# Antiviral Double-Stranded RNA Sensing Immunity in Plants

Manfred Heinlein

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Manfred Heinlein. Antiviral Double-Stranded RNA Sensing Immunity in Plants. Annual Review of Virology, 2025, 12, pp.421-449. 10.1146/annurev-virology-092623-101447. hal-05289995

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*Annual Review of Virology*

# Antiviral Double-Stranded RNA Sensing Immunity in Plants

Manfred Heinlein

Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, Strasbourg,
France; email: manfred.heinlein@ibmp-cnrs.unistra.fr

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Annu. Rev. Virol. 2025. 12:421–49

The *Annual Review of Virology* is online at
virology.annualreviews.org

https://doi.org/10.1146/annurev-virology-092623-101447

Copyright © 2025 by the author(s). This work is
licensed under a Creative Commons Attribution 4.0
International License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are credited.
See credit lines of images or other third-party
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Keywords

virus, plant, recognition, RNA silencing, pattern-triggered immunity, plasmodesmata

Abstract

Plant viruses, which can cause devastating plant diseases, are obligate in-
tracellular pathogens that replicate their genomes inside cells and spread
infection by cell-to-cell movement through cell wall nanochannels called
plasmodesmata (PD). Double-stranded RNA, which occurs as a replica-
tion intermediate of RNA viruses, triggers adaptive and innate host defense
responses that are controlled by virus-encoded effector proteins. These de-
fenses include RNA silencing and RNA decay, which target viral RNA and
inhibit virus accumulation, and pattern-triggered immunity (PTI), which
targets PD and inhibits virus movement. This review discusses the role of
RNA silencing, RNA decay, PTI, and effector-triggered immunity as antivi-
ral defense mechanisms, how they are interrelated, and how viruses interact
with these mechanisms to ensure their successful replication and spread
throughout the plant organism.
PD: plasmodesmata

dsRNA:
double-stranded RNA

INTRODUCTION

Viruses are the most abundant entities in the biosphere and important drivers of evolution. Their ability to modify, replicate, and transfer genetic information has contributed to the diversity of life. Continuous interaction and coevolution with their hosts have resulted in compatible mechanisms for viral replication and spread and a constant struggle for survival, driving the evolution of host defenses and viral countermeasures to control viral spread (1–4). The plant virome is dominated by RNA viruses (5). Together with various DNA viruses, they infect economically important crops and cause global yield losses (6). Viruses infect plants by replicating and spreading their genomes between cells through gated nanochannels in plant cell walls called plasmodesmata (PD) (7, 8) (Figure 1). Now that the cellular mechanisms of viral replication and movement have been extensively studied, current research is focusing on the mutual host-virus defenses that control these processes. Virus replication triggers antiviral RNA silencing, and viruses control this adaptive host immune response with suppressor proteins. In addition, virus replication triggers host responses that lead to the closure of PD and inhibit virus movement. Recent studies have placed the regulation of PD and virus movement in the context of innate immunity. The interaction of viruses with the adaptive and innate immune systems determines host susceptibility and resistance, and further understanding of their nature and interactions will facilitate the development of innovative strategies for virus control in agriculture.

CONTROL OF VIRUS INFECTION BY RNA SILENCING AND DECAY

Plants detect the presence of viruses by recognizing double-stranded RNA (dsRNA), which is pro-
duced as an essential intermediate molecule during viral replication. These viral dsRNA molecules

