CRISPR-Cas9-Mediated Gene Editing of the Plant Pathogenic Oomycete Phytophthora palmivora
- 20 Downloads
Phytophthora palmivora is a destructive oomycete pathogen that infects a large number of plant species. An effective functional genomic tool is required to understand the molecular mechanisms underlying its pathogenicity and broad host range. Gene knockout using homologous recombination has been unviable in oomycetes due to their ploidy (diploidy to polyploidy) and low rates of homologous recombination. CRISPR-mediated genome editing offers a boon for oomycete functional genomics allowing accelerated dissection of gene functions. Agrobacterium-mediated transformation (AMT) represents an advantageous and efficient gene delivery system over other transformation methods. In this chapter, we describe a detailed protocol for generating P. palmivora mutants via AMT to deliver single-guide RNA and Cas9, the components required for gene editing. This protocol can be used directly for CRISPR-Cas9-mediated gene editing of P. palmivora and modified for other culturable oomycete species.
Key wordsPhytophthora palmivora Gene editing CRISPR-Cas9 Oomycete transformation Agrobacterium-mediated transformation
- 1.Erwin DC, Ribeiro OK (1996) Phytophthora diseases worldwide. APS Press, St. Paul, MNGoogle Scholar
- 2.Ali SS, Shao J, Lary DJ, Kronmiller B, Shen D, Strem MD, Amoako-Attah I, Akrofi AY, Begoude BA, Ten Hoopen GM, Coulibaly K, Kebe BI, Melnick RL, Guiltinan MJ, Tyler BM, Meinhardt LW, Bailey BA (2017) Phytophthora megakarya and P. palmivora, closely related causal agents of cacao black pod rot, underwent increases in genome sizes and gene numbers by different mechanisms. Genome Biol Evol 9(3):536–557CrossRefGoogle Scholar
- 10.Im H, Sambrook J, Russell DW (2011) The Inoue method for preparation and transformation of competent E. coli: “ultra competent” cells. Bio-Protocol 1(20):e143Google Scholar