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Fungal Genet Biol. 2007 Oct;44(10):1035-49. Epub 2007 May 18.

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

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1
Department of Plant Sciences, Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ 85721-0036, USA.

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.

PMID:
17600737
DOI:
10.1016/j.fgb.2007.05.001
[Indexed for MEDLINE]
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