Purification of effector-target protein complexes via transient expression in Nicotiana benthamiana

Methods Mol Biol. 2011;712:181-94. doi: 10.1007/978-1-61737-998-7_15.

Abstract

Effectors of plant pathogens play important roles in not only pathogenesis but also plant immunity. Plant pathogens use these effectors to manipulate host cells for colonization, and their activities likely influence the evolution of plant immune responses. Analyses of genome sequences revealed that oomycete pathogens, such as Phytophthora spp., possess hundreds of RXLR effectors that are thought to be delivered into the host cells and hence function inside the cells by interacting with the host protein complexes. This article describes a co-immunoprecipitation protocol aimed at identifying putative target complexes of the effectors by transiently overexpressing the tagged effectors in planta. The identification of the eluted protein complexes was achieved by LC-MS/MS mass spectrometry and peptide spectrum matching.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics
  • Chromatography, Liquid / methods
  • Gene Transfer Techniques
  • Immunoprecipitation / methods*
  • Phytophthora / genetics*
  • Phytophthora / pathogenicity
  • Plant Immunity
  • Tandem Mass Spectrometry / methods
  • Tobacco / genetics*
  • Tobacco / metabolism
  • Tobacco / microbiology*