RNA Interference in Plant Defense Systems

Abstract

Plant pests and pathogens, including viruses, reduce the biological potential of crops and undermine food safety. The methods of crop protection based on natural systemic and cellular plant immunity are currently being developed and special attention is given to a unique mechanism known as RNA interference (RNAi). It creates one of the levels of evolutionary conserved and yet highly species-specific plant immunity. This review deals with the role of small, noncoding RNA and DCL, AGO, and RDR proteins involved in RNAi machinery in the infection by pathogens, viruses, and pests, and the prospects for application of this phenomenon in the creation of preparations designed to protect crops against pests and diseases, including artificial switching off of the genes known as spray-induced gene silencing (SIGS).

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ISSN 1021-4437, Russian Journal of Plant Physiology, 2021, Vol. 68, No. 4, pp. 613–625. © Pleiades Publishing, Ltd., 2021.
Russian Text © The Author(s), 2021, published in Fiziologiya Rastenii, 2021, Vol. 68, No. 4, pp. 356–370.
RNA Interference in Plant Defense Systems
I. V. Maksimova, *, M. Yu. Sheina, and G. F. Burkhanovaa
a Institute of Biochemistry and Genetics, Ufa Federal Research Center, Russian Academy of Sciences, Ufa, Russia
*e-mail: igor.mak2011@yandex.ru
Received August 27, 2020; revised September 29, 2020; accepted October 2, 2020
Abstract—Plant pests and pathogens, including viruses, reduce the biological potential of crops and under-
mine food safety. The methods of crop protection based on natural systemic and cellular plant immunity are
currently being developed and special attention is given to a unique mechanism known as RNA interference
(RNAi). It creates one of the levels of evolutionary conserved and yet highly species-specific plant immunity.
This review deals with the role of small, noncoding RNA and DCL, AGO, and RDR proteins involved in
RNAi machinery in the infection by pathogens, viruses, and pests, and the prospects for application of this
phenomenon in the creation of preparations designed to protect crops against pests and diseases, including
artificial switching off of the genes known as spray-induced gene silencing (SIGS).
Keywords: RNA interference, plant immunity, DCL, AGO, RDR
DOI: 10.1134/S1021443721030134
INTRODUCTION
RNA interference (RNAi) is one of the outstanding
discoveries in biology, which was made in 1998 by
A. Fire and C. Mello with nematode Caenorhabditis
elegans as the test subject [1]. The RNAi mechanism
occurs in essentially all living organisms and governs
the activity of the genes through the formation of short
double-stranded RNA (dsRNA) and via synthesis of
special ribonucleases (RNases) that induce selective
degradation of target RNA (viral, messenger, and
transposon) and/or inhibition of their translation or
replication. Target dsRNA are produced by RNA-
dependent RNA polymerases (RdR) from an initial
single-stranded RNA molecule (viral or messenger
RNA transcribed from DNA and encoding a target pro-
tein or RNA transcribed from transposon DNA and
premicroRNA containing specific hairpin structures).
The role of RdR in crop protection against viruses
consists in the formation of replicative viral dsRNA
that interacts with DCL proteins and induces its own
specific degradation [2]. A low level of StRdR expres-
sion in potato (Solanum tuberosum) plants was accom-
panied by the accumulation of potato virus Y [3]. Sim-
ilarly, in Nicotiana benthamiana tobacco plants mutant
by gene rdr1 virus, the titer considerably rose and
resistance to viruses decreased accordingly [4]; trans-
genic plants expressing gene MtRdR1 from Medicago
truncatula alfalfa were resistant to tobacco mosaic
virus [4]. At the same time, there are indications that
the suppression of gene StRdR1 expression did not
exert a strong influence on the resistance of S. tubero-
sum potato plants to viruses X and Y [5].
The level of gene AtRdR1 transcripts was higher in
Arabidopsis thaliana pretreated with salicylic acid (SA),
whereas gene AtRdR2 was insensitive to it [4]. In
S. tuberosum, gene StRdR1 was very sensitive to SA [5].
In pepper plants (Capsicum annuum), the expression
of gene CaRdR1 was stimulated by SA, abscisic acid
(ABA), hydrogen peroxide (Н2О2), and infection by
TMV; its silencing induced a susceptibility to the virus in
plants [6]. Joint exposure to TMV and SA of tomato
plants Solanum lycopersicum promoted the accumula-
tion therein of transcripts of SlRdR1 and SlRdR2 [7].
In the same way, transgenic plants of tobacco N. ben-
thamiana after treatment with SA accumulating pro-
tein MtRdR1 showed resistance to TMV [4]. In silico
analysis of the promoter region of genes from the fam-
ily RdR1-6 in different plant species has shown that
their main transcription factors were proteins MYB44,
AS1/AS2, and WRKY1 [8]. Down-regulated expres-
sion of NaRdR1 and NaRdR2 (but not of NaRdR3)
elevated the susceptibility of Nicotiana attenuata tobacco
plants to the F. brachygibbosum fungus [9].
Protein RdR6 is involved in the system of resis-
tance to viruses. For instance, tobacco lines expressing
gene RdR6 were resistant to PVY, and, on the con-
trary, the lines of rice Oryza sativa with down-regu-
Abbreviations: CMV—cucumber mosaic virus; dsRNA—double
stranded RNA; HIGS—host-induced gene silencing; nt—
nucleotide residues; PVY—potato virus Y; RdDM—RNA-
directed DNA methylases; RISС—RNA-induced silencing
complex; RITS—RNA-induced transcriptional silencing com-
plex; RNAi—RNA interference; SAR—systemic acquired resis-
tance; SIGS—spray-induced gene silencing; siRNA—small
interfering RNA; ssRNA—single-stranded RNA; ta-siRNA—
trans-acting siRNA; TMV—tobacco mosaic virus; VD-siRNA—
virus-derived siRNA.
