Zoospore-derived extracellular vesicles in the flagellated stramenopile Phytophthora parasitica

Zoospores are unicellular, wall-less and flagellated cells produced by a number of eukaryotic microorganisms. They allow microbial dispersion, enable the search and location for new sources of nutrients where they aggregate, initiate pathogen-host interactions and communication within microbiota. Little is known about their ability to release bioactive extracellular vesicles (EVs) supporting these adaptations. Here we used electron microscopy to establish that in the biflagellate zoospores of the heterokont and phytopathogenic species Phytophthora parasitica, EV biogenesis occurs from vesicles budding either at cell body plasma membrane or at the front flagellum, in particular from the tubular mastigonemes. Zoospore-conditioned water supernatant was fractionated and characterized by means of morphological, immunochemical, proteomic and lipidomic analyses. Three fractions enriched in cell body, flagella and EVs were obtained by differential ultracentrifugation at 3,000g, 31,000g and 100,000g, respectively, with the EV fraction consisting of small vesicles (100-150 nm). Using mass spectrometry, label-free proteomic analysis of the EV fraction (1,470 proteins) revealed a collection of proteins involved in lipid transport, vesicle membrane and cell wall organization, and tubular mastigoneme architecture, but not in virulence. Label-free quantitative lipidomic analysis (191 lipids) revealed enrichment of EVs in sphingolipids, particularly ceramides compared with the cell body. These findings provide a first hallmark of features characteristic of zoosporic EVs, with ceramides, mastigoneme proteins and the EV-related protein PPTG_13069 containing two tetraspanin MARVELous domains among molecular markers. They define the molecular and cellular principles in understanding zoosporic EV biogenesis within Stramenopiles and intercellular communication between self-aggregating zoospores, with host plant cells or microbiota.