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Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita

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Abstract

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

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Data availability

The AaeSDI1 gene sequence employed in this study is available from the NCBI (National Center for Biotechnology Information) GenBank® repository via accession number KU984434. DNA sequence information of AaeSDI1 was obtained via web-based genome browser-mediated access to the in silico-annotated genome sequence of A. aegerita AAE-3 (http://www.thines-lab.senckenberg.de/agrocybe_genome), and the parental dikaryon of the monokaryons A. aegerita AAE-3-13 and A. aegerita AAE-3-32 (Herzog et al. 2016) which were used throughout this study. The genome sequence data of A. aegerita AAE-3 have also been deposited in the European Nucleotide Archive (ENA) under the BioProject accession number PRJEB21917.

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Acknowledgements

This work was funded by the Senckenberg Gesellschaft für Naturforschung and the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE) by the State of Hesse’s Ministry of Higher Education, Research and the Arts. Dr. Michael Bölker was supported by the DFG-funded SFB 987 ‘Microbial Diversity in Environmental Signal Response’. DNA sequence data were obtained from the genome sequence of Agrocybe aegerita AAE-3 (Gupta et al. 2018; http://www.thines-lab.senckenberg.de/agrocybe_genome), the parental dikaryon of the monokaryons A. aegerita AAE-3-13 and A. aegerita AAE-3-32, which were used throughout this study. We thank Dr. Björn Sandrock (Philipps-University Marburg, Germany) for fruitful exchange of ideas on the modes of plasmid-chromosomal integration of pSDI1E3 (carboxin resistance cassette-containing plasmid created in this study) in A. aegerita.

Funding

This study was funded by the Senckenberg Gesellschaft für Naturforschung and the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE) by the State of Hesse’s Ministry of Higher Education, Research and the Arts. Dr. Michael Bölker was supported by the DFG-funded SFB 987 ‘Microbial Diversity in Environmental Signal Response’.

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Authors and Affiliations

  1. Junior Research Group Genetics and Genomics of Fungi, Senckenberg Biodiversity and Climate Research Centre (SBiK-F), Senckenberganlage 25, 60325, Frankfurt am Main, Germany

    Robert Herzog, Irina Solovyeva & Florian Hennicke

  2. Institute of Ecology, Evolution and Diversity, Goethe-University Frankfurt, Max-von-Laue-Str. 13, 60438, Frankfurt am Main, Germany

    Robert Herzog & Florian Hennicke

  3. LOEWE Cluster of Integrative Fungal Research, Senckenberganlage 25, 60325, Frankfurt am Main, Germany

    Robert Herzog, Irina Solovyeva, Michael Bölker & Florian Hennicke

  4. Department of Environmental Biotechnology, TU Dresden, Markt 23, 02763, Zittau, Germany

    Robert Herzog

  5. Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, 35032, Marburg, Germany

    Michael Bölker

  6. Department of Biology, Microbiology, Utrecht University, Utrecht, The Netherlands

    Luis G. Lugones & Florian Hennicke

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  1. Robert Herzog
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  2. Irina Solovyeva
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  3. Michael Bölker
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  4. Luis G. Lugones
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Contributions

Conceived and designed the experiments: RH, IS, and FH. Performed the experiments: RH, IS, and FH. Analysed the data: RH, IS, LGL, and FH. Wrote the paper: RH, IS, MB, LGL, and FH.

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Correspondence to Florian Hennicke.

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Communicated by Reinhard Fischer.

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Herzog, R., Solovyeva, I., Bölker, M. et al. Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita. Mol Genet Genomics 294, 663–677 (2019). https://doi.org/10.1007/s00438-018-01528-6

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