REVIEWS

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MAKSIMOV et al.
lated expression of this gene were susceptible to the
Chinese wheat streak mosaic virus, rice dwarf virus,
and rice stripe virus [10]. In tobacco N. benthamiana
plants NIbV3 NahG deficient in SA and in double
mutants NIbV3 NahG/RdR6i, there existed a high
level of transcripts of plum pox potyvirus genes, which
suggests that this signal molecule and the product of
gene RdR6 play an important role in plant defense
against viral infection [11]. It was found that protein
RdR6 is associated with the mechanism of silencing
expression of the genes encoding receptor toll-like
nucleotide-binding proteins containing leucine-rich
repeats (NB-LRR) and a number of PR-proteins as
well as operation of the mechanism triggered by
pathogen (pathogen-triggered immunity, PTI) and/or
elicitor (effector-triggered immunity, ETI) via genera-
tion of siRNA (ta-siRNA or trans-acting siRNA)
affecting transcription and resistance to bacterial
infection [12]. Protein RdR6 triggers RNA-dependent
DNA-methylation in transcribed DNA regions (trans-
posons) and causes epigenetic silencing of target
genes, which RdR2 cannot realize. Knock-out
mutants of S. lycopersicum and A. thaliana by gene rdr6
were susceptible to bacteria Pseudomonas syringae and
Xanthomonas oryzae, respectively [13]. It was detected
that protein RdR6 accumulates in plants under the
influence of effector avrRpt2 from bacterium P. s yr i n-
gae. It participates in the production of long-chain
siRNA-1 (bacterial-induced siRNA-1) and natural
antisense transcript (NAT) associated with siRNAs,
which plays a key role in regulation of plant resistance
to bacterial infection.
Subsequently, long dsRNA synthesized by RdR are
recognized by ribonucleases of class III called Dicer-
(in vertebrates) or Dicer-like (DCL) (in plants, fungi,
and lower invertebrates) proteins and cleaved to trans-
form into short dsRNA (20–25 nt). DCL proteins in
plants form a multidomain family of class III RNases
(RNaseIII) initiating processing of dsRNA [3]. Indi-
rect evidence of participation of DCL proteins in plant
immunity and general plant physiology is their diver-
sity and lack of vitality in plant lines knocked-out by
protein DCL and especially by DCL1. It is assumed
that the current diversity of this group of proteins in
plants represented by four types of DCL (A. thaliana)
arose in the course of evolution owing to a necessity
for organizing a defense against various viruses,
pathogens, and pests [14].
There are reports about the role of DCL in plant
resistance to viral pathogens, protection against trans-
posons, suppression of amplification of viruses/viroids,
regulation of expression of its own genes, and repres-
sion of transgenes [14], which makes it possible to use
them in transgenosis with the purpose of improving
plants’ resistance to viruses/viroids. Differential con-
secutive activation of genes VvDCL1 and VvDCL3 was
shown in vine Vitis vinifera L. upon fungal pathogene-
sis [15]. Protein DCL4 triggered local RNAi but sys-
temic protective response was induced by a combina-
tion of products of expression of genes DCL2 and
DCL4 [16]. At the same time, A. thaliana plants
mutant by genes Atdcl2, Atdcl3, and Atdcl4 and treated
with SA did not acquire resistance to cucumber mosaic
virus (CMV) and tobacco mosaic virus (TMV) [17].
However, SA treatment of tomato plants S. lycopersicum
and their subsequent infection with TMV promoted the
accumulation of proteins DCL1 and DCL2 [18].
Suppression of expression of gene NaDCL3 (but not
NaDCL2/4) elevated the susceptibility of N. attenuata
to fungus Fusarium brachygibbosum [9]. In the system
of antiviral plant defense, protein DCL4 mediates the
suppression of viral RNA replication [3, 19]. It was
shown that affinity of particularly the DCL4 protein to
viral RNA rose (in contrast to other DCL) in the
course of RNAi pathway evolution [19].
A special role in RNAi belongs to Argonaute (AGO)
proteins that are one of the major catalytic components
of RNA-induced silencing complex (RISC) and RNA-
induced transcriptional silencing (RITS); they ensure
posttranscriptional gene silencing (PTGS) and tran-
scriptional gene silencing (TGS), respectively [20].
The principal functions of AGO proteins in plants
are the binding of siRNA and miRNA generated with
the participation of DCL proteins and operation as a
traffic controller in the course of recognition and sub-
sequent cleavage of target genes’ RNA on transcrip-
tional, posttranscriptional, and translational levels.
Antiviral properties of plant AGO proteins are sum-
marized in a review by Carbonell and Carrington [21].
The products of genes AtAGO1, AtAGO2, and AtAGO7
of A. thaliana are involved in viral resistance [16, 22].
Proteins AGO1 and AGO7 were shown to actively
participate in protection of turnip Brassica rapa
subsp. rapifera against turnip crinkle virus [23]. Pro-
tein NbAGO2 in N. benthamiana plants is an import-
ant component that ensures resistance to tomato
ringspot virus [24], tomato bushy stunt virus [25], and
tomato mosaic virus [26]. Protein AGO2 of A. thaliana
was more efficient against potato virus X as compared
with a counterpart gene from N. benthamiana [27].
Protein AGO4 played an important role in plant resis-
tance to CMV and PVХ, and mutants by gene ago4
also became susceptible to tobacco rattle virus and beet
leafroll virus [28]. Similarly, protein AGO4 turned out
to play an important role in the formation of defense in
N. attenuata plants against fungus F. b ra c hy g i b bo s u m ,
and silencing of its synthesis impaired operation of the
jasmonate signal system [9]. It was found that such a
disturbance is caused by cessation of jasmonic acid
(JA) synthesis, and after treatment of plants with JA
their resistance to the fungus was restored.
Protein AGO1 is looked upon as the first level of
defense exposed to the effect of viral suppressors, and
protein AGO2 is considered to be the next line of
defense preventing accumulation of the virus and it is
jointly controlled by protein AGO1 and miR403 [29].
Functions of protein AGO2 may be performed by pro-

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RNA INTERFERENCE IN PLANT DEFENSE SYSTEMS 615
tein AGO5 when its synthesis is impaired. However,
double mutant ago2ago5 showed a greater susceptibility
to viruses as compared with single mutants ago2 and
ago5 [27]. In addition to transcripts of genes OsAGO1
and OsAGO3, O. sativa plants infected with rice curly
top virus accumulated a transcript of gene OsAGO18.
