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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 9

Beyond plant-microbial/parasite interactions, cross-kingdom RNAi or cross-organismal RNAi has also been
observed between mammalian and insect hosts and their pathogens/parasites. In insects, the fungal
pathogen Beauveria bassiana transports a milRNA into mosquito cells and uses the same mechanism as the
plant fungal pathogen by hijacking mosquito host AGO1 to silence the expression of a mosquito Toll
receptor ligand, thereby attenuating mosquito immunity . Conversely, in this interaction, B. bassiana
[120]
infection induces the expression of two mosquito miRNAs, let-7 and miR-100. These miRNAs move into
the fungal cells and specifically silence the sec2p and C6TF fungal genes, both of which are essential for
fungal growth and pathogenicity . In mammals, the gastrointestinal nematode Heligmosomoides polygyrus
[121]
transports miRNAs into mouse intestinal epithelial cells to suppress innate immune responses and
[42]
eosinophilia . As well, human monocyte cells export miRNAs into the fungal pathogen Candida albicans
[122]
to silence the cyclin-dependent kinase inhibitor Sol1 . Human and mouse gut epithelial cells also secrete
miRNAs that can regulate gene expression and growth of gut bacteria, such as Fusobacterium nucleatum
[44]
and Escherichia coli . Although bacteria do not possess RNAi machinery, bacterial endoribonucleases may
mediate this cross-kingdom gene regulation. Alternatively, host sRNAs could be transported together with
host AGO proteins to silence bacterial genes in trans. There have also been reports about the possible
functions of dietary sRNAs and their potential role in modulating endogenous gene expression in
mammals. Although different studies using different systems with varying experimental conditions have
produced contradictory results, this nascent area of research into the therapeutic effects of dietary sRNAs is
worth pursuing . These observations suggest that cross-kingdom RNAi occurs throughout many branches
[123]
of life between various parasites/microbes and their hosts.

EXTRACELLULAR VESICLES IN CROSS-KINGDOM SMALL RNA TRAFFICKING
Mammalian exosomes from different cell types have been found to carry different RNAs, such as miRNAs,
mRNAs, and long non-coding RNAs, throughout the body for various functions in disease, cancer, and
immune responses [3,17] . For example, human breast cancer cells produce EVs containing miRNA precursors
and DICER and AGO2 proteins that function together to perform gene silencing in naive non-cancerous
[124]
cells . Human T-cells also produce exosomes with specific exosome-enriched miRNAs that are
transported into infected antigen-presenting cells to modulate their gene expression as part of the immune
[125]
response . Renal cancer cells secrete EVs loaded with a long non-coding RNA that acts in tandem with
miRNAs in cells to modulate gene expression and overcome the effects of anticancer drugs . Cancer-
[126]
derived EVs carrying intact mRNAs also facilitate the spread of ovarian cancer into the peritoneal cavity .
[127]
There is a much greater currently characterized diversity of types and functions of RNAs in mammalian
EVs compared to plant or microbial EVs.

EVs can be isolated from uninfected healthy plants [29,31] but are more enriched in plants after pathogen
infection or treatment with the plant defense hormone salicylic acid [29,31,56] , suggesting that EVs play an
important role in plant immunity. Comparative analysis on sRNA profiles of purified fungal cells and EVs
isolated from the infected tissue show that more than 70% of transferred plant sRNAs found in fungal cells
[29]
could be detected in plant EVs pelleted at 100,000 × g . This suggests that EVs are one of the major
pathways for cross-kingdom sRNA trafficking. Using density gradient analysis and direct immunoaffinity
[56]
capture, EV-enriched sRNAs are present in the fractions of TET8-positive EVs . Furthermore, double
mutants of Arabidopsis tet8 and its close homolog tet9 (tet8/tet9) deliver much fewer sRNAs into fungal
cells and show increased susceptibility to B. cinerea infection . These biochemical and genetic results
[29]
further support that TET8-positive EVs are the major class of EVs that transport sRNAs to fungal cells
[Figure 1] [29,56] . Similarly, Arabidopsis sends phasing siRNAs using EVs into oomycete pathogen
Phytophthora capsici to induce cross-kingdom RNAi .
[43]