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

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

doi:10.1016/j.fgb.2007.05.001

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

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

Research output: Contribution to journal › Article › peer-review

66 Scopus citations

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 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.

Original language	English (US)
Pages (from-to)	1035-1049
Number of pages	15
Journal	Fungal Genetics and Biology
Volume	44
Issue number	10
DOIs	https://doi.org/10.1016/j.fgb.2007.05.001
State	Published - Oct 2007

Keywords

Agrobacterium tumefaciens-mediated transformation
CaCl/PEG-mediated transformation
Insertional mutagenesis
Magnaporthe oryzae
Pathogenicity

ASJC Scopus subject areas

Microbiology
Genetics

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10.1016/j.fgb.2007.05.001

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Betts, M. F., Tucker, S. L., Galadima, N., Meng, Y., Patel, G., Li, L., Donofrio, N., Floyd, A., Nolin, S., Brown, D., Mandel, M. A., Mitchell, T. K., Xu, J. R., Dean, R. A., Farman, M. L., & Orbach, M. J. (2007). Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae. Fungal Genetics and Biology, 44(10), 1035-1049. https://doi.org/10.1016/j.fgb.2007.05.001

Betts, MF, Tucker, SL, Galadima, N, Meng, Y, Patel, G, Li, L, Donofrio, N, Floyd, A, Nolin, S, Brown, D, Mandel, MA, Mitchell, TK, Xu, JR, Dean, RA, Farman, ML & Orbach, MJ 2007, 'Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae', Fungal Genetics and Biology, vol. 44, no. 10, pp. 1035-1049. https://doi.org/10.1016/j.fgb.2007.05.001

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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 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.",

keywords = "Agrobacterium tumefaciens-mediated transformation, CaCl/PEG-mediated transformation, Insertional mutagenesis, Magnaporthe oryzae, Pathogenicity",

author = "Betts, {Melania F.} and Tucker, {Sara L.} and Natalia Galadima and Yan Meng and Gayatri Patel and Lei Li and Nicole Donofrio and Anna Floyd and Shelly Nolin and Doug Brown and Mandel, {M. Alejandra} and Mitchell, {Thomas K.} and Xu, {Jin Rong} and Dean, {Ralph A.} and Farman, {Mark L.} and Orbach, {Marc J.}",

note = "Funding 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. ",

year = "2007",

month = oct,

doi = "10.1016/j.fgb.2007.05.001",

language = "English (US)",

volume = "44",

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journal = "Fungal Genetics and Biology",

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AU - Betts, Melania F.

AU - Tucker, Sara L.

AU - Galadima, Natalia

AU - Meng, Yan

AU - Patel, Gayatri

AU - Li, Lei

AU - Donofrio, Nicole

AU - Floyd, Anna

AU - Nolin, Shelly

AU - Brown, Doug

AU - Mandel, M. Alejandra

AU - Mitchell, Thomas K.

AU - Xu, Jin Rong

AU - Dean, Ralph A.

AU - Farman, Mark L.

AU - Orbach, Marc J.

N1 - Funding 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.

PY - 2007/10

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N2 - 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.

AB - 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.

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KW - Pathogenicity

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

Abstract

Keywords

ASJC Scopus subject areas

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