The product encoded by this gene is assumed to play an
important role in viral resistance and contribute to effi-
cient expression of gene OsAGO1 [30]. In S. lycopersi-
cum, protein AGO4 is a necessary component of RNA-
related DNA methylation upon formation of resistance
to bacterium P. s y ri n ga e , strain DC3000 [31].
An important role is performed by miR403a in the
course of SA-induced accumulation of transcripts of
gene NbAGO2 [26]. It was shown that protein AGO2,
together with miR393b, is necessary for regulation of
A. thaliana resistance to bacterium P. s y ri n ga e pv.
tomato showing in regulation of synthesis of protein
MEMB12 from the family of N-ethylmaleimide-sen-
sitive proteins (SNARE) located in the Golgi apparatus
and responsible for secretion into the apoplast of patho-
gen-induced proteins, for instance, of PR-1 [18].
The defense system described above cannot oper-
ate without leading characters of RNAi in plants—
small noncoding RNA belonging to three classes:
(a) 21–22-nt small interfering RNA (siRNA,), (b) 20–
24-nt micro RNA (miRNA), and (c) 24-nt repeat-
associated siRNA (rasiRNA) [32]. Complementary
strands of dsRNA interacting with RISC or RITS and
called siRNA and miRNA are the main components
that bind with target (foreign or host) RNA and block-
ade its subsequent operation (RNA strand is cleaved or
the process of translation is impaired). At the same
time, 21–22-nt siRNA participate in direct degrada-
tion of viral RNA and some endogenous mRNA play-
ing an important role in the system of defense against
viruses [33], whereas 24-nt rasiRNA are unique in
plants and participate in RNA-directed DNA methyl-
ation necessary for the maintenance of genome stabil-
ity via silencing the expression of transposons and
repeated DNA sequences [34].
Biogenesis of numerous small RNA was decoded
and described in detail [35], but, as a rule, the
researchers focused their attention on miRNA accu-
mulating in plants in response to environmental stress
agents, including biotic factors. miRNA is believed to
be an important component involved in host-induced
gene silencing (HIGS) [36]. Formation of plant
immunity to pathogens of different etiology specifi-
cally depended on changes in the content of miR160a,
miR396a, miR398b, miR482, miR1444, miR2118,
and miR7695 whose targets were messenger RNA
responsible for the synthesis of transcription factors,
receptor proteins NB-LRR, and proteins of RNAi
machinery as well as the enzymes of pro- and antioxi-
dant system regulating the level of reactive oxygen spe-
cies [35]. S. tuberosum plants hyper-expressing miR160
and those with its reduced level showed an extreme
susceptibility to the pathogen of late blight of potato,
which suggests that this miRNA plays a regulatory
role [37]. Production of miR159 and miR166 and their
export to the cells of fungus Verticillium dahliae in cot-
ton-plants in response to infection correlated with the
formation of plant resistance to the fungus [38].
miR172 showing a homology with a region of gene
encoding transcription factor AP2/ERF was identified
as a regulator of tomato plant resistance to oomycete
Phytophthora infestans [39]. Similarly, A. thaliana
plants excrete siRNA to extracellular medium within
vesicles that travel to the sites of fungal infection for
inhibiting expression of the genes responsible for
Botrytis cinerea virulence [40]. Excretion in plants of
such extracellular vesicles occurs not only in the case
of infection but also upon abiotic stress exposure and
phytohormonal treatment suggesting an inherent oper-
ation of RNAi mechanism in response to external influ-
ences. Therefore, such RNA transport in exosomes may
be used in the future for developing the means of deliv-
ery of siRNA or miRNA to the infection site.
Both sense and antisense sequences of miR393b
played an important role in formation of A. thaliana
resistance to bacterium P. s y r i n ga e pv. tomato regulat-
ing expression of protein MEMB12 from the SNARE
family. Suppression of MEMB12 synthesis in mutant
plants accumulating miR393∗ and AGO2 induces exo-
cytosis of protein PR1. The complementary strand of
miR393 also contributes to antibacterial responses via
interaction with protein AGO1 [18]. miR393 sup-
presses the expression of mRNA encoding F-box
auxin receptors TIR1, AFB2, and AFB3 that usually
promote expression of the genes of auxin response.
The results obtained with a pair of complementary
fragments miRNA∗/miRNA and proteins AGO1 and
AGO2 point to an efficient operation of plant immu-
nity on the basis of RNAi [18].
A special, finely tuned and multifunctional role in
regulation of plant resistance to viral [41], bacterial,
and fungal [42] infection is performed by isoforms of
miR168. It was shown that a predicted target of 21-nt
miR168 is protein Argonaute (AGO1), which is cor-
roborated by the accumulation of free mRNA of the
target in the mutants deficient by gene ago1 [20]. At
the same time in infected plants, tombusvirus protein
P19 that suppresses silencing and shows a high affinity
to 21-nt isoform of miR168 impedes its interaction
with AGO1 and reduces immune response [41]. In
plants of rice O. sativa infected with a hemibiotrophic
fungus Magnaporthe oryzae, miR319 showed comple-
mentarity to a key enzyme of JA synthesis (lipoxygen-
ase), which suppressed its synthesis and deteriorated
plant resistance [43].
There are interesting data concerning the regula-
tion in expression of defense genes by the miR482 gene
family of an exclusive origin targeted at the nucleo-
tide-binding domain of NB-LRR proteins [44]. Reg-
ulating activity of NB-LRR genes by means of miRNA

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MAKSIMOV et al.
(for instance, miR482f, miR825*, and miR5300) pro-
duces a dramatic effect on the development of plant
immunity [35]. In the case of bacterial infection, the
level of these miRNAs is reduced [45]. A suppression
of basal and RPS5-mediated resistance in Arabidopsis
plants to bacterium P. sy r i n g ae pv. tomato DC3000 via
control over expression of NB-LRR genes was detected
upon simultaneous accumulation in the tissues of pro-
tein RDR6 and miR472 [46]. RNAi mediated by
miR482/2118 is assumed to act as a sensor of pathologi-
cal process and a tuner of plants' immune response [30].
