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Cai et al. Extracell Vesicles Circ Nucleic Acids 2023;4:262-82 https://dx.doi.org/10.20517/evcna.2023.10 Page 3

Recently, plant EVs have been successfully isolated from the apoplastic fluids of leaves, roots, and seeds [29-34] .
Research has attempted to characterize different classes of plant EVs, protein markers, and lipid
composition, mirroring the work done to characterize mammalian EVs. Emerging evidence indicates that
plant EVs play an essential role in the cross-kingdom transport of plant sRNA into fungal cells to silence
virulence-related genes [29,35,36] . This phenomenon, termed "cross-kingdom RNAi", has been observed in
interactions between numerous plant and animal hosts with their associated microbes and parasites [35,37-41] .
In some host-pathogen/parasite interactions, RNA translocation has been linked to EV trafficking [29,42-46] .
The wide range of organisms capable of RNA exchange highlights the importance of research into the
mechanisms of RNA translocation and the potential role of EVs in these interactions. Recent research has
also shown that for plant-infecting microbes such as bacteria, fungi, and oomycetes, EVs could play a
prominent role in pathogenesis by delivering proteins, RNAs, and metabolites into their plant host cells [47-52]
[Figure 1]. In this review, we highlight and discuss the current state of research on plant-derived EVs and
cross-kingdom RNAi, with an emphasis on the role of EVs in cross-kingdom RNA trafficking. We also
discuss plant-interacting pathogen-derived EVs and their biological functions. Finally, we include the
potential applications of plant EVs for agricultural crop protection and advances in human medicine.

PLANT EXTRACELLULAR VESICLES
EVs provide protection for their biological cargo from the abundant nucleases and proteases within the
extracellular environment [53,54] . EV-mediated trafficking is one of the major pathways for RNA transport.
Mammalian EVs can be isolated from the extracellular environment of biological fluids or culture
supernatants [9,10] . Similarly, plant EVs from various sources can be isolated from extracellular apoplastic
washing fluid and cell culture medium [29,30] [Figure 2]. Plant EVs have been observed in numerous plant
[5]
[4]
species such as cotton , carrot , and rice and have been isolated from Arabidopsis thaliana [29-31,56] ,
[55]
sunflower [32,57] , olive [58,59] , and Nicotiana benthamiana [56,60] [Figure 2].
Heterogeneous EV groups have been isolated in plants, which may perform different functions [61,62] . In
Arabidopsis, five EV subtypes have been reported that are either isolated and/or characterized by different
protein markers, sizes and/or biogenesis origins, as described in Figure 2. These subtypes include
tetraspanin (TET)-positive EVs [29,56] , penetration 1 (PEN1)-positive EVs , exocyst-positive organelle
[63]
[45]
[59]
[64]
(EXPO)-derived EVs , autophagy-related EVs , and pollensomes . TET-positive EVs are the class of
plant EVs derived from MVBs and most similar to mammalian exosomes. In Arabidopsis, the secretion of
TET8-positive EVs increases after the infection of fungal pathogen Botrytis cinerea [29,56] . These plant
exosomes are enriched in the supernatant after centrifugation of leaf apoplast fluid at 40,000 × g (P40
fraction), and can be collected by centrifugation at 100,000 × g (P100-P40), the same speed used to collect
mammalian exosomes. Plant exosomes are mainly responsible for transporting sRNAs, including
microRNAs (miRNAs) and small interfering RNAs (siRNAs), from plants to fungal pathogens to induce
cross-kingdom RNAi [29,56] . TET8 is a homolog of the mammalian EV-enriched tetraspanin proteins CD9,
CD63, and CD81. The CD9-, CD63-, and CD81-labeled mammalian EVs are generated from MVBs . In
[65]
mammalian cells, Rab GTPases are essential for intracellular vesicle movement and strongly influence MVB
morphology . TET8-labeled plant EVs colocalize with the Arabidopsis MVB-marker Rab5-like GTPase
[9]
ARA6 and accumulate at fungal infection sites, suggesting that TET8-positive EVs are likely originated from
MVB trafficking [29,56] . Similarities between plant and mammalian exosomes suggest a conserved mechanism
in exosome biogenesis between mammalian and plant cells. In mammals, ceramide plays an important role
in exosome biogenesis and release . Interestingly, sphingolipids in Arabidopsis EVs are nearly pure
[66]
glycosyl inositol phosphoryl ceramides (GIPCs) . GIPCs are only produced by plant and fungal species
[67]
and are not present in mammalian EVs . The amount of cellular GIPCs is lower in the tet8 knockout
[68]
mutant than in the wild type, and the tet8 mutant secretes fewer EVs . Exogenous application of GIPCs
[67]