Overview
Small RNA (sRNA)-mediated RNA interference (RNAi) is a conserved regulatory mechanism
for gene expression throughout the domain Eukarya. Recent studies have shown that
sRNAs can move between a host and an interacting organism to induce gene silencing
in trans, a mechanism termed “Cross-Species RNAi” or, in many cases, “Cross-Kingdom
RNAi.” Pathogens and parasites transport sRNAs into host cells during infection and
silence host defense genes to suppress immunity, whereas hosts can also deliver their
sRNAs into interacting microbes or parasites to suppress infection. Recent studies
of different plant and animal hosts and their interacting organisms have unveiled
extracellular vesicles (EVs) as vehicles of sRNA exchange in cross-species and cross-kingdom
RNAi. The discovery of the pivotal role of sRNAs and EVs in cross-species and cross-kingdom
communication offers innovative tools for pathogen and pest control in agriculture
and biomedicine.
Cross-kingdom RNAi
sRNAs—including microRNAs (miRNAs) that are processed by Dicer-like (DCL) proteins
from single-stranded stem-loop–forming RNA precursors and small interfering RNAs (siRNAs)
that are processed by DCL proteins from double-stranded RNA (dsRNA) precursors—are
loaded into Argonaute (AGO) proteins to induce silencing of genes with complementary
sequences [1]. Some sRNAs from diverse classes of pathogens and parasites are transported
into host cells and induce cross-kingdom or cross-species RNA silencing to facilitate
infection (Fig 1). Fungal pathogens, including ascomycete and basidiomycete species,
can deliver sRNAs into their respective hosts [2–6]. In detail, Botrytis cinerea,
the grey mold fungal pathogen that infects over 1,000 plant species, delivers sRNAs
into plant cells and hijacks host RNAi machinery by loading its sRNAs into the Arabidopsis
AGO1 protein to trigger silencing of host immunity genes, including mitogen-activated
protein kinases (MAPKs), cell-wall–associated kinases, and other defense and signaling
proteins [2]. A panel of sRNAs from Verticillium dahliae, which causes Verticillium
wilt in many plant hosts, also move into plant cells and associate with the host AGO1
protein to silence host genes involved in plant defense [4]. A genome-wide association
study shows that the white mold fungal pathogen Sclerotinia sclerotiorum produces
sRNAs that, to facilitate infection, can target plant genes associated with quantitative
disease resistance [5]. A miRNA-like sRNA from Puccinia striiformis, the causal agent
of the destructive wheat stripe rust, targets wheat pathogenesis-related genes and
suppresses host immunity to achieve successful infection [3]. Likewise, the parasitic
plant Cuscuta campestris (dodder) transports several miRNAs into A. thaliana and Nicotiana
benthamiana to promote invasion [7].
10.1371/journal.ppat.1008090.g001
Fig 1
Cross-species and cross-kingdom RNAi between host and coinhabitants.
(A) Cross-species RNAi between mammals and parasites. Parasites produce EVs containing
parasitic sRNAs, which are internalized by mammalian cells to silence host genes involved
in inflammation and innate immunity. Animal cells can deliver sRNAs into interacting
organisms. They also secrete EVs (e.g., exosomes or MVs) containing host sRNAs. It
is likely that animal hosts may also transport sRNAs using EVs into parasites to suppress
parasitic genes. (B) Cross-kingdom RNAi between plants and fungal pathogens. Fungal
sRNAs translocate into plant cells and hijack host AGO protein of the RNAi machinery
to suppress plant immune responses. It is still unclear how pathogens transport sRNAs.
Conversely, plants secrete EVs to transport host sRNAs into pathogens to silence fungal
genes involved in virulence. The “?” indicates a prediction that has not been validated
experimentally. AGO, Argonaute; EE, early endosome; ER, endoplasmic reticulum; EV,
extracellular vesicle; MV, microvesicle; MVB, multivesicular body; RNAi, RNA interference;
sRNA, small RNA; TGN, trans-Golgi network.