The RNAi pathway is very conservative and highly
selective: each recognizes and silences only its own
target RNA. The RNAi mechanism plays an import-
ant role in regulation of development, epigenetic mod-
ification, and plant responses upon exposure to differ-
ent stress factors [47], including pathogenic agents
[40]. In the first place, the basic protective role of
RNAi consists in formation of a unique inherent
defense strategy of the plant organism against viruses,
different pathogens, and even pests; it is described as
HIGS. Since RNAi is a product of coevolutionary
development of organisms, this effective defense
instrument may be successfully bypassed by patho-
gens, including viruses [40].
Suppression of host RNAi. The same as all other
defense systems, RNAi machinery has its weak points.
This efficient defense mechanism may be hacked by
pathogens, including viruses; i.e., plant metabiome
(including viruses and pests) may silence this process
at the expense of available potential in different stages
of development of the plant’s immune process. This
confrontation is an example of a difficult and violent
evolutionary struggle between viruses, pathogens, and
phytophages on the one hand and plants on the other
hand. Coevolution of suppressors and RNAi machin-
ery in plants also points to an extremely intricate
nature of adaptation of mutualists, symbiotrophs, and
pathogens to the plant’s defense system.
Suppressor activity of some proteins encoded in the
genome of viruses is described in detail in reviews [32,
48]. In contrast to HIGS, this phenomenon was called
virus-induced gene silencing (VIGS) [49]. Currently
accumulated data show that viral silencing suppressors
(VSR) possessing a wide range of biochemical proper-
ties are necessary for host RNAi both on transcrip-
tional and posttranscriptional levels [3] and at the
stage of signal transduction about the onset of the
infection process [50].
Protein HC-Pro (helper-component proteinase) is
the first discovered VSR encoded by viruses from the
family Potyviridae, which efficiently suppresses
defense reactions and promotes a rise in TMV and
CMV titers; this was shown in transgenic tobacco plants
producing this protein. In S. tuberosum plants infected
with the virus, protein HC-Pro formed a stable complex
with the enzymes of methionine cycle S-adenosyl-L-
methionine synthetase 1 and S-adenosyl-L-homocys-
teine hydrolase, ribosomal proteins, and viral protein
VPg–Pro as well as a key protein of RNAi—AGO1 [51].
Protein HC-Pro acted as a negative regulator of salicy-
late-dependent defense system via a direct interaction
with protein SABP3 [52]. Mutation by gene HC-Pro in
PVY restored resistance of potato S. tuberosum pos-
sessing gene Ny [53]. In the same way, protein C4 of
cotton leaf curl virus interacted with S-adenosyl-L-
methionine synthetase 1 of N. benthamiana inhibiting
the enzyme activity. In mutant tobacco plants with its
reduced activity, a suppression of the mechanisms of
gene silencing was detected, which stimulated devel-
opment of cotton leaf curl virus and tomato yellow leaf
curl China virus [54]. It is known that protein 2b of
CMV, protein P0 of potato leafroll virus, protein P38
of turnip crinkle virus capsid, and protein P1 of yam
mottle virus inhibited the RISC complex disturbing
operation of proteins from the AGO family [50]. Pro-
tein 2b of CMV is actively involved in the regulation of
plant defense systems, although it induces accumula-
tion of SA and JA in infected plants and suppresses
development of systemic resistance via salicylate- and
jasmonate-induced pathways [17]. Similarly, in trans-
genic plants of A. thaliana with hypersynthesis of pro-
tein 2b, it was shown to impair signal antistress pro-
gram also induced by ABA [55]. One may assume that
such an effect of CMV protein 2b agrees with the cur-
rent opinion that signal pathways induced by SA, JA,
and ABA interfere with one another. In addition, it
was detected that a disturbance of the jasmonate signal
system by protein 2b occurs via its interaction with
JAZ proteins, which makes host plants more attractive
for insects and facilitates virus transmission in arabi-
dopsis [56].
Beet western yellows virus encodes protein P0 that
interacts with a homolog of S-phase kinase (SKP) (a
component of the SCF complex) ubiquitin E3 ligase.
It was shown that F-box SKP interacts with the PAZ
domain in protein AGO1 and prepares it for degrada-
tion, and cessation of SKP gene expression ensures
resistance to viruses [57]. Hyperaccumulation of pro-
tein Р0 in transformed A. thaliana plants impaired
plant development and raised the siRNA level of target
transcripts indicating that protein P0 operates on the
level of the RISC complex. The ability of P0 to cause
degradation of AGO1 is an example of viral adaptation
to the RNAi defense mechanism. It was found that pro-
tein Р0 that expresses in S. tuberosum plants infected
with potato leafroll virus causes suppression of the
RNAi defense mechanism not only in plants but also in
green peach aphid that transmits this virus; this suggests
a similarity between the mechanisms of suppression of
this defense system in plants and their pests [58].
Protein P6 encoded by cauliflower mosaic virus
(CaMV) and requiring 35S RNA for translation sup-
pressed defense response in A. thaliana plants coin-
fected with the virus and pathogenic bacterium
P. sy r i n g ae pv. tomato; the defense reaction showed in
the suppression of oxidative burst and a reduced level

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RNA INTERFERENCE IN PLANT DEFENSE SYSTEMS 617
of SA and salicylate-dependent autophagia [59]. In
addition, it was detected that the suppressing effect of
protein P6 influenced both salicylate- and jasmonate-
induced defense systems affecting their key signaling
protein NPR1, which raised susceptibility to biotrophs.
At the same time, transgenic plants expressing protein
P6 were notable for a greater resistance to jasmonate-
sensitive pathogens and a susceptibility to salicylate-
sensitive pathogens [60].
Protein P19 of tomato bushy stunt virus from the
family Tombusviridae participating in the processes of
reproduction, translocation, RNA packaging, and
vector transmission of the virus suppressed defense
reactions in N. benthamiana plants. It was shown that
this protein is utterly necessary for systemic penetra-
tion and propagation of the virus in pepper (Capsicum
annuum) and spinach (Spinacia oleracea) plants. For-
mation of a complex between P19 dimers and ds-
siRNA both in vivo and in vitro correlated with the
intensity of viral disease symptoms in plants [61].
Thus, the role of protein P19 is to bind viral siRNA
that circulate in abundance during infection, make
them inaccessible for the RICS protein complex, and
interfere with protective methylation of siRNA.
