An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola

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 th...

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Autores principales:	Oliver Voigt, Nicole Knabe, Sarah Nitsche, Eileen A. Erdmann, Julia Schumacher, Anna A. Gorbushina
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/471308865aca4a36a71a2fde8c2b123a
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spelling	oai:doaj.org-article:471308865aca4a36a71a2fde8c2b123a2021-12-02T12:42:27ZAn advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola10.1038/s41598-020-79120-52045-2322https://doaj.org/article/471308865aca4a36a71a2fde8c2b123a2020-12-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79120-5https://doaj.org/toc/2045-2322Abstract 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.Oliver VoigtNicole KnabeSarah NitscheEileen A. ErdmannJulia SchumacherAnna A. GorbushinaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-14 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Oliver Voigt Nicole Knabe Sarah Nitsche Eileen A. Erdmann Julia Schumacher Anna A. Gorbushina An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
description	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.
format	article
author	Oliver Voigt Nicole Knabe Sarah Nitsche Eileen A. Erdmann Julia Schumacher Anna A. Gorbushina
author_facet	Oliver Voigt Nicole Knabe Sarah Nitsche Eileen A. Erdmann Julia Schumacher Anna A. Gorbushina
author_sort	Oliver Voigt
title	An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
title_short	An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
title_full	An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
title_fullStr	An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
title_full_unstemmed	An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola
title_sort	advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus knufia petricola
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/471308865aca4a36a71a2fde8c2b123a
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An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola

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