|Paz, Zahi -|
|Garcia-Pedrajas, Maria -|
|Andrews, David -|
|Montanez, Baeza -|
|Gold, Scott -|
Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 18, 2011
Publication Date: April 2, 2011
Citation: Paz, Z., Garcia-Pedrajas, M.D., Andrews, D.L., Klosterman, S.J., Montanez, B., Gold, S.E. 2011. One step construction of agrobacterium recombination-ready plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genetics and Biology. 48:677-684. Interpretive Summary: A tool and a procedure were developed and applied for the generation gene deletion constructs for the study of the genetics and biology of fungi. The method is referred to as One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR). The DNA constructs are transferred to the bacterium, Agrobacterium tumefaciens, which then mediates the transfer of the construct into the fungus. Replacement of the gene coding sequence with a selectable marker sequence occurs through the process of homologous recombination in the fungus. In this study, eight constructs for fungal gene deletion were prepared and one gene deletion was confirmed using this method in the soilborne fungal plant pathogen, Verticillium dahliae. The development of tools to efficiently analyze gene function in fungi is critical to our understanding of the genetic interaction between fungi and plants and will facilitate future analyses of the Verticillium dahliae-lettuce interaction.
Technical Abstract: Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Previously we reported a method called DelsGate for rapid preparation of deletion constructs for protoplast-mediated fungal transformation systems, which is based on Gateway® technology. However, over the past several years Agrobacterium tumefaciens-mediated transformation (ATMT) has become the preferred genetic transformation method for an increasing number of fungi. Therefore we developed a method for One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), to rapidly create deletion constructs for ATMT systems. The OSCAR methodology involves PCR amplification of the upstream and downstream flanks of the gene of interest, using gene specific primers each with a 5' extension containing one of four different attB recombination sites, modified from the Invitrogen MultiSite Gateway® system. Amplified gene flanks are then mixed with specifically designed marker and binary vectors and treated with BP clonase, generating the deletion construct in a single cloning step. The entire process of deletion construct preparation can be accomplished in just 2 days. Using OSCAR we generated eight targeted deletion constructs and used one of them to generate a deletion mutant in Verticillium dahliae by ATMT. In summary, OSCAR methodology combines PCR and Gateway® technology to rapidly and robustly generate precise deletion constructs for fungal ATMT and homologous gene replacement.