Abstract
Magnaporthe grisea is the causal agent of rice blast disease and represents a model organism for the study of fungal plant-pathogen interactions. Pathogenicity is a complex phenotype, which is carefully orchestrated by the fungus and begins with recognition and infection of the host plant, followed by growth within the plant cells, and finally dissemination to the next host and continuation of the fungal life cycle. Certain genes must condition the ability of a pathogenic fungus to infect and cause disease symptoms. To learn more about the infection process and the genes that are involved in the complex interplay between M. grisea and rice, we used an insertional mutagenesis approach to attempt to randomly disrupt all genes in the fungal genome. Two transformation approaches were used to build a library of insertion strains in M. grisea. Polyethylene glycol/CaCl2-mediated protoplast transformation was the initial method we used and resulted in the generation of just more than 17,000 insertion strain lines. Later Agrobacterium tumefaciens-mediated transformation was adopted and the final number of insertional mutant strains of M. grisea strain 70-15 generated was more than 57,000. Here, we describe the methods used for A. tumefaciens-mediated transformation of M. grisea and the optimized protocols we have developed to enable high-throughput fungal transformation. Further details of this optimization can be found elsewhere.
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Acknowledgments
This project was funded by the National Science Foundation Plant Genome Program, award DBI no. 0115642. We thank Dr. Jin-Rong Xu from Purdue University for the AGL1 strain and Dr. Yong-Hwan Lee from Seoul National University for discussions about the in vitro appressorium assay. This project was conducted in collaboration with Dr. Mark Farman of the University of Kentucky, whose laboratory produced half of the insertion mutant library and Dr. Ralph Dean, of North Carolina State University.
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Tucker, S.L., Orbach, M.J. (2007). Agrobacterium-Mediated Transformation to Create an Insertion Library in Magnaporthe grisea. In: Ronald, P.C. (eds) Plant-Pathogen Interactions. Methods in Molecular Biology, vol 354. Humana Press. https://doi.org/10.1385/1-59259-966-4:57
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DOI: https://doi.org/10.1385/1-59259-966-4:57
Publisher Name: Humana Press
Print ISBN: 978-1-58829-448-7
Online ISBN: 978-1-59259-966-0
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