<figure><img src="image_5.png" /><figcaption>**Figure 1**</figcaption></figure>

Local and systemic virus spread in infected plants. Viruses infect plants by replication and movement through PD. By local PD-mediated cell-to-cell movement in the inoculated leaf, the virus reaches the phloem, which allows long-distance transport to other plant organs. After unloading from the phloem, the virus moves again from cell to cell through the PD to further spread infection. Images show local and systemic infection by green fluorescent protein–tagged tobacco mosaic virus in *Nicotiana benthamiana*. Abbreviation: PD, plasmodesmata.
activate adaptive immunity through an antiviral RNA silencing mechanism in which the dsRNA is recognized by DICER-LIKE (DCL) enzymes (DCL2 and DCL4), which convert the dsRNA into 21-nt and 22-nt small interfering RNA (siRNA) duplexes. These viral siRNA (vsiRNA) duplexes are then loaded into the AGONAUTE (AGO)-associated RNA-induced silencing complex (RISC), in which one of the two strands directs specific targeting of the viral genome for endonucleolytic cleavage or translational repression in a nucleotide sequence-specific manner. This antiviral host defense response is enhanced by RNA-dependent RNA polymerase 6 (RDR6), which uses the viral RNA cleavage products as templates for the production of new dsRNAs, which are also processed by DCL2 and DCL4, leading to the production of secondary vsiRNAs (9) (Figure 2a). To support virulence, viruses have independently evolved viral suppressors of RNA silencing (VSRs) that interfere with specific components of the host RNA silencing pathway (10,

siRNA: small interfering RNA

RISC: RNA-induced silencing complex

VSR: viral suppressor of RNA silencing

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(Caption for Figure 2 appears on following page)
<caption>**Figure 2** (*Figure appears on preceding page*)</caption>

Host antiviral defense through RNA silencing and decay. (a–c) Host antiviral RNA silencing and suppression by VSRs. (a) RNA viruses induce host antiviral RNA silencing by dsRNA produced during viral replication. The dsRNA is recognized and cleaved by DCL4 and DCL2 into 21- and 22-nt siRNAs, respectively, which are loaded onto AGO-RISCs to direct viral RNA cleavage in a sequence homology-dependent manner. Antiviral RNA silencing is enhanced by RDR6, which uses cleaved viral RNA molecules as templates to produce new dsRNA. This secondary dsRNA is processed by DCL enzymes into secondary siRNAs, which direct the amplified cleavage of viral RNA. Viruses encode VSR proteins that interfere with specific steps of the antiviral RNA silencing pathway. Antiviral RNA silencing is also inhibited by RNA decay, which is subject to substrate competition with RDR6. (b–c) DNA viruses are controlled by PTGS and TGS. PTGS (b) targets viral RNA transcripts by converting them into dsRNAs by RDR6, followed by dsRNA processing into 21- and 22-nt siRNAs by DCL4 and DCL2. As in RNA viruses, siRNA-directed AGO-RISC-mediated RNA target cleavage is amplified by RDR6, presumably in competition with RNA decay. TGS (c) targets the viral genome for transcriptional inhibition and depends on RdDM pathways that may be directed either by PTGS-derived 21- and 22-nt siRNAs that enter the nucleus or by 24-nt siRNAs derived from nuclear Pol IV transcripts that are converted to dsRNA by RDR2 and further processed by DCL3. These siRNAs are loaded onto AGO4 (or a related AGO protein) and guide AGO4 to nascent Pol V transcripts in chromatin, leading to the recruitment of a DNA methyltransferase (DRM1/2) to methylate cytosines at the target site. The methylated, silenced state of the DNA is maintained by the proteins SUVH2 and SUVH9, which bind to methylated DNA and recruit Pol V. RdDM is also supported by SHH1, which binds to H3K9me2 and recruits Pol IV with the help of the CLSY1 protein. Like RNA viruses, DNA viruses also encode VSR proteins that interfere with specific steps of the antiviral RNA silencing pathways. (d–e) Viral infection triggers the production of host siRNAs. (d) RNA decay is an RNA quality control mechanism that degrades aberrant RNA molecules. After decapping and deadenylation, aberrant RNA molecules are degraded by 5'-3' exonucleases such as XRN4 and in the 3'-5' direction by the exosome. Viral RNA that accumulates during viral infection can overload the RNA degradation pathway and cause aberrant host RNAs to escape RNA degradation. These RNAs are subjected to PTGS by conversion to dsRNA and DCL-mediated cleavage into 21- and 22-nt ct-siRNAs. (e) Virus infection can lead to the production of vasiRNAs from coding genes. The example shows the production of vasiRNAs from the *Brassica napus* NBR1 gene induced by TuMV infection. By inducing vasiRNA production from this gene, TuMV can downregulate NBR1 expression and autophagy of its VSR. In addition, the NBR1-derived vasiRNAs can target several other *B. napus* genes for PTGS. Thus, NBR1 acts as an important gene regulatory hub during TuMV infection in this host. Abbreviations: AGO, Argonaute; AGO-RISC, ARGONAUTE-associated RNA-induced silencing complex; CBP, CAP-binding protein; CLSY1, CLASSY 1; ct-siRNA, coding transcript-derived siRNA; DCL, DICER-LIKE; DRM, DOMAINS REARRANGED METHYLTRANSFERASE; dsRNA, double-stranded RNA; H3K9me2, histone 3 lysine 9 dimethylation; mRNA, messenger RNA; NBR1, NEIGHBOR OF BRCA1; ORF, open reading frame; PABP, polyadenylate-binding protein; Pol, polymerase; PTGS, post-transcriptional gene silencing; RdDM, RNA-directed DNA methylation; RDR, RNA-dependent RNA polymerase; SHH1, SAWADEE HOMEOPROTEIN HOMOLOG 1; siRNA, small interfering RNA; SUVH, SU(VAR)3-9 homolog; TGS, transcriptional gene silencing; TuMV, turnip mosaic virus; vasiRNA, virus-activated siRNA; vRDR; viral RNA-dependent RNA polymerase; VSR, viral suppressor of RNA silencing; XRN4, exoribonuclease 4.

