Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola

We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene...

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Bibliographic Details
Published in:Current genetics 2005-12, Vol.48 (6), p.380-388
Main Authors: Flowers, J.L, Vaillancourt, L.J
Format: Article
Language:English
Subjects:
Agrobacterium
Agrobacterium tumefaciens
Agrobacterium tumefaciens - genetics
Colletotrichum - genetics
Colletotrichum graminicola
Deoxyribonucleic acid
DNA
Fungi - genetics
Gene Transfer Techniques
genetic transformation
Glomerella graminicola
green fluorescent protein
Green Fluorescent Proteins
Hygromycin B - metabolism
Hygromycin B - pharmacology
Microbiology
Mutagenesis, Insertional
Mutation
Plant Tumor-Inducing Plasmids - drug effects
Plant Tumor-Inducing Plasmids - metabolism
plasmid vectors
plasmids
Protoplasts - cytology
reporter genes
Spores, Bacterial - genetics
Ti-plasmid
Transformation, Bacterial
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Summary:We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation. Various experimental parameters were tested for their effects on transformation efficiencies. The parameters with the greatest influence were the A. tumefaciens strain used and the Ti-plasmid it carried, the ratio of bacterium to fungus during cocultivation, and the length of cocultivation. Southern analysis demonstrated that most transformants (80%) contained tandem integrations of plasmid sequences, and at least 36% had integrations at multiple sites in the genome. In a majority of cases (70%), the whole Ti-plasmid, and not just the T-DNA, had integrated as a series of tandem repeats. Tandem integrations, especially of the whole plasmid, make it difficult to rescue DNA from both flanks of the integrations with standard PCR-based approaches. Thus, ATMT may be unsuitable for insertional mutagenesis of C. graminicola without further modification.
ISSN:0172-8083
1432-0983
DOI:10.1007/s00294-005-0034-1