Cross-kingdom sRNA trafficking from a fungal pathogen to an animal host was also observed
recently. Beauveria bassiana, an insect fungal pathogen, exports a miRNA-like RNA
(bba-milR1) to the host mosquito, which induces cross-kingdom RNAi to suppress host
immunity [6]. Strikingly, this insect fungal pathogen-derived bba-milR1 also binds
to host AGO1 and silences mosquito target gene Toll receptor ligand Spätzle 4 [6],
which is consistent with the mechanism used by transported sRNAs from plant fungal
pathogens [2, 4].
In addition to eukaryotic pathogens, prokaryotic microbes can also use cross-kingdom
RNA trafficking to manipulate gene expression in the hosts. Specifically, the root-nodule
bacterium Rhizobium delivers tRNA-derived sRNA fragments (tRFs) into soybeans to suppress
host genes involved in nodule formation and root development, which enhances nodulation
efficiency [8]. Surprisingly, these Rhizobium tRFs also function through host AGO1
[8], just like fungal pathogen-derived sRNAs that are bound with host AGO1 to silence
host target genes [2, 4, 6]. Furthermore, it has long been known that virus- or viroid-derived
sRNAs can target various host protein-coding genes to facilitate infection in both
plant and animal hosts [9–14]. A recent study revealed that the targeting of a long
noncoding RNA in tomato by tomato yellow leaf curl virus-derived sRNAs contributes
to disease symptoms [15].
Cross-kingdom RNAi is bidirectional. Plant hosts also transport sRNAs into fungal
pathogens to suppress the expression of virulence-related genes, which contributes
to plant defense responses. Translocation of plant endogenous sRNAs into fungi was
clearly demonstrated by sRNA profiling of fungal cells purified from infected plant
tissue [16]. Cai and colleagues developed an innovative sequential protoplastation
method, which allowed for the removal of all plant cells and the purification of B.
cinerea protoplasts/cells from infected Arabidopsis tissue [17]. These purified fungal
cells contain host miRNAs and siRNAs, including Trans-acting siRNAs, also called secondary
phasing siRNAs (phasiRNAs) [16]. These Arabidopsis sRNAs are delivered into interacting
B. cinerea cells to induce silencing of fungal genes that are involved in pathogenicity,
many of which are related to vesicle trafficking [16]. Mutated B. cinerea strains
with a deletion in these target genes displayed reduced pathogenicity on plant hosts
[16]. Another study found that cotton miRNA166 and miRNA159 accumulated in the mycelium
of V. dahliae grown on artificial agar medium 30 days post re-isolation from infected
tissue, which suggests that cotton miRNAs can translocate into V. dahliae [18]. Both
cotton miRNAs trigger silencing of V. dahliae genes involved in virulence, Ca
2+
-dependent cysteine protease (Clp-1), and isotrichodermin C-15 hydroxylase (HiC-15),
which enhances disease resistance against this vascular pathogen [18]. Similarly,
the wheat miRNA1023 suppresses an alpha/beta hydrolase gene in Fusarium graminearum,
which is important for fungal infection [19]. Plant sRNA-induced silencing of pathogen
genes is not restricted to fungi. A similar phenomenon was later observed in the interaction
between plants and an oomycete pathogen, Phytophthora capsici. Arabidopsis may use
secondary sRNAs to silence Phytophthora genes during infection [20].
Cross-species RNAi also exists in animal–parasite interactions. Some mammalian parasites
use cross-species RNAi strategies to silence host genes and enable infection. For
instance, the gastrointestinal nematode Heligmosomoides polygyrus (also known as H.
bakeri) secretes sRNAs, including miRNAs, which suppress type II innate immune response
in the murine host [21]. Conversely, some animal hosts also deliver sRNAs into parasites.