Therefore, one may assume that the ability of P19 to
bind siRNA may impede the operation of enzyme
HEN1 responsible for methylation of siRNA. Full
inactivation of the gene encoding P19 results in a com-
plete degradation of viral RNA and makes impossible
the synthesis of viral proteins. Similarly, pretreatment
of tobacco plants with siRNA efficiently interacting
with protein P19 ensured defense against tomato bushy
stunt virus [61].
Protein P21 (21 kD) of beet western yellows virus
inhibits RNA-induced suppression of protein GFP
expression. In beet Beta vulgaris subsp. vulgaris plants
infected with the virus, protein P21 is detected in the
cytoplasm in a soluble form and as insoluble protein
bodies at the periphery of the cell. Another homolog
of protein P21 in citrus tristeza virus suppresses RNAi
on intracellular and intercellular levels [62] interacting
with dsRNA and siRNA in vivo and impeding their
methylation but does not affect the activity of the
RISC complex. In addition, short noncoding RNA
(low-molecular-weight tristeza 1, LMT1) were dis-
covered in the genome of citrus tristeza virus; they
accounted for its virulence in respect to tobacco plant
N. benthamiana, which showed in a sharp decline in the
level of SA in infected plants and in the activation of
alternative oxidase that suppresses oxidative burst [63].
Protein P38 (CP or p38) of turnip crinkle virus
capsid is responsible for systemic propagation and
intercellular translocation of the virus. This protein is
a powerful suppressor of RNAi, which is related to its
interaction with viral dsRNA irrespective of the size of
molecules. This means that interaction between P38
and dsRNA makes the substrate inaccessible to DCL
(for instance, to DCL4); this results in a decreased
accumulation of 21-nt siRNA by nuclease. At the
same time, protein P38 did not interfere with the
activity of protein DCL2 that produces 22-nt siRNA
but efficiently inhibited operation of protein complex
RISC interacting with AGO [64]. Such a mechanism
of suppression of the defense system is interesting
because, as was noted above, 22-nt siRNA showing an
affinity for protein AGO10 inhibited accumulation of
AGO1 [41].
Cysteine-rich 17 kD protein γb of barley stripe
mosaic virus (BSMV) is not necessary for replication
and translocation of the virus; however, it consider-
ably affects pathogenesis, and interaction of viral pro-
tein with RNA is a key function of protein γb in sup-
pression of RNAi. The first indirect indication of a
possible participation of protein γb in RNAi suppres-
sion was obtained in the experiments with a mutant of
tobacco rattle virus with a defective gene encoding
protein Р16. It was found that protein γb interacted
with dsRNA via three Zn-binding regions located in
the N terminus of protein and was activated in the
presence of Zn ions [65].
The product of gene p122 of TMV in S. tuberosum
plants infected with wild lines of the virus repressed
transcription of the genes Pidcl2 and NtDCL2 encod-
ing proteins and of RNA-dependent RNA-poly-
merase genes Pirdr1 and NtRdR1, respectively, in
tobacco N. tabacum plants infected with oomycete
Ph. infestans. Knock-out mutation by gene p122 in
TMVcr-Δ122 line restored the ability of the men-
tioned genes of plant and oomycete to express [66].
Viral genes encoding RNases belonging to family III
were detected to have a high suppressor activity against
antiviral RNAi [67]. In the family of potyviruses that
comprise plum pox virus, turnip mosaic virus, SMV,
and PVY, an important protein responsible for replica-
tion of viral RNA and suppression of the host’s
immune system is RNA-dependent RNA-polymerase
NIb encoded by the viral genome, which produces
replication complexes with participation of host pro-
teins and suppresses NPR1-mediated immune
response [68].
Phytohormones. Operation of the RNAi mecha-
nism in plants heavily depends on phytohormones.
Since SA is one of the key signal molecules responsible
for the induction of plant SAR to biotrophic patho-
gens, it is interesting to look into its ability to partici-
pate in the operation of RNAi machinery. Coordina-
tion between proteins involved in SAR and proteins
participating in RNAi is not completely decoded. The
main components of RNAi, such as endonucleases
DCL2, DCL3, or DCL4, were not associated with
SAR induced by SA or its functional analogs [69],
whereas transcriptional activity of gene RdR1 intensi-
fied under the effect of SA and depended on the
expression of protein NPR1 [4]. Accordingly,
SA-induced resistance to viruses was related to the
RNAi via gene RdR1 and was regulated by its product.

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In turn, expression of gene RdR1 intensified expres-
sion of the genes encoding RdR6 (RNAi system) and
activated alternative oxidase (pro- and antioxidant
system). Apparently, such coordination is flexible
since expression of gene AGO2 became salicylate-sen-
sitive in transgenic plants of A. thaliana accumulating
viral effector protein 2b [17]. It was detected quite
recently that SA and miR403a complementary to a
fragment of gene NbAGO2 efficiently regulated the
expression of gene NbAGO2 in tobacco plants N. ben-
thamiana [26]. Protein AtAGO2 from A. thaliana was
more efficient against PVX than NbAGO2 from
N. benthamiana [27]. Thus, the obtained data clarify
the issue of how defense systems responsible for devel-
opment of SAR and RNAi can cooperate in plants as
was earlier shown in transgenic NahG plants express-
ing another RNAi viral effector TEV P1/HC-Pro [70].
Potato line NahG-Désirée deficient in SA turned
out to be more susceptible to viruses than original cv.
Désirée; this was associated with a decline in the level
of miR164, miR167, miR169, miR171, miR319,
miR390, and miR393 [71]. Plant resistance and
miRNA level were restored after treatment with an SA
analog 2,6-dichloroisonicotinic acid [72]; i.e., the
salicylate system was involved in RNAi regulation via
expression of pre-miRNA.
Therefore, a relationship between RNAi and the
salicylate signal system in plants is not absolute. Since
proteins DCL2, DCL3, and DCL4 did not play a sig-
nificant role in SA-induced resistance of tobacco
plants to TMV and CMV [17], Lee et al. [4] assumed
that the antivirus defense system in tobacco plants
involving SA depends on other mechanisms, for
instance, on signal processes related to the operation
of alternative mitochondrial respiratory systems. Such
a relationship was mediated by inducing the expres-
sion of gene RDR1 regulated by SA [73]. This suggests
that SA in plants triggers several redundant or concur-
rent defense mechanisms related to DCL proteins or
independent of them.
