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Rutter et al. Extracell Vesicles Circ Nucleic Acids 2023;4:90-106 https://dx.doi.org/10.20517/evcna.2023.04 Page 96

can be inaccurate owing to human error and shrinkage of particles that occurs while preparing samples.
NTA struggles to measure particles smaller than 100 nm, and while DLS can detect particles over a much
wider range (10 nm to 1 µm), it has less resolution than NTA.

Protein biomarkers are a common tool used to verify the presence of EVs in a pellet. For example, small
(< 200 nm in diameter) mammalian EVs are enriched for specific proteins, including tetraspanins such as
[70]
CD9 and CD81, which play a role in membrane organization , and members of the endosomal sorting
complex required for transport (ESCRT), such as Tumor Susceptibility Gene 101 (TSG101) and Alix, which
are required for the budding of intralumenal vesicles into the interior of multivesicular bodies [71,72] .
Unfortunately, there are currently no standard biomarkers for fungal EVs. While fungi encode proteins
with a very similar structure to mammalian tetraspanins (i.e., with four transmembrane domains and a
pattern of conserved cysteines in one of two extracellular loops), they do not possess true orthologs, and
[73]
fungal “tetraspanins” have not yet been detected in any available fungal EV proteome . Fungi do possess
orthologs to ESCRT proteins, but these proteins are either not detected or present at very low levels in
fungal EV proteomes .
[52]
Work on Candida albicans identified 22 potential protein markers enriched in EVs compared to the whole
cell lysate. The list includes three members of the Sur7 family . Members of the Sur7 family possess four
[74]
transmembrane regions, similar to tetraspanins, and, in S. cerevisae, function as members of a large complex
associated with endocytosis . Sur7 family proteins have since been detected in several fungal EV
[75]
proteomes, including those of Cryptococcus, Z. tritici and C. higginsianum [56,59] .

Our research found that Sur7 family proteins were not abundantly present in C. higginsianum EVs and
instead chose to focus on the v-SNARE ChSnc1, the t-SNARE ChSso2 and the 14-3-3 protein ChBmh1 .
[59]
Members of the Snc1, Sso1/2, and Sec9 SNARE complex function in vesicle docking and fusion at the
plasma membrane . Orthologous proteins have been linked to the unconventional secretion of effectors in
[76]
the phytopathogens Magnoportha oryzae and V. dahliae [77,78] , as well as phytotoxin secretion in
F. graminearum . As a 14-3-3 protein, ChBmh1 facilitates protein-protein interactions. Orthologs of this
[79]
protein have been detected in EVs from other phytopathogens, including Fov and Z. tritici [52,56] . 14-3-3
[22]
proteins and SNARES are also common components of plant EV proteomes .

We could generate transgenic strains of fungi expressing fluorescently labeled versions of each protein. We
further demonstrated that the fusion proteins were protected from protease degradation inside membrane
vesicles . ChSnc1 and ChSso2 were also enriched in EVs compared to cell lysate, suggesting they may make
[59]
excellent EV markers .
[59]
Whether a universal marker for fungal EVs can be developed remains to be seen. Variability in EV protein
cargo undoubtedly arises with different species of fungi, growth conditions and methods of isolation. More
standardization of methods may be required before a universal fungal EV marker can be established.

PROTEIN CONTENT
Fungal EVs are associated with a diverse range of proteins, including enzymes involved in metabolism,
oxidation/reduction, cell wall synthesis, signaling, translation and transport [80,81] . Many of these proteins are
[81]
sequestered from the cytoplasm, but a high proportion are associated with the plasma-membrane .
Additionally, because EV secretion represents a form of unconventional secretion, a lower percentage of EV
proteins possess signal peptides.