Patients who suffer from sickle cell anemia show abnormal erythrocyte development
but exhibit resistance to the malaria parasite Plasmodium falciparum. One of the reasons
for malaria resistance is that these patients accumulate higher levels of a specific
panel of miRNAs, which are transported into the parasite and suppress P. falciparum
virulence [22]. Though P. falciparum lacks canonical RNAi components, such as DCLs
and AGOs, the authors demonstrated that cross-kingdom RNA regulation occurs through
impaired ribosomal loading by the fusion of host miRNAs with the parasite target mRNAs.
This chimerization blocks target mRNA translation and causes an inhibition of parasite
growth [22]. Anti-Plasmodium cross-kingdom RNA regulation was also reported based
on the human miR-451/140 targeting the P. falciparum antigen erythrocyte membrane
protein-1 (PfEMP1). Human miR451 was found in the parasitic cell in complex with human
AGO2, providing the first example of cross-kingdom delivery of an sRNA–AGO complex
[23].
In the mammalian gut, miRNAs secreted by human and mouse intestinal epithelial cells
were shown to influence gene expression even in gut bacteria that lack canonical RNAi
machinery, suggesting a regulatory role of host miRNAs in gut microbiome homeostasis
[24]. Furthermore, dietary plant miRNAs can also enter gut bacteria through plant-derived
exosome-like nanoparticles, further shaping the gut microbial community [25]. RNAi
does not exist in prokaryotes per se; however, bacteria have various ribonucleases,
including type III ribonucleases [26], which may interact with the host or dietary
miRNAs to interfere with bacterial mRNA expression. The increasing number of discovered
cases of cross-species and cross-kingdom RNAi or RNA Trans-regulation across diverse
host–microbe and host–parasite systems has made it clear that cross-species and cross-kingdom
RNA communication is likely a ubiquitous mechanism in host–microbe and host–parasite
interactions.
EVs in animal–parasite interactions
In mammals, RNAs circulating through body fluids are often encapsulated in extracellular
vesicles (EVs). EVs are membrane-surrounded vesicular compartments released by cells
to the extracellular environment to transport proteins, RNAs, lipids, and other molecules
to other cells or to interacting organisms [27]. EVs are categorized into multiple
classes based on their biogenesis pathways and associated protein markers. In mammalian
systems, multiple classes of EVs have been shown to carry sRNAs. In particular, exosomes,
which are derived from multivesicular bodies (MVBs) and have tetraspanin proteins
as one of the key protein markers [28], play an important role in sRNA trafficking
[29]. Microvesicles, which bud from the plasma membrane, can also transport sRNAs
into recipient cells [30]. Both types of EVs are involved in cell-to-cell communication
in homeostasis, immune signaling, and neural networks [31, 32]. While exosomes and
microvesicles are secreted during normal cellular processes, apoptotic bodies are
formed during programmed cell death [33]. Functional molecules, including RNAs, can
be detected in apoptotic bodies [34, 35]. Some reports have shown that apoptotic bodies
can transport these functional molecules into recipient cells [35, 36], though whether
they are also involved in cross-kingdom communication between parasites/microbes and
animal hosts remains to be explored.
It is not surprising that pathogens and pests would evolve to exploit or target these
natural cell-to-cell communication pathways. Diverse parasites have been shown to
use EVs to deliver sRNAs to host cells and modulate host gene expression (Fig 1A)
[37]. The miRNA-containing EVs that are released by the gastrointestinal nematode—or
helminth—H. polygyrus are internalized by host mouse cells and suppress inflammation
and innate immune responses during infection [21]. Many of the nematode miRNAs share
common ancestry and identical seed sites with miRNAs of the mouse host, such that
they would be expected to be able to tap into existing miRNA target networks in the
mouse cell. However, the RNAi mechanisms used between these two animals are complex,
as the nematode packages a nematode-specific AGO protein (extra cellular worm Argonautes
[exWAGO]) into the EVs bound to siRNAs from rapidly evolving nongenic regions of the
parasite genome [38]. Indeed, these studies suggest that different parasites and pathogens
might have diverse tools for RNA-mediated suppression of host genes. The study of
these pathogen RNA transmission mechanisms may guide new strategies for effective
therapeutic delivery of RNAs (for example, delivering RNA–AGO complexes, rather than
RNA alone) [39]. Since the EVs from helminths are immune suppressive, the EVs and
their RNA cargoes also represent another potential therapy for treating colitis and
allergies in humans [21, 40, 41].
