Abstract
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. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This 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.
Highlights
Surfaces of desert rocks and other sun-exposed materials challenge their settlers with extreme and rapidly changing environmental c onditions[1]
Bright fluorescence was observed indicating that the dyes as well as sufficient light passed through the melanised cell walls
Cell walls were successfully stained with calcofluor white (CFW), nuclei with 4′,6-diamidin2-phenylindol (DAPI), mitochondria with MitoTracker Green (MTG), and membranes with FM4-64 (Fig. 1b)
Summary
Surfaces of desert rocks and other sun-exposed materials challenge their settlers with extreme and rapidly changing environmental c onditions[1]. Slow yeast-like or meristematic growth is accompanied by physiological adaptations including the absence of specialised reproductive structures; multi-layered cell walls and the production of characteristic secondary metabolites including 1,8-dihydroxynaphthalene (DHN) melanin, carotenoids, mycosporines and extracellular polymeric substances (EPS) All help these black fungi ( called black yeasts) to resist environmental stresses including extreme temperatures, desiccation (and rehydration), low nutrient availability and intense solar radiation. K. petricola strain A95 (CBS 123872) was isolated from marble of the Philopappos Monument in Athens (Greece) and possesses all the characteristics of microcolonial black fungi It grows moderately well in culture and is already intensively used in studies of mineral weathering and symbiotic interactions with the cyanobacterium Nostoc punctiforme[24,25,26,27,28,29,30,31,32,33]. Different genetically encoded fluorescent protein variants were visualised, and genes of synthetic pathways were modified by the application of traditional gene replacement, CRISPR/Cas9-mediated gene editing and replacement, as well as gene silencing (RNA interference) demonstrating the functionality of these methods in K. petricola
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