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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 11

bacterial outer membrane, generating outer membrane vesicles (OMVs) [136-138] . It has been debated how
vesicles can be released or taken up by organisms with thicker cell walls compared to the ease of vesicle
movement within mammalian cells lacking cell walls. A recent review highlighted the plasticity of the fungal
cell wall, allowing for the passage of vesicles as large as 200 nm in and out of fungal cells, despite the original
measurement of fungal cell wall pores being much smaller [139,140] . Recent proteomics data showed that fungal
EVs are enriched with cell wall-modifying enzymes [50,141] , which could allow for movement of fungal EVs
through the plant cell wall. This emphasizes the dynamic and flexible nature of the cell wall and provides
information on how vesicles could move in and out of the cells of thicker-walled organisms. As well, EV
membranes are malleable and have sufficient structural plasticity to facilitate passing through the cell walls.

Microbial EVs contain specific cargoes, such as DNA, RNA, metabolites, and proteins, that could be
involved in pathogenesis and modulation of host immunity [135,137,141,142] . Most microbial EVs are isolated from
cellular cultures by differential centrifugation at a predetermined time point to avoid capturing dying
cells [134,141,143] . Bacteria in the human gut have been shown to release bacterial EVs that can enter into the
bloodstream and lymphatic system and travel to distant organs throughout the body . Bacterial EVs can
[144]
have beneficial or detrimental effects on human cells, such as inducing dendritic cell polarization or
stimulating cancer proliferation . The human fungal pathogen Cryptococcus neoformans also produces
[144]
[145]
EVs that contain components of its fungal capsule, a cellular structure essential for virulence . Proteomics
analysis of EVs from five species within the genus Candida identified 36 common proteins enriched for
[146]
orthologs of biofilm mediators of the mammalian pathogen C. albicans . This study also discovered that
vesicles from one Candida species can confer function to other Candida species, suggesting an important
role of EVs in the development of microbial biofilm communities . Scanning electron microscopy and
[146]
cryo-transmission electron microscopy allowed the observation of EVs with an average size of 100 nm on
the surface of the Candida biofilm, corroborating that EVs can easily move across the fungal cell wall .
[146]

Over the past decade, studies of microbial EVs have mainly focused on mammalian-infecting pathogens,
with limited reports on EVs derived from plant pathogens. To date, most of the plant-interacting bacteria
from which EVs were isolated are Gram-negative bacteria, including Xanthomonadaceae family
bacteria [147-150] , Xylella fastidiosa [151,152] , and Pseudomonas syringae [153-155] . Proteomic analysis of Gram-negative
bacterial EVs reveals virulence factors and signaling molecule contents that may play a role in plant
[143]
infection . Direct evidence that phytopathogen-derived EVs contribute to virulence was provided by the
study of the xylem-colonizing plant pathogenic bacterium, X. fastidiosa . X. fastidiosa-derived EVs block
[152]
X. fastidiosa cells from interacting with the xylem wall, which increases its systemic spread within the plant
host and promotes virulence . It has also been observed that animal pathogenic bacteria produce EVs that
[152]
[156]
fuse with the host plasma membrane . The opportunistic human pathogen Pseudomonas aeruginosa
produces EVs that fuse with the host cell plasma membrane at lipid raft microdomains for long-distance
[157]
delivery of bacterial virulence factors . Similarly, the phytopathogen Xanthomonas campestris generates
EVs that fuse directly with the Arabidopsis plasma membrane. This process depends on the clustering of the
plant lipid raft proteins remorin 1.2 and remorin 1.3 . The observation that phytobacterial EVs fuse with
[150]
the host plasma membrane suggests that EVs facilitate cross-kingdom delivery of pathogen cargo to plant
cells. However, whether phytobacterial EVs also contain RNA or DNA molecules is still unknown. As
indicated above, symbiotic Rhizobium bacteria transport tRFs into host root cells to silence host nodulation
genes, suggesting that bacterial tRNA may act as an sRNA source to mediate cross-kingdom RNAi . It is
[113]
interesting to pose whether Rhizobium RNA trafficking is mediated by EVs [Figure 1].

More than twenty species of yeast and filamentous fungi have been observed to secrete EVs . Fungal EVs
[141]
are thought to be derived from MVBs or to bud directly from the plasma membrane [135,142] . In the