Advertisement

CRISPR-Cas9-Mediated Gene Editing of the Plant Pathogenic Oomycete Phytophthora palmivora

  • Miaoying TianEmail author
  • Natasha Navet
  • Dongliang Wu
Protocol
  • 20 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

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 words

Phytophthora palmivora Gene editing CRISPR-Cas9 Oomycete transformation Agrobacterium-mediated transformation 

References

  1. 1.
    Erwin DC, Ribeiro OK (1996) Phytophthora diseases worldwide. APS Press, St. Paul, MNGoogle Scholar
  2. 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
  3. 3.
    Sharma N (1990) Aseptic germination of oospores of Phytophthora palmivora. Can J Bot 68:2548–2552CrossRefGoogle Scholar
  4. 4.
    Pan C, Ye L, Qin L, Liu X, He Y, Wang J, Chen L, Lu G (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6:24765CrossRefGoogle Scholar
  5. 5.
    Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu YG, Zhao K (2016) Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One 11(4):e0154027CrossRefGoogle Scholar
  6. 6.
    Fang Y, Tyler BM (2016) Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol Plant Pathol 17(1):127–139CrossRefGoogle Scholar
  7. 7.
    Fang Y, Cui L, Gu B, Arredondo F, Tyler BM (2017) Efficient genome editing in the oomycete phytophthora sojae using CRISPR/Cas9. Curr Protoc Microbiol 44:21A.1.1–21A.1.26CrossRefGoogle Scholar
  8. 8.
    Wu D, Navet N, Liu Y, Uchida J, Tian M (2016) Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora. BMC Microbiol 16(1):204CrossRefGoogle Scholar
  9. 9.
    Gumtow R, Wu D, Uchida J, Tian M (2018) A Phytophthora palmivora extracellular cystatin-like protease inhibitor targets papain to contribute to virulence on papaya. Mol Plant Microbe Interact 31(3):363–373CrossRefGoogle Scholar
  10. 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
  11. 11.
    Mersereau M, Pazour GJ, Das A (1990) Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90(1):149–151CrossRefGoogle Scholar
  12. 12.
    Peng D, Tarleton R (2015) EuPaGDT: a web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens. Microb Genom 1(4):e000033PubMedPubMedCentralGoogle Scholar
  13. 13.
    Reuter JS, Mathews DH (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11:129CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Plant and Environmental Protection SciencesUniversity of Hawaii at ManoaHonoluluUSA

Personalised recommendations