Homologous recombination as the main mechanism for DNA integration and cause of rearrangements in the filamentous ascomycete Ashbya gossypii

Abstract

A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resistance gene expressed from a strong A. gossypii promoter. Transformations with circular plasmids yielded slowly and irregularly growing geneticin-resistant mycelia in which 1% of nuclei contained plasmid sequences. Occasionally, fast growing sectors appeared which were shown to be initiated by homologous integration of the transforming DNA. Transformants obtained with plasmids linearized within the homology region immediately exhibited fast radial growth. In all 28 transformants analyzed plasmid DNA was integrated homologously. Such apparent lack of nonhomologous recombination has so far not been observed in filamentous ascomycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, mainly in the older mycelium: excision of whole plasmid copies and rearrangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lacking sequence repetitions were constructed and used for stable one-step gene replacement in A. gossypii.