic tools, it is now possible to manipulate the genome of the rock-inhabiting model fungus Knufia petricola. Thus, gene functions and the cell biology of black fungi can be studied using CRISPR/Cas9-based genome editing and live-cell imaging with genetically encoded fluorescent proteins. Here, we are addressing the question to which extent constitutive pigment formation (melanin and carotenoids) and responses mediated by the stress-activated mitogen-activated protein (MAP) kinase contribute to the observed extremotolerance of K. petricola. The mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively. The other putative melanogenic genes were identified in the genome, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Sak1 encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. Growth of the obtained single, double and triple deletion mutants was tested by droplet tests on media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even without environmental pressure and are hypersensitive to different stresses: e.g. osmotic, oxidative, membrane, pH and heat stress. Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress.
Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But to cope with sunlight-associated stresses [e.g. high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses] it is important for fungi to accurately sense and respond to changes in light. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs.
Sunlight is an important environmental factor is almost all ecosystems by being a source of energy, information, and stress. All organisms must protect themselves from the harmful effects of light such as UV radiation, ROS accumulation, heat, and desiccation. Finally, light qualities and quantities can be used for decision making, timing and as guide for directed growth when they are sensed and transduced into intracellular signals. Botrytis cinerea and other plant pathogens infecting the sun-exposed parts of the plant must cope with the high light conditions the host plant seeks. Further they experience an altered light spectrum (‘green gap’) when they colonize shaded parts of the plant; it is depleted for blue and red light that is absorbed by the plant chlorophyll and enriched for green and far-red light that is reflected or transmitted by the plant tissue. As these ambient light conditions trigger the shade avoidance response in the plant, the pathogens may trigger their own ‘shading response’ such as the upregulation of virulence determinants and inoculum production. B. cinerea maintains a highly sophisticated light signaling machinery that senses different light qualities to trigger a variety of responses, that are protection, morphogenesis, positive and negative tropisms, and entrainment. These characteristics render B. cinerea a valuable model to enlighten the role of light in parasitic fungus-plant interactions and beyond. The vegetative mycelium – the core of all infection and developmental programs – is not visibly pigmented and thus considered to be sensitive to biotic and abiotic stresses. However, the vegetative hyphae have a very limited half-life and are usually restricted to the invasive growth phase in which they are protected from light by the plant tissue. Fast colonization of host tissues and by this proper nutrient acquisition enables the rapid formation of long-lasting reproduction structures (melanized conidiophores with conidia, sclerotia) on the surfaces of rotted plant tissues. Depending on the light and temperature conditions, conidiation or sclerotial development is initiated. Taken together, B. cinerea uses light-regulated signaling networks to avoid light whenever possible; for example, by minimizing the half-life of sensitive cells that are hiding in plant tissues and by scheduling critical steps such as conidiogenesis, conidial germination and penetration of plant tissues for the night.
Black fungi also called black yeasts, rock-inhabiting fungi or microcolonial fungi are a group of Ascomycetes [Eurotiomycetes, Arthoniomycetes and Dothideomycetes] that exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. They dominate a range of hostile natural and man‑made environments – from desert rocks and salterns to dishwashers, roofs, and solar panels. Due to their slow growth and the lack of sexual cycles and genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We consider the rock inhabitant K. petricola [Eurotiomycetes, Chaetothyriales] a suitable model for studying the phenotypic characteristics of black fungi. With K. petricola the regulation of pigment synthesis, general stress responses and the unusual modes of cell division can be dissected by advanced reverse and forward genetics approaches. The genome of K. petricola strain A95 was sequenced using a combination of short high quality Illumina reads and long PacBio reads. The final assembly consists of twelve contigs: five complete chromosomes and six contigs with one telomer each. Gene annotation supported by transcriptomics and proteomics data was manually curated. Recently, we developed a set of genetic tools to manipulate the genome for analyzing gene functions and studying the cell biology. This set includes CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering in the laboratory. Mutants defective in DHN melanogenesis, carotenogenesis or both processes are currently studied to elucidate the role of these protective pigments in tolerance of natural and man-made stresses, weathering of olivine, penetration of marble, and adhesion to surfaces. Further, the established protocols and knowledge gained from K. petricola form a starting point for making other extremotolerant black fungi accessible to genetic manipulation.
Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. Sunlight-associated stresses are however multiple: high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses. Ascomycota dominating light-flooded habitats accurately sense and respond to changes in light using it as a cue to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships. Two species from two light-flooded habitats – phyllosphere and sun-exposed solid surfaces – were analysed for their photoreceptor distribution.
In both habitats phototroph-associated and black [dihydroxynaphthalene (DHN) melanin-containing] fungi are prevalent. This diversity was sampled with the plant-associated fungus Botrytis cinerea (Leotiomycetes), while Knufia petricola (Eurotiomycetes) was included as a typical biofilm-former on sun-exposed solid surfaces e.g. rocks, building facades, roofs, and solar panels. The analysis has shown that genomes of black fungi contain more photoreceptors than animal pathogens and saprophytes such as Aspergillus nidulans and Neurospora crassa1,2. B. cinerea that causes the grey mould disease by infecting the above-ground parts of more than 200 dicots has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts1.
Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers of photoreceptors along with the same set of protective metabolites i.e. melanin, carotenoids and mycosporines2. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for sun-stressed fungi that receive nutrients through cooperation with phototrophs. CRISPR/Cas9-based genetic tools for manipulating K. petricola were established3 and are currently used for elucidating the functions of the different photoreceptors in the biology of rock-inhabiting fungi.
This work was supported by the grant SCHU 2833/4-1 from the German Research Foundation (DFG) and internal funds of the BAM.
Rock-EATING FUNGI - Genetic Dive into the Biology of the Microcolonial Black Fungus Knufia petricola
(2021)
Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast‑like growth and constitutive melanin formation. They dominate a range of hostile natural and man‑made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock‑inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. The state‑of‑the‑art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.