title: Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae.
abstract: Towards the goal of disrupting all genes in the genome of Magnaporthe oryzae and identifying their function, a collection of >55,000 random insertion lines of M. oryzae strain 70-15 were generated. All strains were screened to identify genes involved in growth rate, conidiation, pigmentation, auxotrophy, and pathogenicity. Here, we provide a description of the high throughput transformation and analysis pipeline used to create our library. Transformed lines were generated either by CaCl(2)/PEG treatment of protoplasts with DNA or by Agrobacterium tumefaciens-mediated transformation (ATMT). We describe the optimization of both approaches and compare their efficiency. ATMT was found to be a more reproducible method, resulting in predominantly single copy insertions, and its efficiency was high with up to 0.3% of conidia being transformed. The phenotypic data is accessible via a public database called MGOS and all strains are publicly available. This represents the most comprehensive insertional mutagenesis analysis of a fungal pathogen.
authors.affiliation: Department of Plant Sciences, Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ 85721-0036, USA.
authors.forename: Melania F
authors.initials: MF
authors.lastname: Betts
authors.forename: Sara L
authors.initials: SL
authors.lastname: Tucker
authors.forename: Natalia
authors.initials: N
authors.lastname: Galadima
authors.forename: Yan
authors.initials: Y
authors.lastname: Meng
authors.forename: Gayatri
authors.initials: G
authors.lastname: Patel
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Nicole
authors.initials: N
authors.lastname: Donofrio
authors.forename: Anna
authors.initials: A
authors.lastname: Floyd
authors.forename: Shelly
authors.initials: S
authors.lastname: Nolin
authors.forename: Doug
authors.initials: D
authors.lastname: Brown
authors.forename: M Alejandra
authors.initials: MA
authors.lastname: Mandel
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Mitchell
authors.forename: Jin-Rong
authors.initials: JR
authors.lastname: Xu
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Dean
authors.forename: Mark L
authors.initials: ML
authors.lastname: Farman
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Orbach
mesh_terms: D024509:3' Flanking Region; D024506:5' Flanking Region; D016960:Agrobacterium tumefaciens / Q000235:genetics; D020082:Magnaporthe / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010641:Phenotype; D011523:Protoplasts; D014170:Transformation, Genetic