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Page 97 Rutter et al. Extracell Vesicles Circ Nucleic Acids 2023;4:90-106 https://dx.doi.org/10.20517/evcna.2023.04

Historically, identifying fungal EV proteins has been a struggle, with most published proteomes containing
[81]
fewer than 100 proteins . This is likely due to the challenge of isolating sufficiently high numbers of fungal
EVs, as well as differences in methodology used in isolation and proteomic analysis. Despite these
challenges, research into fungal phytopathogen EVs has produced EV proteomes with larger numbers of
proteins, ranging from 420 to 710 proteins; the one exception being the EV proteome of A. infectoria, which
contains only twenty proteins [Table 1]. To date, there are published proteomes for A. infectoria, Fov,
F. graminearum, Z. tritici and C. higginsianum [52,55,56,59,60,62] .

When analyzed for gene ontology (GO) terms, fungal phytopathogen EV proteomes are enriched for
proteins involved in general metabolism, translation, the cytoskeleton, GTPase/ATPase binding/activity,
vesicle-mediated transport, cell redox homeostasis, protein folding and regulation of transcription, as well
as proteins associated with the membrane and cell wall [Figure 2]. The percentage of proteins with
predicted transmembrane regions varies with the species and the growth conditions and ranges from
~6%-35% [52,55,56,59,62] . The same is true for proteins with signal peptides, ranging from ~6%-30% [52,55,56,59,62] .

A common feature of EVs from fungal phytopathogens is the presence of virulence-related proteins,
especially those involved in the production of secondary metabolites (SMs). Proteins belonging to SM-
producing biosynthetic gene clusters have been identified in EVs isolated from every phytopathogen tested
so far, with the exception of Z. tritici. Such proteins are often enriched in vesicles compared to the whole
cell lysate and include polyketide synthases for the production of melanin in A. infectoria, fusarubin in Fov
and higginsianins in C. higginsianum [52,59,60,62] . Enzymes for the production of mycotoxins are also present in
Fov EVs [52,62] , while EVs from F. graminearum are associated with several SM-related proteins, including
those involved in the synthesis of aflatoxins . These metabolites not only contribute to the virulence of the
[55]
fungus on its host plant, but their presence in food can have damaging effects on livestock and human
health [82,83] .

It has been suggested that SM-related proteins may allow fungal EVs to function as “mini-factories” for the
production and delivery of toxins . Indeed, SMs have been identified in association with fungal
[55]
phytopathogen EVs. A purple pigment co-fractionated with Fov EVs presents an iodixanol density gradient.
This pigment possibly stimulates cell death in cotton leaves and, due to the presence of a fusarubin cluster-
[84]
esterase in Fov EVs, it was initially thought to be purpurfusarin or 8-O-methyl anhydrofusarubin .
[82]
However, the pigment’s UV-vis absorbance spectra better match a naphthoquinone . The same study also
[52]
detected a polyketide bikaverin, which has known antibiotic properties and may indicate that fungal EVs
play a role in microbial competition [52,85] . Still, it is unknown whether these metabolites are merely associated
with fungal EVs, packaged inside of them before their release or synthesized in vesicles after their release.

Proteases and cell wall modifying proteins are also present in fungal phytopathogen EVs and may
contribute to virulence. Proteases are some of the most abundant proteins in Fov EVs and have also been
detected in EVs from F. graminearum and C. higginsianum [52,55,59,62] . Proteins involved in cell wall
maintenance and modifications, such as chitinases, glucanases and a variety of synthases, are equally
abundant [52,55,59,60,62] . Together, these enzymes may promote penetration into host tissues or facilitate the
[62]
release of EVs past the fungal cell wall . The presence of cell wall-associated enzymes may also indicate
that fungal EVs play a role in the maintenance of the cell wall, as has been shown for Aspergillus fumigatus
and S. cerevisiae [66,86] .

Other virulence-related proteins include oxidoreductases, which could protect fungi from reactive oxygen
species produced early on during the plant immune response. Putative effectors have also been detected in