11). siRNAs can move through PD and phloem (12–14), and the systemic mobility of vsiRNAs has been implicated in immunizing uninfected cells and preventing viral entry into the shoot apical meristem (SAM), thereby maintaining a virus-free SAM stem cell niche required for plant development (15). (See the sidebar titled Meristem Exclusion Is Not the Rule.) By immunizing cells ahead of the viral infection front, mobile vsiRNAs may allow virus-infected plants to recover from symptoms in newly grown leaves, which is usually caused by antiviral RNA silencing associated with reduced virus titers (16). (See the sidebar titled Symptom Recovery Can Also Be Caused by Induced Tolerance.)

DNA viruses such as cauliflower mosaic virus (CaMV) or geminiviruses are controlled by post-transcriptional gene silencing (PTGS) (Figure 2b) targeting viral RNA transcripts for

**PTGS:**
post-transcriptional
gene silencing

MERISTEM EXCLUSION IS NOT THE RULE

The ability of viruses to enter the apical meristem of plants is highly relevant for their vertical transmission by seeds. Thus, meristem exclusion is not the rule, as several viruses and viroids can invade the meristems of their host plants (162). In cases of stem cell exclusion, further research should clarify how the meristem, in contrast to antiviral RNA silencing in the rest of the plant, can evade viral suppressor of RNA silencing-mediated repression.
SYMPTOM RECOVERY CAN ALSO BE CAUSED BY INDUCED TOLERANCE

Although recovery is often caused by antiviral RNA silencing associated with reduced virus titers, it may also reflect mechanisms involved in tolerance and virus-host symbiosis (159, 163). The viral suppressors of RNA silencing (VSRs) of several viruses, including those of tobamoviruses, inhibit RNA silencing by sequestration of small RNAs. Sequestration of microRNAs (miRNAs) may interfere with miRNA-mediated gene regulation during plant development, thus causing disease symptoms. Consistently, in a study addressing symptoms recovery of *Arabidopsis* plants infected with oilseed rape mosaic virus (ORMV), symptoms recovery correlated with the inactivation of VSR activity (159). In this case the recovered leaves showed normal viral accumulation, indicating that recovery occurred by achievement of a tolerant state rather than through virus silencing. Because recovery was restricted to young leaves and depended on nuclear and cytoplasmic small interfering RNA (siRNA) synthesis, it has been proposed that recovery occurred upon saturation of the VSR siRNA binding activity by siRNAs recruited via the phloem from the highly infected mature source leaves (159). This case of induced tolerance observed in ORMV-infected plants may serve as a model for studying the molecular mechanism of tolerance and for creating crops that are more tolerant to viruses in the environment.

