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

Producción científica: Article › revisión exhaustiva

70 Citas (SciVal)

Resumen

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.

Idioma original	English
Páginas (desde-hasta)	1035-1049
Número de páginas	15
Publicación	Fungal Genetics and Biology
Volumen	44
N.º	10
DOI	https://doi.org/10.1016/j.fgb.2007.05.001
Estado	Published - oct 2007

Nota bibliográfica

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.

Financiación

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.

Financiadores	Número del financiador
National Science Foundation (NSF)	0115642

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

@article{d7fe7660608c4388bb4dd5e16dcbc712,

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",

volume = "44",

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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, n.º 10, pp. 1035-1049. https://doi.org/10.1016/j.fgb.2007.05.001

TY - JOUR

T1 - Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae

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

Y1 - 2007/10

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.

KW - Agrobacterium tumefaciens-mediated transformation

KW - CaCl/PEG-mediated transformation

KW - Insertional mutagenesis

KW - Magnaporthe oryzae

KW - Pathogenicity

UR - https://www.scopus.com/pages/publications/34548200824

UR - https://www.scopus.com/pages/publications/34548200824#tab=citedBy

U2 - 10.1016/j.fgb.2007.05.001

DO - 10.1016/j.fgb.2007.05.001

M3 - Article

C2 - 17600737

AN - SCOPUS:34548200824

SN - 1087-1845

VL - 44

SP - 1035

EP - 1049

JO - Fungal Genetics and Biology

JF - Fungal Genetics and Biology

IS - 10

ER -

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

Resumen

Nota bibliográfica

Financiación

ASJC Scopus subject areas

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