Journal
FUNGAL GENETICS AND BIOLOGY
Volume 44, Issue 10, Pages 1035-1049Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.fgb.2007.05.001
Keywords
Magnaporthe oryzae; Insertional mutagenesis; Agrobacterium tumefaciens-mediated transformation; CaCl2/PEG-mediated transformation; pathogenicity
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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. (C) 2007 Elsevier Inc. All rights reserved.
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