Isolation and proteomic analysis of intracellular vesicles from the potato late blight pathogen Phytophthora infestans

Abstract

Abstract The oomycete Phytophthora infestans is a filamentous plant pathogen and the causal agent of potato late blight resulting in annual crop losses of billions of dollars worldwide. Key to the success of this pathogen are the effector proteins it secretes during infection, whose functions include breaching the plant cell wall and suppression/evasion of plant immunity. Currently, little is known regarding the intracellular trafficking of effectors enroute to secretion. Here, we developed a robust density gradient ultracentrifugation method to isolate and separate diverse populations of intracellular vesicles from P. infestans based on buoyancy for the purpose of identifying vesicle-associated proteins by DIA-mass spectrometry. Gene Ontology Enrichment Analysis of buoyant fractions revealed enrichment for proteins associated with membranes and vesicle trafficking. Buoyant fractions were also enriched in RXLR effectors, carbohydrate-active enzymes, and signal peptide containing secretory proteins, all representing potential vesicle cargo. Recently identified P. infestans extracellular vesicle markers were also present. Unravelling how effector proteins are trafficked for secretion is a critical step in developing robust strategies for combating potato late blight disease; the proteomics dataset and method presented here are valuable resources from which potential biomarkers for P. infestans vesicles can be identified for future studies towards this end.