Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14‐3‐3 protein (Bmh1) as potential EV markers and generated transgenic strains expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.

BackgroundCell biology approach using membrane protein markers tagged with fluorescent proteins highlights the dynamic behaviour of plant cell membranes, not only in the standard but also in changing environmental conditions. In the past, this strategy has been extensively developed in plant models such as Arabidopsis.ResultsHere, we generated a set of transgenic lines expressing membrane protein markers to extend this approach to rice, one of the most cultivated crop in the world and an emerging plant model. Lines expressing individually eight membrane protein markers including five aquaporins (OsPIP1;1, OsPIP2;4, OsPIP2;5, OsTIP1;1, OsTIP2;2) and three endosomal trafficking proteins (OsRab5a, OsGAP1, OsSCAMP1) were obtained. Importantly, we challenged in roots the aquaporin-expressing transgenic lines upon salt and osmotic stress and uncovered a highly dynamic behaviour of cell membrane.ConclusionWe have uncovered the relocalization and dynamics of plasma membrane aquaporins upon salt and osmotic stresses in rice. Importantly, our data support a model where relocalization of OsPIPs is concomitant with their high cycling dynamics.Electronic supplementary materialThe online version of this article (10.1186/s12284-018-0209-2) contains supplementary material, which is available to authorized users.