A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani

Mikkel Rank Nielsen, Anna Karolina Rilana Holzwarth, Emmett Brew, Natalia Chrapkova, Samba Evelyne Kabemba Kaniki, Kenneth Kastaniegaard, Trine Sørensen, Klaus Ringsborg Westphal, Reinhard Wimmer, Teis Esben Sondergaard, Jens Laurids Sørensen

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Abstract

Background: Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation. Results: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin. Conclusion: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094. Graphic abstract: [Figure not available: see fulltext.]

Original languageEnglish
Article number25
JournalFungal Biology and Biotechnology
Volume6
Issue number1
ISSN2054-3085
DOIs
Publication statusPublished - 2019

Bibliographical note

© The Author(s) 2019.

Keywords

  • Agrobacterium tumefaciens-mediated transformation
  • Bostrycoidin
  • Fluorescence
  • Fusarium
  • Fusarubin
  • Heterologous expression
  • Polyketides
  • Secondary metabolites
  • Transformation

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