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Page 8 Cai et al. Extracell Vesicles Circ Nucleic Acids 2023;4:262-82 https://dx.doi.org/10.20517/evcna.2023.10

of both DCL genes without knocking out expression, resulted in reduced pathogenicity of B. cinerea [88,100,105] ,
[104]
[106]
F. graminerum , and P. viticola . Mutation of DCL genes has also shown reduced virulence and growth
[107]
rate of the anthracnose pathogen Colletotrichum gloeosporioides . TEs are major components of
eukaryotic genomes and give rise to many siRNAs [89,108] . In phytopathogens, the LTR retrotransposons are
associated with virulence in host-adapted subpopulations of B. cinerea . This phenomenon may be due to
[109]
sRNA effectors being generated from these LTR regions. Thus, cross-kingdom transported sRNAs
originating from these TEs could explain the rapid variation of sRNA species produced during the co-
evolutionary arms race with the host.

Beyond fungal pathogens and plants, cross-kingdom RNAi is a conserved mechanism across many diverse
species. The parasitic plant dodder Cuscuta campestris delivers 22-nucleotide miRNAs into Arabidopsis,
which can act as virulence factors during parasitism . Cross-kingdom RNA trafficking also occurs in host-
[110]
symbiontic interactions. For example, to stabilize a symbiotic interaction, the ectomycorrhizal fungus
Pisolithus microcarpus transports miRNAs into Eucalyptus grandis cells to silence host NB-ARC domain-
containing genes. This attenuates the superfluous immune response of the plant to accommodate the
symbiont . As well, in the symbiosis between the arbuscular mycorrhizal fungi Rhizophagus irregularis
[111]
and its host plant Medicago truncatula, fungal sRNAs are predicted to target host mRNAs of genes whose
expression is modulated in roots after mycorrhizal colonization . Furthermore, sRNA trafficking between
[112]
hosts and microbes is not limited to eukaryotes with RNAi machinery. The plant symbiotic bacterium
Rhizobium transports 21-nucleotide tRNA-derived sRNA fragments (tRFs) into its soybean host, which are
loaded into soybean AGO1 and cleave nodulation-related mRNAs, contributing to the symbiotic
interaction . It remains to be determined whether cross-kingdom transport of tRFs into plant hosts occurs
[113]
during pathogenic bacterial infections.

Cross-kingdom RNAi is bidirectional, in which sRNAs are sent from the microbe into the host and also
from host cells into interacting microbes. As part of the plant defense response, some plant sRNAs,
generated by DCL proteins , are transported into interacting microbes/pests and silence target genes
[114]
[38]
necessary for pathogen/pest infection, thereby reducing plant disease . In Arabidopsis, trans-acting small
interfering RNAs (tasiRNAs) are trafficked into interacting B. cinerea cells and silence fungal vesicle
trafficking-related genes essential for virulence, such as vacuolar protein sorting 51 (VPS51), dynactin (
[29]
DCTN1) and suppressor of actin(SAC1) . The transport of host plant endogenous sRNAs into pathogens
has been observed in other pathosystems. Tomato plants transport multiple miRNAs into B. cinerea cells
that attenuate virulence by suppressing spore germination and silencing virulence-related genes [115,116] .
Cotton plants produce miR159 and miR166 that respectively target genes in V. dahlia that are essential for
[117]
fungal virulence . As well, wheat plants produce a miRNA that silences the expression of a hydrolase
enzyme in the fungus Fusarium graminearum which directly decreases its virulence .
[118]
During the arms race between hosts and pathogens, some pathogens have also developed countermeasures
to suppress cross-kingdom RNAi. A recent study showed that the fungal pathogen V. dahliae has an
adaptive mechanism to evade host-derived cross-kingdom RNAi. V. dahliae secretes protein secretory
silencing repressor 1 (VdSSR1) into plant cells, which interferes with host AGO1-miRNA export from the
nucleus and inhibits antifungal cross-kingdom RNAi . In addition,to promote infection, the oomycete
[119]
Phytophthora capsici produces an effector called Phytophthora suppressor of RNA silencing 2 (PSR2),
which inhibits the biogenesis of Arabidopsis secondary siRNAs that target Phytophthora genes via cross-
[43]
kingdom RNAi .