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Open Access Highly Accessed Methodology article

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

Lisette Quaade Sørensen1, Erik Lysøe3, Jesper Erup Larsen1, Paiman Khorsand-Jamal1, Kristian Fog Nielsen2 and Rasmus John Normand Frandsen1*

Author Affiliations

1 Eukaryotic Molecular Cell Biology Group, Department of Systems Biology, The Technical University of Denmark, Søltofts Plads building 223, DK-2800 Kgs., Lyngby, Denmark

2 Metabolic Signaling and Regulation group, Department of Systems Biology, The Technical University of Denmark, Søltofts Plads building 221, DK-2800 Kgs., Lyngby, Denmark

3 Bioforsk–Norwegian Institute of Agricultural and Environmental Research, Høgskoleveien 7, Ås 1430, Norway

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BMC Molecular Biology 2014, 15:15  doi:10.1186/1471-2199-15-15

Published: 22 July 2014

Abstract

Background

The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed.

Results

The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A.

Conclusion

The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this.

Keywords:
Single step cloning; ATMT; Agrobacterium tumefaciens mediated transformation; Fusarium avenaceum; USER-Brick; Genome modification; Transformation; Fusaristatin; FaPKS6; Mycotoxin; LC-MS; MS-MS; Polyketide; Nonribosomal peptide