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Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae

2007; Academic Press; Volume: 44; Issue: 10 Linguagem: Inglês

10.1016/j.fgb.2007.05.001

1096-0937

Melania Figueroa, Sara L. Tucker, Natalia Galadima, Yan Meng, Gayatri Patel, Lei Li, Nicole Donofrio, Anna Floyd, Shelly J. Nolin, Doug Brown, M. Alejandra Mandel, Thomas K. Mitchell, Jin‐Rong Xu, Ralph A. Dean, Mark Farman, Marc J. Orbach,

Plant tissue culture and regeneration

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 CaCl2/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.