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.
Bibliographical noteFunding Information:
We would like to thank Anath Das for providing Agrobacterium strains and plasmids. We would also like to thank Dan Ebbole, Cari Soderlund, Guo-Liang Wang and Vishal Pampanwar for helpful discussions. We also thank Yong-Hwan Lee for advice on the use of green glass mirror for appressorium analyses and for providing initial glass plates to us. The National Science Foundation Plant Genome Program, award DBI #0115642, funded this project.
- Agrobacterium tumefaciens-mediated transformation
- CaCl/PEG-mediated transformation
- Insertional mutagenesis
- Magnaporthe oryzae