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The rotation of the Earth causes a day/night cycle that is characterized by changing light and temperature conditions. Fungi actively sense the environmental light conditions to induce protective mechanisms against the light-associated stresses and to regulate development. Since fungi adapted to habitats with different light regimes, the complexities of their ‘visual’ systems and photoresponses may vary significantly. Fungi that are associated with plants experience a special light regime because the host seeks optimum light conditions for photosynthesis – and the fungus must cope with them. Moreover, fungi living under the canopy are confronted with an altered spectrum enriched for green and far-red light. Plants sense light to coordinate growth and morphogenesis: a low red:far-red ratio indicates the presence of competitors (shading by other plants) and triggers the shade avoidance response which is accompanied by increased susceptibility to nectrophic pathogens.
Botrytis cinerea, the causal agent of gray mold diseases on many plant species in moderate climate zones (high humidity, temperatures around 20°C, seasons), exhibits striking photoresponses accompanied by a broad action spectrum and a high number of photoreceptors. Light is the most important factor controlling morphogenesis: it induces conidiation for disease spreading (summer cycle) and represses sclerotial development for survival and/or sexual recombination (winter cycle). In the past years, we identified several cellular components involved in photoperception and regulation of photomorphogenesis and virulence indicating that the same signaling pathways are of relevance for both processes, propagation and infection. Notably, we recognized the role of the phytochromes (red/far-red light sensors) for coordinated responses to light and elevated temperatures (photo-/thermomorphogenesis) – similar to the function of Arabidopsis PhyB. BcPHY2-like thermosensors are likely restricted to the Leotiomycetes, which include several plant pathogens of the moderate climate zones.
Sunlight, the major source of energy, drives life but in excess it provokes UV-induced DNA damage, accumulation of reactive oxygen species, desiccation, osmotic stresses and so on. Biological strategies to survive excess light include protection, avoidance and active utilisation. Phototrophic organisms including cyanobacteria, green algae and plants use all three strategies. Fungi occupying phototrophic niches may profit from the surplus photosynthetic products which also obliges them to cope with the same light-induced stresses. One way of handling these stresses would be to use similar signalling pathways to sense the presence and to interact with their phototrophic partners. Obviously, the levels of adaptation and responses to light will depend on the environment as well as fungal genotype and phylogenetic position. Black fungi – a polyphyletic group – accumulate the dark pigment DHN melanin in their cell walls and often occupy light-flooded habitats from phyllosphere to rock surfaces. Here we compare two sequenced melanised fungi of different lifestyles in their response to light.
The Leotiomycete Botrytis cinerea is an aggressive pathogen that primarily infects the above-ground parts of plants. It possesses large numbers of photoreceptors that respond to a broad spectrum of light. As a consequence, light controls morphogenesis in which it induces conidiation for disease spreading and represses sclerotial development for survival and/or sexual recombination. Cellular components involved in photo-perception and regulation of morphogenesis, stress responses and virulence have been identified and appear to regulate propagation, survival and infection. These include phytochromes, a group of photoreceptors which are particularly enriched in the Leotiomycetes and that mediate coordinated responses to light and elevated temperatures. Assuming that photo-regulation may be equally important for fungi that live in mutualistic relationships with phototrophs either by forming composite organisms or biofilms, we investigate the role of light in the rock-inhabiting Eurotiomycete Knufia petricola. Like other black yeasts, K. petricola grows slowly, does not form specialised reproductive structures and constitutively produces DHN melanin as well as carotenoids. Combining K. petricola with the cyanobacterium Nostoc punctiforme, we developed a model system for studying biofilm formation and bio-weathering. A genetic toolbox to manipulate this model system is being developed. K. petricola strain A95 possesses ten putative photoreceptors, more than found in filamentous Eurotiomycetes suggesting that light plays an important role for abiotic and biotic interactions in extremo-tolerant and symbiosis-capable fungi.
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.
Schwarze mikrokoloniale Pilze besiedeln zunehmend von Menschen geschaffene Habitate, wie schadstoffbelastete Böden, Statuen, Gebäudefassaden und Dächer. Sie verfärben und zersetzen die Oberflächen von anfälligen Materialien oder reduzieren die Lichtausbeute von Solaranlagen. Die Biologie dieser Pilze und ihre Relevanz für die Materialforschung stehen im Fokus unserer Studien an der Bundesanstalt für Materialforschung und -prüfung (BAM). Mit einer Kollektion schwarzer Pilze, die von Solaranlagen isoliert wurden, bringen wir klima- und materialrelevante Biodiversität in den Stammbaum des Lebens. Die Überlebensstrategien dieser Organismen versuchen wir mit molekularbiologischen und genetischen Untersuchungsansätzen zu entschlüsseln.
Dihydroxynaphthalene (DHN) melanin is produced by different 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, survival and/or infection structures. Remarkable is the regulation of the DHN melanogenesis in the foliar plant pathogen Botrytis cinerea: it involves two differently expressed PKSs providing the precursor in conidia and sclerotia, respectively (Schumacher 2016, Mol Microbiol). In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) 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. As part of our continuing research on microcolonial rock-inhabiting fungi, we chose the genetically amenable Knufia petricola strain A95 (Nai et al. 2013, Fungal Genet Biol; Noack-Schönmann et al. 2014, AMB Express) for detailed studies. DHN-deficient mutants generated by targeted mutation of biosynthetic genes were studied with regard to the architecture of the cell wall and the EPS (extracellular polymeric substances) matrix, attachment to and weathering of olivine, as well as the tolerance to abiotic and biotic stresses. We will discuss the critical role of the outer cell surface (DHN melanin and EPS) in adhesion to the substrate and subsequent damage of the colonized surface.
Microcolonial black fungi, a polyphyletic group of ascomycetes, exhibit constitutive melanin formation, yeast-like growth and high stress tolerances. They dominate – often together with bacteria and algae in sub-aerial biofilms – a range of hostile environments including natural and man-made ones, from salterns to dishwashers, roofs and solar panels. Because of lacking genetic tools and the slow growth of most isolates, the genetic bases for these specific properties are largely unknown. The rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales) exhibits all characteristics of microcolonial black fungi and was selected as recipient for genetic engineering to study gene functions and genetic interactions. Different variants of green and red fluorescent proteins were successfully expressed indicating that fluorescence microscopy using genetically encoded fluorescent proteins and fluorescent dyes enables various cell biology approaches. Furthermore, genes of biosynthetic pathways (DHN melanin, carotenoids, uracil, adenine) were successfully mutated by applying traditional gene replacement and plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9 or silenced by RNA interference (RNAi). The availability of this advanced and efficient genetic toolbox and the annotated genome sequence of strain A95 makes K. petricola an excellent model for exploring the secrets of microcolonial black fungi.
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.
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.
Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi.
Fungi that share light-flooded habitats with phototrophs may profit from excess photosynthetic products. But to cope with sunlight-associated stresses it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The filamentous foliar 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 Dothideo- 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 photoregulation are important for fungi that avoid loss of energy and nutrients through cooperation with phototrophs.