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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 7

immunoaffinity purification using an antibody that recognizes a specific protein marker of the particular
EV class. A TET8 native antibody that recognizes an extracellular domain 2 (EC2) of TET8 has been
successfully generated to isolate plant TET8-positive EVs [30,56] . Plant miRNAs and siRNAs were detected in
[56]
immunoaffinity purified TET8-positive EVs , supporting that plant sRNAs are mainly transported by
exosomes. PEN1-positive EVs and EXPO-derived EVs have not been successfully separated from
heterogeneous EV classes. The preparation of antibodies that recognize the extracellular domains of PEN1
and EXO70E2 to purify PEN1-positive EVs and EXPO-derived EVs will help determine the RNA cargoes
and other molecules in these subclasses of EVs. It is highly desirable that immunoaffinity capture be used to
properly assess specific cargo of different vesicle classes. Recent technical advances in flow field-flow
fractionation chromatography may make it possible to sort different classes of EVs by different sizes or
[84]
fluorescence labeling .

CROSS-KINGDOM RNAI
sRNAs are short non-coding regulatory RNAs that silence genes with complementary sequences [37,85] . In
eukaryotes, sRNAs are generated by the ribonuclease III-like enzyme Dicer or Dicer-like (DCL) proteins.
sRNAs are then incorporated into Argonaute (AGO) proteins to form an RNA-induced silencing complex,
which performs gene silencing in a sequence-specific manner by mRNA cleavage and degradation,
translational inhibition, or transcriptional gene silencing. During microbial infection, host RNAi machinery
is critical for reprogramming gene expression to induce plant immunity [37,86,87] . Recent studies demonstrate
that, in addition to their endogenous functions, sRNAs travel between hosts and their interacting organisms
and induce ‘cross-kingdom or cross-organismal RNAi’ in trans [35,38,88-90] [Figure 1].

Cross-kingdom RNAi was first observed in the interaction between Arabidopsis and the pathogen
[91]
Botrytis cinerea . During infection on both Arabidopsis and tomato host plants, B. cinerea delivers sRNAs
into plant cells that silence plant immunity genes by binding to the host AGO1 protein [91,92] . Pathogen
sRNAs associate with host AGO proteins and target genes involved in plant immune responses, including
plant kinase genes, which are often involved in signal transduction pathways that mediate plant defense .
[93]
B. cinerea sRNAs specifically target mitogen-activated protein kinase (MAPK) pathway genes and cell wall-
associated kinases . Like B. cinerea, sRNAs from the fungal wilt pathogen Verticillium dahliae also bind to
[91]
Arabidopsis AGO1 for host gene silencing [88,94] . In the fungal rust pathogen of wheat, Puccinia striiformis, a
micro-like RNA (milRNA) serves as an important pathogenicity factor that silences pathogenesis-related 2 (
pr2) gene, encoding a β-1,3-glucanase, which is a key wheat immunity gene . As well, other P. striiformis
[95]
sRNAs are predicted to target wheat kinase genes . On tomato hosts, the fungal pathogen Fusarium
[96]
oxysporum generates mil-R1, which is loaded into tomato AGO4 to silence the tomato CBL‐interacting
protein kinase gene . In addition to fungal pathogens, the oomycete pathogen Hyaloperonospora
[97]
arabidopsidis sends sRNAs into Arabidopsis cells that associate with Arabidopsis AGO1 to silence defense-
related genes: with no lysine kinase (AtWNK)2 and apoplastic, enhanced disease susceptibility-dependent
(AtAED)3 . sRNAs from the biotrophic powdery mildew pathogen, Blumeria hordei, have also been
[98]
predicted to target possible barley host immunity-related genes .
[99]
Fungal sRNA effectors play an important role in fungal pathogenicity and virulence. The majority of fungal
sRNA effectors are derived from long terminal repeat (LTR) retrotransposons and are generated by fungal
[91]
DCLs (DCL1 and DCL2) . Mutation or silencing of fungal DCL genes can attenuate the virulence and
growth of fungal pathogens. Mutation of both DCL genes reduced the virulence and growth of the grey
[101]
mold pathogen B. cinerea [88,91,100] , the soilborne pathogen Fusarium graminearum , the post-harvest decay
pathogen Penicillium italicum , the apple canker pathogen Valsa mali , and the grapevine downy
[103]
[102]
[104]
mildew pathogen Plasmopara viticola . Furthermore, even silencing of DCL genes, reducing expression