An important role in SA-regulated resistance is
performed by miRNA, for instance, by miR160.
Potato lines with a suppressed expression of miR160
did not start up a systemic acquired resistance (SAR)
regulated by SA. Since signal systems regulated by SA
and auxins are antagonistic, one can suppose that
miR160 participates in their interplay [37].
Participation of ABA in plant defense against viral,
bacterial, and fungal infection was discussed earlier
[74]. This hormone usually regulates plant responses
to abiotic stress and participates in phytoimmune pro-
cesses preventing pathogens from colonizing plant tis-
sues via regulation of stomatal movement, expression
of genes of the pro- and antioxidant system, and cal-
lose synthesis. Eventually, ABA was detected to play
an important role in regulation of the RNAi mecha-
nism and, accordingly, in the formation of plant viral
immunity [55]. In A. thaliana plants hyper-producing
ABA, the accumulation of transcripts of genes AtAGO4
and AtAGO10 is induced. Similarly, in Rsv3 soybean
lines resistant to mosaic virus, ABA induced accumu-
lation of transcripts of gene GmDCL2 [75]. The role of
ABA in the formation of resistance to viruses with par-
ticipation of RNAi mechanisms was summarized in a
review by Alazem and Lon [22]. For instance, the
role of this hormone in resistance to bamboo mosaic
virus was associated with induced expression of genes
AtAGO1, AtAGO2, and AtAGO3 and repressed accumu-
lation of transcripts of genes AtAGO4 and AtAGO10.
In physiological reactions, SA and ABA are often
considered to act as antagonists; however, they can
jointly modulate different defense responses (includ-
ing RNAi) specifically to viruses on the level of regu-
lating expression of the genes encoding transcription
factors (trans-factors) and the major proteins involved
in the RNAi pathway. Since ABA induced expression
of the genes AGO1 and RdR1 only in mutant by SA
level plants, one can assume that ABA triggers expres-
sion of the genes encoding a cascade of signals of salic-
ylate-dependent expression and SA regulates intensity
of accumulation of protective product [22]. It was
revealed that the plants with active AGO1 synthesis
are low-sensitive to ABA, while, vice versa, they
develop a hyper-sensitivity to ABA upon a disturbance
of AGO1 synthesis. In soybean plants, resistance to
mosaic virus caused by ABA treatment was accompa-
nied by a deposition of callose in the zone of plas-
modesmata and accumulation of transcripts of genes
AGO1, AGO4, AGO9, RdR2, and RdR6 [22]. It was
shown that regulation of AGO1 synthesis with the par-
ticipation of ABA much depends on miR168 that reg-
ulates plant immunity to viruses [41]. The obtained
data point to a close relationship between RNAi
mechanisms and ABA-mediated signal pathway of
formation of plant resistance to viruses.
Jasmonates induce a resistance in plants to necro-
trophs and pests. In respect to viral infections and
biotrophic and hemibiotrophic pathogens, their role is
contradictory. Analysis of expression of AGO genes in
O. sativa plants infected with rice stripe virus (RSV)
showed that the virus considerably induced genes
OsAGO1a, OsAGO1b, OsAGO3, and OsAGO18 whose
transcripts in jasmonate-insensitive coi1-13 plants
accumulated much weaker, which correlated with the
extent of virus spreading [30]. In tobacco plants, the
formation of resistance to the fungus F. br a c h y g i bb o -
sum based on the jasmonate signal pathway critically
depended on the presence of protein NaAGO4 [9].
Accumulation of jasmonic acid (JA)—triggered by
capsid (coat) protein of rice stripe virus—that ensures
resistance of rice O. sativa plants to RSV is associated
with activation of gene OsAGO18 [30]. Reduction in
JA accumulation caused by induction of miR319 in
O. sativa plants infected with rice ragged stunt virus
(RRSV) created a susceptibility to the virus [76]. It was
revealed that activation by bacterium Bacillus amylo-
liquefaciens FZB42 of A. thaliana resistance via a jas-

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RNA INTERFERENCE IN PLANT DEFENSE SYSTEMS 619
monate signaling pathway is controlled by miR846.
This was corroborated by the fact that both overex-
pressing miR846 and knockdown miR846 lines of
A. thaliana, as well as the plants treated with an inhib-
itor of JA synthesis diethyldithiocarbamic acid, turned
out to be susceptible to P. s yr i ng a e DC3000 [77].
In order to properly comprehend the mechanism of
molecular relationships between the defense system of
the organism and viruses, thorough molecular, bio-
chemical, and structural investigations of viral sup-
pressors are necessary. However, available data point
to a serious coevolutionary arms race between host
plants and pests. In time, this knowledge may be put
into practice for realization of efficient strategies of
creating plants resistant not only to viruses but also to
other pathogens and pests.
RNAi and crop protection. One of the progressive
lines in this area is the application of RNAi-based
methods of genetic engineering and working out new
approaches to crop protection against various patho-
gens and pests. A unique RNAi mechanism discovered
by Fire gave biologists a new instrument of control
over the plant phenotype and estimating the biological
function of proteins. Strategies for application of
RNAi mechanisms rest on a basic knowledge about
the formation of inherent hairpin structures by viral
RNA and the creation of artificial siRNA (on the basis
of pathogenic mRNA) and ta-siRNA (on the basis of
mRNA of the genes encoding suppressors of host
immune response). At present, attention is focused
on constructing genetically modified (GM) and
genetically edited (GE) cultivars and hybrids with
improved productivity and elevated quality, includ-
ing CRISPR/CasN technology of gene editing inte-
grated with components of RNAi technology [78].
