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Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

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https://ir.library.oregonstate.edu/concern/articles/j6731549r

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  • Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9-induced DNA double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end-joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology-directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR-mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.
  • This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by John Wiley & Sons, Ltd and British Society for Plant Pathology. The published article can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291364-3703. Supporting information available online at: http://onlinelibrary.wiley.com/doi/10.1111/mpp.12318/suppinfo
  • Keywords: Avr4/6, RXLR effector, Phytophthora sojae, CRISPR/Cas9, gene replacement, oomycetes, genome editing
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  • Fang, Y., & Tyler, B. M. (2016). Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Molecular Plant Pathology, 17(1), 127-139. doi:10.1111/mpp.12318
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  • 17
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  • 1
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  • This work was supported in part by grant 2011-68004-30104 from the Agriculture and Food Research Initiative of the US Department of Agriculture (USDA) National Institute for Food and Agriculture.
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