Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker.
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 800bp and was linearized in the middle. Using a genomic sequence of approximately 500bp 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 400bp 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.
Maier
Frank J
FJ
Molekulare Phytopathologie und Genetik, Biozentrum Klein Flottbek, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany.
Malz
Sascha
S
Lösch
Anke P
AP
Lacour
Thierry
T
Schäfer
Wilhelm
W
D001483:Base Sequence; D003001:Cloning, Molecular; D005670:Fusarium / Q000201:enzymology / Q000235:genetics*; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010641:Phenotype; D048630:Polyketide Synthases / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D048630:Polyketide Synthases