The development of a transformation system for the dimorphic plant pathogen Holleya sinecauda based on Ashbya gossypii DNA elements.
We have developed a transformation system for the dimorphic plant pathogenic fungus Holleya sinecauda based on an electroporation protocol used for the closely related filamentous fungus Ashbya gossypii. DNA-mediated transformation of the dominant selection marker kanMX generated H. sinecauda transformants that were resistant to the antibiotic drug G418/geneticin. Freely replicating plasmids could be established in H. sinecauda using an A. gossypii autonomously replicating sequence (ARS) element, whereas Saccharomyces cerevisiae ARS elements, which are functional in A. gossypii, were not functional in H. sinecauda. In addition, centromeric DNA of A. gossypii stabilized the maintenance of plasmids in H. sinecauda under non-selective conditions. We isolated a fragment of the HsLEU2 gene and used this locus for targeted integration of kanMX3, consisting of the kanMX gene flanked by direct repeats. This allowed the construction of a Hsleu2 strain which became G418 sensitive after direct repeat-induced marker excision. The Hsleu2 strain can be complemented by the ScLEU2 gene. Finally, we constructed high- and low-copy shuttle vectors for H. sinecauda.
Schade
D
D
Junior Research Group: Growth-control of Fungal Pathogens, Hans-Knöll Institut für Naturstoff-Forschung, Jena, Germany.
Walther
A
A
Wendland
J
J
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D018274:Electroporation; D005819:Genetic Markers; D016681:Genome, Fungal; D005839:Gentamicins; D007613:Kanamycin Resistance; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010944:Plants / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D004718:Saccharomycetales / Q000235:genetics*; D012727:Sex Characteristics; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005819:Genetic Markers; D005839:Gentamicins; C010680:antibiotic G 418