Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae.
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.
Betts
Melania F
MF
Department of Plant Sciences, Division of Plant Pathology and Microbiology, University of Arizona, Tucson, AZ 85721-0036, USA.
Tucker
Sara L
SL
Galadima
Natalia
N
Meng
Yan
Y
Patel
Gayatri
G
Li
Lei
L
Donofrio
Nicole
N
Floyd
Anna
A
Nolin
Shelly
S
Brown
Doug
D
Mandel
M Alejandra
MA
Mitchell
Thomas K
TK
Xu
Jin-Rong
JR
Dean
Ralph A
RA
Farman
Mark L
ML
Orbach
Marc J
MJ
D024509:3' Flanking Region; D024506:5' Flanking Region; D016960:Agrobacterium tumefaciens / Q000235:genetics; D020082:Magnaporthe / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010641:Phenotype; D011523:Protoplasts; D014170:Transformation, Genetic