Abstract
In filamentous fungi such as Fusarium graminearum, 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-loxP system. The fusion genes contain commonly used C-terminal drug resistance markers, either nptII (G418), nat1 (nourseothricin), or hph (hygromycin B). These resistance genes are fused to the sequence encoding Herpes simplex 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 Trichoderma pyruvate kinase (PKI) promoter also confer antibiotic resistance in Escherichia coli, allowing straightforward construction of disruption plasmids. For removal of the loxP 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 Fusarium by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the FgPKS12 gene via integration of the loxP-flanked HSVtk-nptII 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 loxP site. We also found that it is possible to remove several unselected loxP-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 in situ with genomic DNA containing a different allele.
Highlights
Fusarium graminearum is a filamentous plant pathogenic fungus that causes Fusarium head blight in wheat, barley and other economically relevant small grain cereals, as well as ear rot in maize
We constructed a series of fungal transformation vectors consisting of an N-terminal Herpes simplex virus thymidine kinase (HSVtk) gene fused to one of three different resistance markers: neomycin phosphotransferase II (nptII) (G418), nat1 and hph
Custom polylinkers with unique sites were added on both sides of the resistance cassette to facilitate digestion/ligation cloning of flanking regions used for fungal gene disruption
Summary
Fusarium graminearum is a filamentous plant pathogenic fungus that causes Fusarium head blight in wheat, barley and other economically relevant small grain cereals, as well as ear rot in maize. Due to the ubiquitous presence and the broad host range of the pathogen, these diseases, which cause major yield losses and mycotoxin contamination of harvested grains, are a matter of worldwide concern. Besides trichothecenes, Fusarium is seemingly capable of producing many other secondary metabolites. Based on the genome sequence, Sieber et al (2014) predicted 67 gene clusters in F. graminearum with significant enrichment of putative secondary metabolism related enzymatic functions. Elucidation of the metabolites corresponding to these clusters might give further insight into the virulence mechanisms of the pathogen. Knockouts of individual secondary metabolite biosynthesis genes can have little impact on virulence (Gaffoor et al, 2005), and multiple gene deletions might be required to reveal redundancies. In F. graminearum, disruption of the genes necessary for production of the three different siderophores malonichrome (NPS1), ferricrocin (NPS2), and triacetylfusarinine (NPS6) led to drastically reduced virulence (Oide et al, 2014)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
