P-302 - A modular cloning system for fungal transformation

Molecular genetics is a powerful tool to examine the function of genes. Many fungi, including plant pathogens, are amenable to genetic modification by both, ectopic and in-locus transformation. Genetic modifications including gene deletion, complementation and generation of in-frame fusion proteins are widely used to study fungal cellular processes. Magnaporthe oryzae is an ascomycete fungus causing blast disease on cereals and wild grasses and serves as a model organism for plant-pathogen interactions. While M. oryzae has been used as a genetic model system for almost 40 years, a streamlined cloning system had been missing. Here, we present a homologous recombination based fungal transformation toolbox which utilizes the modular Golden Gate cloning system for its efficient assembly. Our system facilitates standardized processes for gene deletion, safe-harbor integration as well as in-locus fluorescence-tagging of proteins, thereby limiting unwanted side-effects which might occur during ectopic integration. Our vector suite is applicable for both, Agrobacterium tumefaciens and Polyethyleneglycol mediated transformation and contains various resistance and fluorescence cassettes, providing flexibility and enabling streamlined screening of transformants. In addition, the system contains commonly used fungal expression cassettes (promoter, terminator, resistance markers), making it generally applicable for standardized genetic studies of fungal plant pathogens.