siRNA-mediated cleavage and also by transcriptional gene silencing (TGS), which targets the viral DNA for RNA-directed DNA methylation (RdDM) (17–22) (Figure 2c). TGS involves 24-nt siRNAs produced by DCL3-mediated processing of dsRNAs derived from short tran- scripts generated by RNA polymerase IV (Pol IV) (23, 24) and converted to dsRNAs by RDR2 (25, 26). The 24-nt siRNAs are loaded into AGO4, which recruits the DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA cy- tosines at target sites that are associated with Pol V-generated transcripts, providing a scaffold for the binding of the AGO4-24-nt siRNA complex (27, 28). The Su(var)3-9-like proteins SUVH2 and SUVH9 play an important role in recruiting Pol V to chromatin regions already marked by DNA methylation. Loci targeted by RdDM tend to associate with histone 3 lysine 9 dimethylation (H3K9me2). The chromatin reader SAWADEE HOMEOPROTEIN HOMOLOG 1 (SHH1) binds to H3K9me2 and, together with the CLASSY1 (CLSY1) protein, recruits Pol IV to further reinforce RdDM (5). RdDM can also occur through siRNA-driven pathways that are independent of DCL3 (Figure 2c). The defensive role of viral chromatin methylation against DNA viruses is supported by the hypersensitivity of methylation-deficient mutant plants to geminivirus infection and the existence of numerous viral countermeasures against viral DNA methylation. Neverthe- less, the mechanisms of Pol IV-mediated RdDM and histone methylation have been inferred from investigations into plant endogenous targets, and numerous mechanistic details pertaining to TGS of DNA viruses remain to be elucidated.

In addition to RNA silencing, plants use exonuclease-mediated RNA decay to degrade viral RNA (11, 29, 30). RNA decay plays an important role in RNA quality control, usually by removing redundant or aberrant messenger RNA (mRNA) molecules. Such mRNAs are either deadenylated and further degraded in the 3'-5' direction by the exosome complex or decapped and degraded in the 5'-3' direction by a 5'-3' exoribonuclease (XRN) (31) (Figure 2d). Attenuation or genetic mutation of components of either pathway allows viral RNAs to accumulate at high levels, and overexpression of such components attenuates viral infection (29). As with RNA silencing, viruses also have evolved counter-defensive mechanisms against RNA decay. These mechanisms include functional interference with essential RNA decay proteins through binding by VSRs (29, 32) or evasion, such as avoiding decay-inducing signals (30) or protecting viral RNA from access by the RNA decay nucleases (11).

TGS: transcriptional gene silencing

RdDM: RNA-directed DNA methylation
**ct-siRNAs:**
coding transcript-derived siRNAs

**PTI:** pattern-triggered immunity

**ETI:**
effector-triggered immunity

**PRR:**
pathogen-recognition receptor

**RLK:** receptor-like kinase

Because RNA decay and RNA silencing both control RNA levels and compete for RNA substrates (Figure 2a,b), their mechanisms are closely linked. Several key components of the RNA decay pathway that eliminate aberrant transcripts have been shown to act as suppressors of gene silencing, i.e., their disruption leads to post-transcriptional silencing of coding genes by converting their transcripts into dsRNA by RDR6, followed by processing of these dsRNAs into coding transcript-derived siRNAs (ct-siRNAs) by DCL2 and DCL4 (33–36). Overloading the antiviral RNA decay pathways with viral RNA during infection can have the same effect, i.e., antiviral RNA silencing and post-transcriptional silencing of coding genes can be triggered when RNA decay fails to degrade viral and host RNA (Figure 2d). However, as long as RNA decay is active, it counteracts antiviral silencing by undergoing substrate competition with RDR6 and cleaving the viral RNA template required for secondary vsiRNA production (29, 34, 35) (Figure 2a,b). Viral infection of *Arabidopsis* and other *Brassicaceae* plants triggers the accumulation of siRNAs derived from coding genes (37–39). Possibly, these virus-activated siRNAs (vasiRNAs) could represent ct-siRNAs generated upon viral saturation of RNA decay pathways, which remains to be investigated. However, rather than being just a consequence of viral infection, the production of vasiRNAs may have evolved as a means of preventing the overexpression of host genes during infection, thus playing an essential role in virus-host compatibility. Many of these genes encode factors with key roles in photosynthesis and stress responses. One of the vasiRNA-producing genes induced by turnip mosaic virus (TuMV) infection in *Brassica napus* encodes a homolog of the *Arabidopsis* autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) (38). In *Arabidopsis*, the NBR1 protein plays an important role in the degradation of viral proteins and particles by autophagy and has also been shown to specifically target the HC-Pro VSR of TuMV (40). Thus, by inducing the conversion of the NBR1 transcript into vasiRNAs, the virus may gain control over the autophagy-mediated host defense response. Strikingly, degradome analyses revealed that NBR1-derived vasiRNAs direct RISC-mediated cleavage of numerous *B. napus* transcripts, suggesting that NBR1 functions as a virus-induced hub for broad control of host gene expression during infection (38) (Figure 2e).

