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Page 101 Rutter et al. Extracell Vesicles Circ Nucleic Acids 2023;4:90-106 https://dx.doi.org/10.20517/evcna.2023.04

produced by an invading fungal phytopathogen (e.g., haustoria) are usually encased in callose and separated
[106]
from the rest of the plant apoplast by a collar around the haustoria neck . Such collars would limit the
diffusion of EVs from the fungal extra-haustorial space into the bulk plant apoplast, potentially stymying
[22]
attempts to isolate fungal EVs from plant apoplastic fluids . Until better methods are developed,
researchers will have to rely on growth media that mimic the environment of the plant and stimulate fungal
virulence. In this manner, it may be possible to examine fungal EVs that are more similar to those produced
during infection.

Another set of long-standing questions involves the production and secretion of fungal EVs. A few studies
have successfully identified genetic components that contribute to EV formation and secretion in yeasts.
Mutations in the SEC6 gene of C. neoformans, which encodes a subunit of the exocyst complex,
[107]
compromises EV secretion . In S. cerevisiae, mutations in several different genes encoding different
regulators of endomembrane trafficking have been shown to affect EV size and composition. These include
SEC4, which encodes a vesicle associated RabGTPase, Golgi Reasssembly Stacking Protein (GRASP; a
regulator of unconventional secretion), and several members of the ESCRT complex [86,108] . Lipid transporters
a l s o a f f e c t E V s i z e s i n C r y p t o c o c c u s n e o f o r m a n s a n d C. gattii [109,110] .
C higginsianum, Fov, F. graminearum U. maydis and Z. tritici are all genetically tractable organisms, so it is
feasible that orthologous candidates of EV biogenesis and secretion in yeasts could be evaluated in fungal
phytopathogens [111-115] .

When and where EVs are secreted from fungal phytopathogens are also important questions to be
answered. Unlike yeasts, filamentous fungal plant pathogens develop different cell types throughout an
infection, including germ tubes, surface hyphae, appressoria, penetration pegs and haustoria/infection
hyphae. EVs could be secreted from any of these cell types. EVs can be secreted from vegetative hyphae, so
it is possible that infection hyphae within plant tissues also secrete EVs. The question then is “which regions
of the hyphae secrete vesicles?” Hyphal tips are sites of active exocytosis, but secretory events can also occur
in regions distal to the tip . Based on the localization of fluorescent EV markers in C. higginsianum, EV
[116]
[59]
secretion may occur at the tips of infection hyphae, but this has yet to be verified .
Last but not least, functionality remains an elusive question in most EV studies. Researchers can predict the
roles and activities of EVs based on their cargo, but experimentally verifying a function is another matter.
While fungal phytopathogen EVs putatively promote virulence, only two studies have demonstrated a direct
cytotoxic effect of fungal EVs in leaves [24,52] . It will be interesting to see if EVs from other fungal
phytopathogens can also induce plant cell death or produce other predicted effects, such as altering host cell
metabolism or suppressing defense responses.

CONCLUSIONS
Phytopathogenic fungi pose a severe threat both ecologically and to global food security. Estimates suggest
that fungi destroy a third of all food crops annually, leading to massive human suffering and economic
loss . Understanding how fungi infect plants is an important step toward developing better systems of
[26]
disease management and engineering more robust crops. Emerging evidence suggests that EVs from fungal
plant pathogens are important mediators of virulence, capable of transporting protein effectors, toxic
secondary metabolites and RNA [52,55,57,59,60,62] . While research into fungal phytopathogen EVs is progressing
quickly, there is still much work to be done. So far, significant efforts have been made to characterize EVs
secreted by multiple pathogens. Standardized methods for fungal growth and EV isolation, as well as greater
inclusion of tests to validate EV cargo, will help clarify EV composition and lead to the development of
important markers for vesicle tracking and isolation. As researchers gain a better understanding of the