The development of extracellular vesicle markers for the fungal phytopathogen Colletotrichum higginsianum

Abstract

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.

Extracellular Vesicles of the Plant Pathogen Botrytis cinerea

“…This corroborates previous observations in the human pathogen C. neoformans [98], including our parallel observation of single vesicles whose origin could be the budding of the plasma membrane. Lastly, BcEVs were also observed in hyphae interacting with plant cells, strengthening the biological meaningfulness of in vitro observations and corroborating observations in another phytopathogenic fungus [61].…”

“…Based on the low variation in abundance between 4 biological replicates, 673 of these proteins were selected as the best candidates of BcEV proteins. The predicted distribution and functions of these proteins are very similar to those of the proteins identified in EVs produced by mammalian cells and fungi [42,43], including human and plant pathogenic species [41,59,61]. First, these proteins are cytosolic or TM and GPIanchored proteins, two categories of EV proteins defined by Théry et al [18].…”

“…In medical mycology, such explorations have become important during the last decade [42,45], but they are still in their infancy in phytopathology. EVs have only been described in the cotton pathogen F. oxysporum [59]; the cereal pathogens F. graminearum, Zymoseptoria tritici, and Ustilago maydis [60,94]; and the polyphagous C. higginsianum [61].…”

“…In this study, 271 membrane and GPI-anchored proteins were identified in BcEVs, among which 16 proteins involved in fungal cell wall biogenesis. Such proteins, identified in other fungal EVs and proposed to play a role in FCW remodeling, could participate in the crossing of FCW by BcEVs [28,31,41,61,104,105]. Furthermore, BcEVs contain cytoskeleton proteins, and these proteins are postulated to play a role in the passage of EVs across cell walls [106].…”

“…On the fungal side, EVs were identified in Fusarium oxysporum and F. graminearum, carrying proteins and effectors involved in virulence [59,60]. EVs were also identified in Colletotrichum higginsianum, the causal agent of anthracnose disease [61].…”