The first plant with artificial protection against viral
infection based on RNAi, which received a license for
application in 1998, was potato with inserted gene
Orf1/Orf2 from potato leafroll virus [79]. Successful
tests were then conducted with GM plants of plum
Prunus domestica carrying a gene-encoding CP of
latent mosaic virus. In the United States, GM and
GE cultivars of pumpkin Cucurbita pepo and pawpaw
Carica papaya resistant to latent mosaic virus and
ringspot virus, respectively, are being actively intro-
duced into agriculture; in China, cultivars of sweet
bell pepper Capsicum annuum and tomato S. lycoper-
sicum are being introduced. In their review [80], Sang
and Kim listed at least 11 efficient technologies of
crop protection based on RNAi methods; for
instance, they described protection of barley Hor-
deum vulgare and wheat Triticum aestivum against
fungus Fusarium graminearum with suppression of
expression of genes FgCYP 51 (gene of ergosterol bio-
synthesis) and FgChs3b (gene of chitin synthase),
respectively. Dow AgroSciences and Monsanto (now
Bayer CropScience) companies inserted SmartStax
PRO (MON87411), a vector expressing dsRNA of
gene snf7 from western corn rootworm Diabrotica
virgifera virgifera, into genome of maize Z. mays, cv.,
in order to protect transgenes in case of formation in
the pests of resistance to Bt toxin (Bacillus thuringien-
sis) [81].
It is known that PTGS of target genes may be
caused by direct introduction of dsRNA into plant
cells by means of transgenosis. New gene editing sys-
tems (GES) extend the list of available instruments
ensuring plant resistance to viruses with the use of
RNAi components. The RNAi method showed the
importance of reception of pathogen-associated
molecular patterns (PAMP) (in particular, chitin) in
subsequent development of oxidative burst in the zone
of infection. For instance, silencing of gene CEBiP
responsible for the binding of chitooligosaccharides in
O. sativa plants by means of RNAi technology inhib-
ited the formation of reactive oxygen species and
expression of the genes participating in immune
response [82]. Singh et al. [78] showed that potato
S. tuberosum lines expressing genetically engineered
construct CRISPR/Cas13a containing siRNA against
RNA of potato virus Y accumulated less viruses in the
tissues and, therefore, the symptoms of disease therein
were less pronounced. A direct positive correlation
was observed between resistance of transgenic lines
and the level of siRNA expression.
Interesting results were obtained for maize Z. mays
protection from fuminosin-producing strains of fun-
gus Fusarium verticillioides using antisense constructs
to respective genes FUM1 and FUM8, which made it
possible to considerably lower the concentration of
toxin [83]; the authors believe that this ensures the
creation of plant forms with respective genes for con-
trol over toxin production by the pathogen. In geneti-
cally modified plants with antisense fragments of
vitally important genes of insects, their harmfulness
was greatly reduced. For instance, by using such frag-
ments people may modify transcriptional activity of
the genes related to the ability of insects to degrade
nicotine (tobacco hornworm Manduca sexta,
CYP6B46) [84]) and affect expression of the genes
encoding trypsin-like serine protease (rice brown plan-
thopper Nilaparvata lugens, Nltry) and transmembrane
hexose transporter protein (N. lugens, NlHT1) [85]. At
the same time, transgenic plants in general (including
those carrying RNAi genes) are negatively accepted by
the public at large, pose many questions, and stir up dis-
putes on legal and ecological levels.
Especially interesting are the works that propose
using RNAi mechanism with a target dsRNA as
sprays, which opens up new horizons in development
of scientific technologies and crop protection strate-
gies for the purpose of disturbing expression of vital
genes in pests and pathogens. This quickly developing
line of crop protection is called spray-induced gene
silencing (SIGS) [80, 86]. This method is unique since
dsRNA themselves can possess PAMP properties and
trigger a defense system of the plant cell related to PTI

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MAKSIMOV et al.
and ETI classic signal systems of defense mediated by
activation of somatic embryogenesis receptor-like
kinases (SERK) and mitogen-activated protein
kinases (MAPK) weakly interacting with RNAi mech-
anisms. On the other hand, dsRNA can get to plant
tissues, undergo amplification, turn into siRNA (as a
rule, 22-nt), induce inherent mechanisms of RNAi,
and cause silencing of target genes in a pest, pathogen,
or virus after application of dsRNA preparations on
the plant surface [86].
There exist examples when preparations containing
dsRNA were used to control gene expression in patho-
gens. For instance, application of a preparation con-
taining a target dsRNA on leaf surface in N. benthami-
ana and Vigna unguiculata induced in the plants RNAi
mechanisms with subsequent degradation of RNA of
mosaic virus not only in the case of mechanical pene-
tration of the virus through the leaf surface but also
upon insect-mediated inoculation [87]. Sammons et
al. [90] showed that exogenous dsRNA can affect the
suppression of genes responsible for resistance to her-
bicide, which may be employed to reduce pesticide
resistance in weeds.
Tenllado et al. [88] were the first to show that exog-
enous application of dsRNA molecules as a spray
makes plants resistant to viral infections. Since then,
the technology of application of dsRNA against viral
infection has improved. In order to elevate dsRNA sta-
bility and prolong antiviral defense, Mitter et al. [89]
loaded dsRNA on layered clay nanosheets 80–300 nm
in size (BioClay). Such a complex turned out to be
more protected from nucleases and did not wash off
from the leaf surface; 330-nt dsRNA targeting CP of
CMV could be detected on sprayed plants even 30 days
after treatment [89]. Similar results were obtained
after spraying the plants of tobacco N. benthamiana
and cow pea Vigna unguiculata with a preparation con-
taining nanoparticles of BioClay and 461-nt dsRNA
targeting the capsid protein of bean mosaic virus aim-
ing to silence CB [87].
Prospects of the fight with transmitters of viruses
on the basis of RNAi are also discussed in the context
of creating different biological pesticides in the form of
RNAi sprays [90], including biological insecticides
(RNAi-insecticides) when the spraying of vegetating
plants with dsRNA suppresses the genes of pathogen
virulence or pest vital functions [91]. Successful works
have been recently performed with the application of
dsRNA preparations against the larvae of Asian corn
borer (Ostrinia furnacalis), insects from the order Dip-
tera (mosquitoes and tsetse f ly) and order Hemiptera
(aphids and whitefly) as well as gall, cyst-forming, and
other plant-parasitic nematodes. It is worth noting
that mechanisms of RNAi triggering in different
groups of insects physiologically differ what makes the
researchers differentially approach the development of
such preparations [92]. BASF published reports about
application of preparations based on RNAi technology
(https://www.researchandmarkets.com/reports/5007781/
agricultural-biotechnology-emerging-technologies).
