Journal
FEMS YEAST RESEARCH
Volume 5, Issue 6-7, Pages 653-662Publisher
OXFORD UNIV PRESS
DOI: 10.1016/j.femsyr.2004.12.008
Keywords
Fusarium graminearum; Gibberella zeae; homologous recombination; gene disruption
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We cloned a polyketide synthase gene (pks12) from Fusarium graminearum, a devastating fungal pathogen of cereals. Transformation-mediated gene disruption led to an easily detectable albino phenotype of the disruptants. We used the disruption of the pks12 gene as a visible marker for transformation-mediated homologous recombination and optimized the transformation procedure to achieve a high rate of homologous recombination. In combination with the published genomic sequence data and the generation of expressed sequence tags (ESTs) for F graminearum, this is a useful tool to investigate this important plant pathogen on a molecular level. Optimized transformation of F graminearum resulted in at least 93% homologous recombination events when the homologous genomic DNA fragment in the vector had a size of approximately 800 bp and was linearized in the middle. Using a genomic sequence of approximately 500 bp in the transformation vector, 70% of the transformants still exhibited homologous recombination. On the Contrary, no more than 10% homologous recombination events were observed when less than 400 bp DNA fragments were used. We co-transformed F. graminearum with two different vectors. One vector harboured a DNA insert homologous to the pks12 gene, while the other vector consisted of the same vector backbone carrying the selection marker specific for F graminearum. About 70% of the transformants had a disrupted pks12 gene, and all of these showed an integration of the second vector into the pks disruption vector. Therefore, the time-consuming construction of a single transformation vector can be avoided; furthermore, it is now easily feasible to express a gene construct at a defined and mutated genomic, site. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
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