In mammalian systems, EVs have been shown to transport sRNAs between cells within
the organism; we speculate that EVs may also be used by the host cells to deliver
sRNAs to its interacting organisms, such as parasites and pathogens.
EVs in plant–microbe interactions
In 1967, plant EVs were initially observed in carrots by electron microscopy [42].
Forty years later, Regente and colleagues isolated plant EVs from extracellular wash
fluids of imbibed sunflower seeds [43]. However, the origin of plant EVs still remained
unknown. In mammals, exosomes are a class of EVs derived from MVBs. Mammalian tetraspanins
cluster of differentiation (CD)63, CD81, and CD9 are enriched in exosome membranes
and are commonly used as biomarkers to isolate and phenotype exosomes [28]. Arabidopsis
encodes 17 members of the TETRASPANIN (TET) family [44], and two Arabidopsis TETs
(TET8 and TET9) are induced upon infection by B. cinerea. Moreover, TET8-associated
vesicles accumulated to a high level at the fungal infection sites [16]. TET8 is colocalized
with Arabidopsis MVB-marker Rab5-like GTPase ARA6 inside the cell, and TET8-associated
vesicles are secreted into the apoplast [16], suggesting that TET8-associated EVs
are derived from MVBs and secreted into apoplastic space and can, therefore, be considered
bona fide plant exosomes. These exosomes contain plant-endogenous sRNAs and are efficiently
taken up by B. cinerea fungal cells. Plant exosomes deliver sRNAs into fungal pathogens
to suppress fungal infection by inducing silencing of fungal virulence-related genes.
Similarly, Arabidopsis also transports secondary phasiRNAs from PPR gene clusters
into an oomycete pathogen, P. capsici, likely also by EVs, which silence target genes
in the pathogen [20]. Thus, plants have adapted EV-mediated cross-kingdom RNAi for
immune responses during the coevolutionary arms race with interacting pathogens (Fig
1B).
In addition to exosomes, PENETRATION (PEN)1-associated EVs, which contain several
stress-response–related proteins, were identified in Arabidopsis [45]. The biogenesis
pathway of PEN1-associated EVs remains unclear, although PEN1 was originally identified
as a plasma-membrane–associated plant-specific syntaxin [46]. PEN1-associated EVs
were purified from the apoplast wash fluid of Arabidopsis leaves using an ultracentrifugation
speed (40,000g) [45, 47], which is slower than that used to isolate TET-associated
exosomes (100,000g) [16]. Secretion of PEN1-assoiated EVs was increased during infection
by a bacterial pathogen (Pseudomonas syringae) or following treatment with the phytohormone
salicylic acid [45]. Baldrich and colleagues analyzed the sRNA population in these
EVs isolated from uninfected Arabidopsis leaves and found that these EVs carry predominantly
“tiny RNAs,” which are 10–17 nucleotides in length and derived mainly from the positive
strand of mRNA transcripts [48]. It is not clear whether these tiny RNAs have any
biological function. Since pathogen-infected samples were not included in this study,
whether this class of EVs is also involved in plant and pathogen interactions and
whether tiny RNAs are delivered into pathogen cells via these EVs remain unclear.
