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Page 12 Cai et al. Extracell Vesicles Circ Nucleic Acids 2023;4:262-82 https://dx.doi.org/10.20517/evcna.2023.10

phytopathogenic fungus Ustilago maydis, five effectors and two membrane proteins form a stable protein
[158]
complex anchored in EV-like structures that may be derived from MVBs . In plant pathosystems, EVs
[51]
h a v e b e e n i s o l a t e d f r o m s e v e r a l f i l a m e n t o u s f u n g i , i n c l u d i n g Fusarium oxysporum ,
[50]
[159]
Fusarium graminearum , wheat pathogen Zymoseptoria tritici , corn smut fungus Ustilago maydis ,
[49]
anthracnose pathogen Colletotrichum higginsianum , post-harvest rot pathogen Penicillium digitatum ,
[47]
[160]
and powdery mildew fungus Blumeria hordei . These EVs were purified from culture supernatants or
[99]
fungi grown on plant tissue and contained proteins, nucleic acids, lipids, metabolites, and polysaccharides.
Proteomic analysis of F. oxysporum EVs detected functional proteins involved in secondary toxin
metabolism, cell wall degradation, and protein degradation . Furthermore, injection of F. oxysporum EVs
[51]
into the leaves of cotton or N. benthamiana plants induced a phytotoxic response in plants, suggesting that
fungal EVs likely play an important role in infection . In addition, critical virulence-related proteins and
[51]
effectors have been identified in fungal EVs. For instance, proteomic analysis of F. graminearum EVs
identified protein effectors, some of which do not have predicted secretion signal peptides . This suggests
[49]
that EVs are an unconventional secretion pathway for fungal effectors. EVs isolated from the fungus Z.
tritici also contain a number of putative virulence-associated proteins that may play a role in the infection of
wheat . Current phytopathogen effector analysis focuses exclusively on predicted secreted proteins with
[50]
signal peptides. Proteomics analysis of pathogen EVs may help identify a novel class of previously
unidentified pathogen effectors.
Studies of RNA in EVs from plant pathogenic fungi are limited. EVs from U. maydis contain mRNAs
encoding metabolic enzymes, known effectors, and virulence protein s[159] . EVs isolated from the infection
site of Blumeria hordei on barley plants were enriched in B. hordei-derived milRNAs, which have a potential
[99]
role in host gene silencing . As mentioned above, the fungal pathogens B. cinerea, V. dahliae, and the
oomycete pathogen H. arabidopsidis have been shown to deliver sRNAs into plant host cells [88,91,98] . The
mechanism of the delivery of these sRNAs is likely via fungal EVs [Figure 1], and many experiments have
been initiated to test this hypothesis. Aside from RNA and proteins, one study found that P. digitatum uses
[47]
EVs to transport phytotoxic metabolites into citrus fruit cells during infection . This is one of the first
characterized reports of cross-kingdom metabolite trafficking in a host-pathogen interaction, demonstrating
the functional diversity of EVs and their potential to traffic a variety of biological cargoes that have yet to be
discovered.

POTENTIAL APPLICATIONS OF PLANT EVS
The significant role of plant EVs in transporting genetic cargo in plant-pathogen interactions has motivated
efforts to develop nanocarrier mimics of these natural EVs for RNAi-based pathogen control strategies in
agriculture and as drug delivery agents in the medical field [Figure 3]. These organic nanocarriers can
consist of various materials, including lipid-based artificial nanovesicles (AVs) and plant-derived
nanovesicles (PDNVs). AVs have recently been used for dsRNA delivery in spray-induced gene silencing
(SIGS) to improve the stability of dsRNA in the environment [39,161-163] . SIGS is a powerful and eco-friendly
method for crop protection. Many pathogens can efficiently take up RNAs from the environment [88,163] ,
which makes SIGS possible. Topical application of exogenous RNA targeting pest/pathogen virulence genes
results in gene silencing and subsequent disease inhibition. AVs formulated with 1,2-dioleoyl 3-
trimethylammonium-propane (DOTAP) + polyethylene glycol (PEG), 1,2-dioleyloxy-3-
dimethylaminopropane (DODMA), or DOTAP alone were found to provide strong and prolonged
protection of dsRNA against B. cinerea on pre- and post-harvest materials . Encapsulation of dsRNA in
[163]
AVs extended RNAi-based protection for up to 10 days on fruit and 21 days on grape leaves . Similarly,
[163]
other reports have investigated the use of liposomes and lipid transfection reagents for SIGS against insect
pests [164,165] .