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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 100

transcripts encoding mainly metabolic enzymes that are enriched in EVs and highly expressed during the
[57]
early stages of infection . The presence of these transcripts led the authors to suggest that fungal EVs
[57]
might function to reprogram plant host metabolism .
While Kwon et al. remains the only study to analyze EV RNA from an axenically grown culture, a recent
pre-print study by Kusch et al. attempted to isolate fungal RNAs from Blumeria hordei-infected barley
plants [57,105] . The study isolated crude EV pellets from the apoplastic wash of infected and non-infected
plants. EV pellets from infected plants were enriched for fungal milRNAs that were predicted to target
barley genes more often than the endogenous fungal genes . Predicted targets of the milRNAs were
[105]
mainly protein kinases, which suggests that fungal EVs may contain milRNAs that have a role in disrupting
[105]
plant immune signaling .

Both studies suggest that coding and non-coding RNAs are packaged into the EVs of phytopathogenic
fungi. Kwon et al. treated vesicles with RNase prior to RNA isolation and showed that significant
[57]
degradation of the RNA occurred only in the presence of both RNase and a detergent to disrupt vesicles .
This suggests that the mRNAs detected are present in the EV lumen. However, extracellular RNAs can also
be associated with protein complexes and lipoproteins, which would be unaffected by the presence of
RNase. The study compared RNA libraries from mock- and RNase-treated EVs but did not include RNA
from detergent- and RNase-treated EVs. It is therefore difficult to rule out the presence of non-vesicular
protein-RNA complexes. Kusch et al. only analyzed crude vesicle pellets without any kind of RNase
treatment. So, it is difficult to determine which detected RNAs are packaged in the EV lumen and which
[105]
may be non-vesicular RNAs merely associated with the EV pellet .
QUESTIONS FOR THE FUTURE
The study of EVs in fungal phytopathogens is a nascent field and thus fraught with unanswered questions.
Much of the work so far has focused on developing methods to isolate EVs and characterize EV content.
Undoubtedly, these methods will continue to be refined. Standardization of procedures and, if possible,
growth conditions for species of fungi will improve comparisons among studies.

There is a growing appreciation of the heterogeneity of fungal EVs. Evidence from A. infectoria suggests
there are two separate populations of differently sized EVs , while EVs from C. higginsianum and
[60]
F. graminearum are separated into two distinct populations on the basis of buoyant density [24,59] . These
separate populations suggest there may be multiple distinct pathways for EV biogenesis, but this has not yet
been shown. Most studies analyze the EV pellet in toto, meaning current proteomes and RNA libraries
reflect a mixture of different vesicle populations and co-pelleting protein complexes. Greater care should be
taken to purify vesicles using methods such as SEC and iodixanol density-gradients and to verify the
packaging of proteins and RNAs inside EVs; this means treating EVs with protease or protease followed by
RNase to confirm EV protein or RNA cargo, respectively.

Fungal EVs have been almost exclusively isolated from cultures grown in liquid or on solid medium. Apart
from the pre-print Kusch et al., no other study has attempted to isolate fungal EVs from an infected
organism . Because growth conditions affect the contents and composition of EVs, it is probable that EVs
[105]
from fungal phytopathogens infecting plants may contain different cargo compared to those isolated from
axenically grown fungi [62,64] . Such cargo may better reflect the virulence-promoting activities of fungal EVs
and could lead to the discovery of new effectors or toxic metabolites. However, detecting EV protein or
RNA signals in a sample mainly containing plant materials will prove challenging and may require the
development of immunoprecipitation protocols to separate fungal EVs. Additionally, infection structures