PEN1 and the ATP-binding cassette (ABC)-transporter PEN3 are incorporated into extracellular
encasements surrounding the haustoria of the powdery mildew fungus, Golovinomyces
orontii, suggesting that PEN1-asociated EVs contribute to defense responses against
powdery mildew [45, 49, 50]. A third type of plant EV, which is derived from a novel
double-membrane–bound exocyst-positive organelle (EXPO) [51], has been reported in
plants. These EXPO-derived EVs were discovered through transient expression of exocyst
subunit exo70 family protein E2 (Exo70E2), a component of exocyst complex, in protoplasts
from Arabidopsis suspension-cultured cells. Whether EXPO-derived EVs contain RNAs
and are involved in cross-kingdom communication remains to be discovered.
Similar to animal EVs, which comprise diverse, heterogeneous, and cell-type–specific
populations with a wide range of biological functions in cell-to-cell communication
[52], the previously cited studies suggest that plant cells also secrete different
classes of EVs that may contain specific cargoes. Establishing plant EV biomarkers
(such as TET8, PEN1, and Exo70E2) will enable immuno-based analysis of EVs to further
understand the biological functions of EVs in complex biological systems such as plant–microbe
interactions.
Though EV-mediated transport is a key mechanism for RNA secretion and delivery between
hosts and microbes/pests, nonvesicular extracellular RNAs have also been discovered.
Specifically, in human plasma, extracellular RNAs were found within RNA–protein complexes,
including AGO proteins and high-density lipoprotein complexes [53–56]. Additionally,
exomeres, extracellular nonmembranous nanoparticles, have recently been discovered
in mammalian systems containing AGO1, AGO2, and AGO3 proteins; amyloid precursor proteins;
RNAs; and DNAs. Notably, these exomeres contained a profile of macromolecules distinct
from exosomes [57, 58]. In a plant system, Baldrich and colleagues found that sRNAs
were still present in apoplastic wash fluid, which they believe was depleted of EVs
by centrifugation at 40,000g [48]. However, small, RNA-containing EVs, such as exosomes,
are mostly collected at higher speeds (between 100,000g and 120,000g) from various
plant and mammalian systems [52, 59–61], as well as from fungi [62, 63]. Furthermore,
plant tetraspanin-labeled exosomes, which transport sRNAs from the hosts to fungal
cells, were much more enriched after centrifugation at a speed of 100,000g than at
40,000g [16]. Therefore, it is unlikely that plant EVs can be depleted at 40,000g,
and, consequently, whether nonvesicular RNAs are secreted by plants requires further
investigation. Furthermore, the origins of nonvesicular RNAs and their potential role
in cross-kingdom RNAi remain to be explored.
RNA and EV-based innovative tools for disease control
Global disease control mainly relies on chemical protection measures using fungicides,
pesticides, and antibiotics, which not only threatens the health of humans and ecosystems
but also generates novel uncontrollable drug-resistant pathogenic strains [64]. We
are in urgent need of innovative, durable, and eco-friendly fungicides and antimicrobial
drugs to avoid a global collapse in our ability to control pathogen/parasite infections
in both plants and animals, including humans.
One direct application of cross-kingdom RNAi is host-induced gene silencing (HIGS),
a promising technology in which transgenic plants express dsRNAs or sRNAs that target
pathogen or insect virulence-related genes to combat plant diseases [65, 66]. This
approach has also made it possible to control multiple pathogens spontaneously by
designing dsRNA and sRNA constructs that target multiple genes from different pathogens
[4]. Although HIGS is effective, it involves the generation of genetically modified
organisms (GMOs), which is not only technically challenging in many crop species but
unfortunately still a concern for many consumers. Furthermore, GMOs are banned in
European agricultural productions, rendering HIGS not practically usable, at least
in the near future.
