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Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus
BACKGROUND: Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leav...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9840287/ https://www.ncbi.nlm.nih.gov/pubmed/36639657 http://dx.doi.org/10.1186/s40694-023-00149-y |
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author | Lutz, Tobias Hadeler, Birgit Jaeckel, Mareike Schulz, Barbara Heinze, Cornelia |
author_facet | Lutz, Tobias Hadeler, Birgit Jaeckel, Mareike Schulz, Barbara Heinze, Cornelia |
author_sort | Lutz, Tobias |
collection | PubMed |
description | BACKGROUND: Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host–pathogen interaction can be studied in more detail. RESULTS: Here, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus, for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette. CONCLUSIONS: The invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40694-023-00149-y. |
format | Online Article Text |
id | pubmed-9840287 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-98402872023-01-15 Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus Lutz, Tobias Hadeler, Birgit Jaeckel, Mareike Schulz, Barbara Heinze, Cornelia Fungal Biol Biotechnol Methodology BACKGROUND: Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host–pathogen interaction can be studied in more detail. RESULTS: Here, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus, for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette. CONCLUSIONS: The invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40694-023-00149-y. BioMed Central 2023-01-13 /pmc/articles/PMC9840287/ /pubmed/36639657 http://dx.doi.org/10.1186/s40694-023-00149-y Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Lutz, Tobias Hadeler, Birgit Jaeckel, Mareike Schulz, Barbara Heinze, Cornelia Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus |
title | Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus |
title_full | Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus |
title_fullStr | Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus |
title_full_unstemmed | Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus |
title_short | Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus |
title_sort | stable overexpression and targeted gene deletion of the causative agent of ash dieback hymenoscyphus fraxineus |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9840287/ https://www.ncbi.nlm.nih.gov/pubmed/36639657 http://dx.doi.org/10.1186/s40694-023-00149-y |
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