New Plasmids for Transformation Allowing Positive-Negative Selection and Efficient Cre- Mediated Marker Recycling.

In filamentous fungi such as , disruption of multiple genes of interest in the same strain (e.g., to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre system. The fusion genes contain commonly used C-terminal drug resistance markers, either (G418), (nourseothricin), or (hygromycin B). These resistance genes are fused to the sequence encoding virus thymidine kinase (HSVtk). Despite the presence of the 1 kb HSVtk gene (about ∼30% increase in total marker size), there is only a slight reduction in transformation efficiency on a molar basis. The fusion genes expressed under the pyruvate kinase (PKI) promoter also confer antibiotic resistance in , allowing straightforward construction of disruption plasmids. For removal of the flanked resistance cassettes, protoplasts of transformants are directly treated with purified Cre recombinase protein. Loss of the HSVtk containing cassette is selected by restoration of resistance to 5-fluoro-2-deoxyuridine (FdU). As a proof of principle, we demonstrated the efficiency of the HSVtk-based marker removal in by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the gene via integration of the -flanked HSVtk cassette into the promoter or the first intron, thereby generating transformants with a white mycelium phenotype. Using Cre recombinase and FdU, the selection marker was subsequently removed, and the resulting transformants regained red pigmentation despite the remaining site. We also found that it is possible to remove several unselected -flanked cassettes with a single Cre protein treatment, as long as one of them contains a negative selectable HSVtk cassette. The negative selection system can also be used to introduce allele swaps into strains without leaving marker sequences, by first disrupting the gene of interest and then complementing the deletion with genomic DNA containing a different allele.