Environmental RNAi, initially discovered in the nematode Caenorhabditis elegans [67],
is the cellular uptake of RNAs from the environment and the induction and spreading
of systemic gene silencing. Forward genetics screening in C. elegans revealed that
Systemic RNA interference deficient (SID)-1 and SID-2 encode for two dsRNA transmembrane
channel proteins, which are required for dsRNA uptake and systemic gene silencing
[68, 69]. In this invertebrate system, there is higher uptake and silencing efficiency
for long dsRNA (>60 bp) than short (<25 bp) or single-stranded RNA [70, 71]. Inspired
by environmental RNAi of C. elegans, Wang and colleagues tested whether fungal cells
can also take up RNAs from the environment and observed rapid RNA uptake by B. cinerea
cells [4]. These RNAs induce silencing of fungal genes in a sequence-specific manner.
Unlike C. elegans, which primarily takes up long dsRNAs, fungal uptake of environmental
RNAs seems less dependent on RNA size, because both short sRNA duplexes and long dsRNAs
are taken up by fungi and induce robust gene silencing in the fungal cells [4]. Fungal
environmental RNAi allowed plant scientists to design spray-induced gene silencing
(SIGS) to control fungal and potentially other pathogens through spray application
of pathogen gene-targeting dsRNAs and sRNAs (Fig 2A) [4, 72, 73]. Wang and colleagues
demonstrated that spray application of long dsRNAs or sRNA duplexes that target B.
cinerea DCL1 and DCL2 genes can effectively suppress grey mold diseases on fruits,
vegetables, and flowers [4]. Koch and colleagues have shown that SIGS can also effectively
control a fungal disease in the monocot barley [73]. Spray application of a long dsRNA
that targets fungal cytochrome P450 lanosterol C-14α-demethylase genes on barley leaves
can inhibit F. graminearum infection [73]. Similarly, application of exogenous dsRNAs
helps protect Brassica napus from infection by S. sclerotiorum and B. cinerea [74].
These pathogen gene-targeting dsRNAs and sRNAs represent a novel class of eco-friendly
fungicides, “RNA fungicides” (Fig 2A).
10.1371/journal.ppat.1008090.g002
Fig 2
SIGS is an efficient disease control strategy in plants and potentially in humans.
(A) Spray application of dsRNAs and sRNAs that target pathogen/pest genes can potentially
control plant diseases. The SIGS-based protection can be prolonged by incorporating
RNAs into artificial vesicles (black circle) or nanoparticles (pink rhombus) to protect
RNAs from degradation or water rinsing. (B) Future RNA-based antifungal drugs have
the potential to control human mycoses. Artificial vesicles/liposomes will likely
facilitate the RNA delivery. Figures were created with BioRender. The “?” indicates
a prediction that has not yet been validated experimentally. dsRNA, double-stranded
RNA; SIGS, spray-induced gene silencing; sRNA, small RNA.
Exogenous RNAs can either be directly internalized into fungal cells [4] or indirectly
via passage through plant tissue before transport into interacting pathogen cells
[75]. Furthermore, Koch and colleagues observed inhibition of F. graminearum growth
in the distal nonsprayed barley leaf tissue [73], suggesting that sprayed dsRNAs taken
up by plant cells moved through vasculature systemically. While the molecular mechanism
of RNA uptake in C. elegans and some nematodes is based on SID proteins, which are
not present in plants or fungi, the mechanisms for uptake of environmental RNAs into
fungi and plants need further investigation.
