Immunoisolation of Plant Endosomal Vesicles to Explore Uptake of Pathogen Effector Proteins During Infection of Nicotiana benthamiana.

Endocytosis is an essential cellular process that uptakes substances into cells at the plasma membrane from the extracellular space and plays a major role in plant development and responses to environmental stimuli. Research has shown that plant membrane-resident proteins are endocytosed and transported into plant endosomes in response to pathogen-secreted elicitors. However, there is no conclusive experimental evidence demonstrating how secreted cytoplasmic effectors from oomycetes and fungi enter host cells during infection. The adapted protocol in this chapter describes endosome isolation using immunopurification with the aim to co-capture Phytophthora infestans RXLR effectors during infection. This protocol can be widely used in the isolation and purification of different subtypes of endosomal vesicles that uptake extracellular molecules during pathogen infection and in response to environmental stimuli.