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

Abstract

The oomycete Phytophthora infestans, a filamentous plant pathogen belonging to the kingdom Stramenopila, is the causal agent of potato late blight resulting in annual crop losses amounting to 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 immune responses. Currently, little is known regarding the intracellular trafficking of effectors enroute to secretion. In this study, we developed a robust density gradient ultracentrifugation method to isolate intracellular vesicles from P. infestans and to separate diverse vesicle populations based on buoyancy for the purpose of identifying vesicle-associated proteins by mass spectrometry. Gene Ontology Enrichment Analysis of proteins identified in buoyant fractions revealed enrichment for membrane-associated proteins and proteins involved in vesicle trafficking. Buoyant fractions were also enriched in RXLR class effectors, carbohydrate-active enzymes, and secretory proteins possessing N-terminal signal peptides, all representing potential vesicle cargo. In addition, previously identified P. infestans extracellular vesicle markers were also present. Unravelling how effector proteins are trafficked for secretion during infection 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.