Obviously, the effectiveness of SIGS relies on extracellular RNA stability and RNA
uptake efficiency of pathogens. To technically improve RNA stability, Mitter and colleagues
docked an antiviral dsRNA onto double hydroxide clay nanosheets, which increased the
efficacy of plant antiviral protection [75]. In addition, the use of artificial vesicles
or liposomes to protect RNAs could be an effective strategy to improve SIGS for plant
protection and to develop potential antifungal drugs for therapy, as some fungi are
capable of taking up EVs efficiently (Fig 2) [16, 76]. Since EV trafficking is also
a natural RNA transport mechanism in mammals, it is exciting to consider the potential
for extension of artificial vesicle-protected RNA-based antifungal strategies in humans
(Fig 2B). Indeed, lipid-based nanoparticles have been used to stabilize therapeutic
compounds, including sRNAs, in biomedical applications [77]. For example, liposomal
amphotericin B, the world’s leading antifungal drug, was based on liposomal formulation
of amphotericin B to reduce toxicity [78]. Moreover, Walker and colleagues have observed
that amphotericin B–containing liposomes remained intact during transit through the
cell walls of phylogenetically distant fungal pathogens, Candida albicans and Cryptococcus
neoformans, although liposomes (60–80 nm) are larger than the theoretical cell wall
porosity (approximately 5.8 nm)[79]. This work suggests that the fungal cell wall
is deformable and viscoelastic to allow liposomes to pass, which makes it possible
to efficiently deliver new generation of antifungal drugs, including RNA-based drugs,
using liposomes/artificial vesicles [79]. In 2018, the Food and Drug Administration
(FDA) approved the very first therapeutic siRNA drug, patisiran, to treat hereditary
transthyretin-mediated amyloidosis, a rare, debilitating and often fatal genetic disease
[80]. Patisiran uses a lipid nanoparticle delivery mechanism to transfer 21-bp siRNA
duplex into cells in the liver [80]. Besides patisiran, there are at least 6 other
RNAi therapeutics already in phase III clinical trials [80].
Although more than 300 human or animal pathogenic fungal species have been recorded
and fungal infections display disproportional high mortality rates, mycoses are rather
neglected in infection biology research [81]. Survival rates of patients suffering
from respiratory and systemic fungal infections often caused by the opportunistic
fungi Candida (candidiasis), Aspergillus (aspergillosis), or Cryptococcus (cryptococcosis)
are low due to limited availability of antifungal drugs. Drug-resistant fungal strains
have already emerged to all the commonly used antifungal drugs [64]. Therefore, innovative
drugs to combat fungal infections are urgently needed, and based on the effects observed
for antifungal SIGS approaches in plants, development of novel antifungal RNA therapeutics
and artificial vesicle/liposome-mediated delivery methods may be effective in the
fight against mycoses.
Future perspectives
The field of cross-species and cross-kingdom communication via RNA is still in its
infancy, yet an increasing number of studies across diverse systems demonstrate that
mobile RNAs are key regulatory molecules that shape the interactions between hosts
and interacting pathogens or organisms. Plants and animals deliver sRNAs into interacting
(micro-)organisms to inhibit infection, and pathogens and parasites can, in turn,
transport sRNAs into the host to suppress host immunity. Current studies show that
EVs play an essential role in transporting sRNAs from the plant hosts to pathogens
and from parasitic nematodes to mammalian hosts, and it is very likely that mammalian
hosts could also utilize EVs to deliver sRNAs into their parasites and pathogens,
though this is currently just speculation. Recent advances in methodology development
for isolating different classes of EVs in mammalian systems provide excellent tools
and guidelines to study RNA delivery in cross-species and cross-kingdom RNAi [82,
83]. Although there is diversity in the properties of EVs based on their cell and
tissue origin (and purification techniques, which can also impact the exact profile
of RNAs and proteins found in EVs), it is clear that small EVs, including exosomes,
play an important role in delivering sRNAs [60, 84, 85].
The discovery of cross-species and cross-kingdom RNAi and fungal RNA uptake has inspired
scientists to design novel disease control strategies against pathogens and pests
in agriculture, such as HIGS and SIGS. Structural and mechanistic studies of EVs in
sRNA trafficking allows for the development of innovative delivery methods of sRNAs
using artificial vesicles, or nanoparticles, which may also be considered for therapeutic
applications in mammalian systems. We speculate that future development and application
of a new generation of RNA-based fungicides and antifungal drugs will be an important
research direction to control diseases caused by eukaryotic pathogens and parasites.