Location: Toxicology & Mycotoxin ResearchTitle: Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs.
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/7/2016
Publication Date: 6/12/2017
Citation: Gold, S.E., Glenn, A.E., Paz, Z. 2017. Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs. Journal of Visualized Experiments. 124:e55239. doi:10.3791/55239.
Interpretive Summary: Our research required that we delete specific genes in target fungi such as Fusarium verticillioides. Gene deletion mutants generated through such procedures is the gold standard for gene function studies. We report here a new procedure referred to the OSCAR method for the rapid and specific generation of deleted genes. Having successfully performed this procedure we substantiated it with modern molecular based methods to confirm the deletions of specific genes in the mutants. This now will allow us to perform experiments used to determine specific actions of genes relative to mycotoxin synthesis.
Technical Abstract: Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene’s function in the living organism. In this protocol we describe the OSCAR method of precise and rapid deletion plasmid construction. OSCAR relies on the Gateway cloning system in which a single BP clonase reaction is carried out containing the purified PCR-amplified 5’ and 3’ flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. We then describe the use of Agrobacterium tumefaciens to mediate introduction of the deletion vector into fungal spores (referred to as ATMT). Finally, we describe PCR assays to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. We have successfully used this approach for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides.