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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 fungus Botrytis cinerea (Botryotinia fuckeliana) infects more than 500 plant species and causes a wide range of symptoms: soft rots, accompanied by collapse and water-soaking of tissues followed by the appearance of gray masses of conidia on leaves and soft fruits (gray mold), and spots that may turn brown to full-scale soft rotting on flower petals (Botrytis blight). In general, B. cinerea is responsible for severe economic losses that are either due to the damage of growing plants in the field or the rot of harvested fruits, flowers, and vegetables during storage under cold and humid conditions. B. cinerea has adapted to the plant host and its environment by evolving strategies to use plant tissues for proliferation in terms of a necrotrophic lifestyle, and to survive biotic stresses (host responses) as well as abiotic factors of the host’s environment such as sunlight and concomitant stresses. B. cinerea maintains a complex regulatory network of light-sensitive proteins and signal transduction pathways to use light for coordinating stress responses, virulence, and reproduction. Different light-controlled reproduction cycles enable B. cinerea to live in moderate climate zones by infecting and propagating in summer and resting in winter when green host tissues are unavailable.
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.
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, microcolonial black fungi exhibit 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 regulation and relevance of 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 deletion mutants are studied for specifying the functions of DHN melanin in K. petricola.
DHN (1,8-dihydroxynaphthalene) melanin is produced by Ascomycetes via slightly differing synthetic routes. Polyketide synthases release YWA1, AT4HN or T4HN. YWA1 and AT4HN are deacetylated by ‘yellowish-green’ hydrolases, and T4HN is converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. The melanogenic genes are tightly, partially or not clustered in the genomes, and are often regulated in a spatial and/or temporal fashion. By contrast, microcolonial fungi/black yeasts – a polyphyletic group of Ascomycetes dwelling in hostile habitats such as bare rock surfaces – feature constitutive DHN melanogenesis. Here, we report on the DHN melanogenic genes of Knufia petricola (Eurotiomycetes/Chaetothyriales).
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.
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.
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.