By the present time, this corporation has taken out
patents for several inventions in the area of control
over nematodes employing the RNAi mechanism. It is
assumed that life time of RNA components of prepa-
rations will last for at least 20–30 days. This pattern
fundamentally differs from the approach to crop pro-
tection based on genetic modification but requires
elaborating the methods of delivery of such RNAi-
insecticides, RNAi-fungicides, or RNAi-viricides and
protection of RNA molecules from sunlight and wash-
ing off.
It was found that intake of dsRNA by insect organ-
isms with feed may inhibit the expression of target
genes and, therefore, induce wanted phenotypic
effects, for instance, death of larvae or grown-up spe-
cies, and reduced prolificacy [92]. For instance, in a
number of dangerous insects (Mythimna separate, Haly-
omorpha halys, Nilaparvata lugens, Lymantria dispar,
Helicoverpa armigera, Bemisia tabaci, Spodoptera exi-
gua, Chilo suppressalis, etc.) oral intake of dsRNA sup-
presses the activity of specific target genes. In Colo-
rado beetle Leptinotarsa decemlineata, it was shown
that inhibition of dsRNA-specific RNases boosts the
effect of RNA-insecticides upon oral intake [93]. In
China, maize Z. mays and soybean Glycine max lines
were produced, which express fragments of ATPase gene
from mirid bug Apolygus lucorum; in these lines, damage
caused by this pest was considerably reduced [94].
Preparations based on RNAi should be produced with
due consideration for the fact that the mechanism of
regulating silencing defense genes in plants colonized
by pests may depend on symbiont microbiomes that
possess their own systems of plant defense suppression
ensuring more active occupation of a feed niche.
CONCLUSIONS
Current molecular and biochemical investigations
of suppressors of plant immune reactions of different
origin considerably broadened our notions about the
complexity of RNAi pathway and promoted under-
standing of the nature of interactions between plants
and pathogens. In addition, thousands of small regu-
latory RNA have been identified and the mechanism
of RNAi has been thoroughly studied in individual
plant objects (for instance, in Arabidopsis). However,
many issues are yet to be clarified in this area. For
instance, we know little about the role of RNAi in reg-
ulation of defense against pathogens in lower terres-
trial plants and algae and about its role in relationships
between plants and their parasitic forms. Physiological
mechanisms of interaction between RNAi and classic
signal phytoimmune systems responsible for systemic
resistance that are regulated by SA and JA remain
unknown. However, one may believe that RNAi
mechanisms in plant cells are independent of PAMP-
induced signal systems of defense and relatively insen-

RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 68 No. 4 2021
RNA INTERFERENCE IN PLANT DEFENSE SYSTEMS 621
sitive to such low-molecular signal molecules as JA
and SA, reactive oxygen species, calcium ions, and
other secondary messengers. Possible investigations
may also concern the operation of RNAi mechanisms
under formation of a multilevel plant microbiome
consisting of at least pathogen and endophyte and/or
symbiont as in the system “host-pathogen-endophytic
bacterium.”
On the one hand, since the RNAi mechanism is
inherent and efficiently operates in all taxons of living
organisms, it is assumed that pathogens (viruses, bac-
teria, fungi, and oomycetes) and insects cannot easily
overcome such a defense. On the other hand, since a
desired effect is produced in the case of exogenous
dsRNA application without modification of the
plant’s genome structure, this technology does not
come under prohibitions related to GMO. Indeed, in
2018 the New Zealand Environmental Protection
Authority issued a decision that the organisms treated
with dsRNA do not belong to GMO [95] and, thereby,
approved the use of preparations containing dsRNA
for induction of RNAi and control over gene expres-
sion (https://www.epa.govt.nz/assets/FileAPI/hsno-ar/
APP203395/APP203395-Decision-FINAL-.pdf). The
Ministry for Primary Industries of New Zealand included
RNA in the list of environmentally safe substances
(https://www.mpi.govt.nz/dmsdocument/5134). How-
ever, this does not settle issues concerning prospects
and safety of application of such a profitable approach
to improvement of plant resistance to viruses, patho-
gens, and pests since the potential and safety of exog-
enous dsRNA application are poorly investigated and
the risks were not completely assessed. At the same
time, one may believe that transgenes on the basis of
RNAi are profitable and ecologically friendly since
such plants do not produce alien proteins and biocidal
substances and do not pollute the environment.
Data concerning the operation of RNAi machinery
make it possible to work out efficient strategies of
struggle against a complex of pathogens and pests in
agrocenosis promoting elevation of crop productivity
and keeping off from ecologically harmful pesticides.
Molecular switching over (activation) of the genes
responsible for resistance to diseases and host-
induced silencing of pathogen and even pest genes also
offer an opportunity to produce a new generation of
pesticides on the basis of dsRNA. It is worth noting
that control over numbers of pests, pathogens, and
viruses in agrocenosis may be implemented either via
classic insertion of vectors with RNAi constructs into
the plant genome and regulation of inherent HIGS
mechanisms or through application of dsRNA as
sprays for direct treatment of plants or attraction of
endophytes as generators of RNAi.
Thus, taking into consideration political and public
pressure for safe solution of current agricultural prob-
lems, exogenous application of dsRNA molecules
capable of triggering RNAi is a powerful instrument in
current platforms of yield protection and improvement.
Available data show that the presence of RNAi pathway
molecules in food is not dangerous to humans [96].
Under experimental conditions, target dsRNA produce
a beneficial protective effect; however, there remain
unsettled issues concerning the selection of dsRNA
hyper-producers, the mass accumulation of target
product, and ensuring its preservation in f ield condi-
tions for crop protection from pathogens and pests.
FUNDING
This work was supported by a joint international grant
given by the Russia Science Foundation of the Russian
Federation no. 19-46-02004 and the Department of Sci-
ence and Technology (DST), Government of In dia, project
no. C/1756/IFD/2019-20.
COMPLIANCE WITH ETHICAL STANDARDS
Conf lict of interests. The authors declare that they have no
conf licts of interest.
Statement on the welfare of animals. This article does not
contain any studies involving animals performed by any of the
authors.
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Translated by N. Balakshina