Rapid Deletion Plasmid Construction Methods for Protoplast and Agrobacterium-based Fungal Transformation Systems

Abstract

The increasing availability of genomic data and the sophistication of ­analytical methodology in fungi have increased the need for functional genomics tools in these organisms. Gene deletion is a critical tool for functional analysis. The targeted deletion of genes requires both a suitable method for the transfer of foreign DNA to fungal cells and the generation of deletion constructs. The deletion constructs should contain the regions flanking the gene of interest, while the ORF is replaced by a DNA fragment harboring a marker that allows selection of cells transformed with this foreign DNA. Deletion mutants are produced upon transformation by integration of this construct into the fungal genome by homologous recombination. Protoplasts have been widely used as starting material for genetic transformation in fungal species. However, a number of fungi have proven to be recalcitrant to protoplast-mediated transformation (PMT). Among the alternative methodologies developed for those species, Agrobacterium ­tumefaciens-mediated transformation (ATMT) has been particularly successful, becoming the preferred genetic transformation method for an increasing number of fungi. Here we describe two methods to rapidly generate plasmid-based gene deletion constructs, namely, DelsGate and OSCAR, which are compatible with PMT and ATMT, respectively. Both procedures are based on PCR of the target gene flanks and Gateway cloning technology, allowing generation of deletion constructs in a very simple and robust manner in as little as 2 days. Gateway vectors have been modified so that a single Gateway cloning step generates the deletion construct itself. The PCR and transformation steps these methodologies involve should be well suited for high-throughput approaches to gene deletion construction in fungal species in which either of the two major DNA transformation methods, PMT or ATMT, is used. We describe here the entire process, from the generation of the deletion constructs to the analysis of the fungal transformants for gene replacement confirmation, with the Basidiomycete fungus Ustilago maydis for DelsGate and PMT, and with the Ascomycete fungus Verticillium dahliae for OSCAR and ATMT.