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United States Department of Agriculture

Agricultural Research Service

Title: Transformation of Colletotrichum Trifolii Protoplasts by Electroporation and Peg Mediated DNA Uptake

Authors
item Oneill, Nichole
item Brooker, Nancy - PITTSBURG STATE-KANSAS
item Lydon, John
item Saunders, James

Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 17, 1997
Publication Date: N/A

Interpretive Summary: Electroporation is a process of moving DNA into cells through the use of a high voltage electrical shock. Electroporation has been extensively used in animal and plant cells for genetic transformation efficiency of electroporation to an older technique which uses polyethylene glycol (PEG) to move DNA into fungal cells. A pathogenic fungus from the genus Colletotrichum that causes anthracnose, a severe disease in many crop plants including beans, corn, sorghum and alfalfa, was used as the model in this study. Using various types of cells from this fungus we were able to develop an electroporation transformation system which was very effective at transferring antibiotic resistance to the fungal cells. These transformed cells were stable for several generations and can be used as a tool for investigating mechanisms of pathogenicity of anthracnose and other fungal diseases. This will enable scientists to distinguish between similar races of the pathogen during the complicated host/pathogen interactions.

Technical Abstract: Comparative transformation studies of the fungal isolate Colleotrichum trifolii, the causal agent of alfalfa anthracnose, were performed using either electrotransformation or polyethylene glycol (PEG) mediated DNA uptake. Transformants derived from protoplasts were obtained using a fungal expression vector that contained a bacterial gene encoding hygromycin B phosphotransferase. Transformed fungal isolates were evaluate for changes in virulence, race specificity, mitotic stability, and hygromycin B phosphotransferase activity and gene integration patterns. Restriction enzyme digests of the putative transformants provided evidence for site-directed, partial site directed, and random integration events in the fungal genome. Transformation by either PEG or electroporation resulted in the isolation of mitotically stable transformants which varied in cultural characteristics and virulence but retained race specificity of the wild-type. The frequency of transformation with either electroporation or PEG mediated DNA uptake resulted in similar numbers of stable transformants.

Last Modified: 4/17/2014
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