# CONTROL OF PLANT VIRUS INFECTION BY INNATE IMMUNITY

## The Innate Immune System of Plants

The plant innate immune system consists of two interrelated layers, termed pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) (Figure 3a). PTI acts as the first layer and is initiated by the recognition of conserved microbe-associated molecules by pathogen recognition receptors (PRRs) at the plasma membrane (PM), which activate further signaling and rapid defense responses. PRRs are mainly represented by PM-resident receptor-like kinases (RLKs) and receptor-like proteins (RLPs) with different extracellular domains [e.g., leucine-rich repeats (LRRs), lysine motifs (LysMs), epidermal growth factor (EGF)-like repeats, lectin motifs, or malectin-like motifs] for the specific recognition of conserved microbe-derived molecules known as microbe- or pathogen-associated patterns (MAMPs, PAMPs). Specific PRRs also recognize danger-associated molecular patterns (DAMPs), which are released by cells when they are damaged by pathogens. DAMPs can include ubiquitous cellular molecules [constitutive DAMPs (cDAMPs)] or molecules that are absent in uninfected cells and induced by the pathogen [inducible DAMPs (iDAMPs)] (41, 42). The perception of DAMPs or MAMPs/PAMPs by PRRs often depends on the recruitment of coreceptors belonging to the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family (43). For example, the PRR-RLKs BRASSINOSTEROID INSENSITIVE 1 (BRI1), FLAGELLIN-SENSING 2 (FLS2), and ELONGATION FACTOR-Tu (EF-Tu) RECEPTOR (EFR) form complexes with BRI1-ASSOCIATED RECEPTOR KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) upon
<figure><img src="image_7.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_9.png" /></figure>

(Caption for Figure 3 appears on following page)
<caption>**Figure 3** (Figure appears on preceding page)</caption>

Innate receptor-mediated immunity. (a) Overview of PTI and ETI. Microbial PAMPs or DAMPs and iDAMPs released by cells are ligands for cognate PRRs on the host cell PM. Ligand binding and dimerization with a coreceptor induce autophosphorylation and phosphorylation of an RLCK and the activation of a MAPK cascade leading to the induction of PTI-responsive genes. Activated RLCK also phosphorylates and induces NADPH oxidases (RBOHs) to produce ROS, and Ca²⁺ channel proteins leading to Ca²⁺ influx. It also activates other downstream genes, including phytohormone-related genes. Recognition of microbial effectors by NLR proteins enhances plant defense by ETI, which involves the multimerization of the NLR proteins leading to the formation of resistosomes that act as Ca²⁺ channels allowing increased Ca²⁺ influx. CC-NLRs, which contain a CC domain, form pentameric resistosomes, with CC domains responsible for the Ca²⁺ channel. TIR-NLRs contain a TIR protein domain and form tetrameric complexes that activate the TIR domain NADase activity. Small molecules generated by this activity act as secondary messengers that allosterically promote the interaction of EDS1-PAD1 or EDS1-SAG101 heterodimers with either the CC-NLR NGR1 or the CC-NLR ADR, respectively, resulting in the formation of resistosome complexes that act as Ca²⁺ channels. Although the consequences of the resistosome-mediated Ca²⁺ influx are not understood, the NLR responses are similar to those of the PRR-mediated responses (e.g., ROS production, Ca²⁺ influx, MAPK activation, defense gene upregulation, phytohormone production), although on a larger scale. The NLR-mediated Ca²⁺ influx therefore likely enhances the Ca²⁺ signaling already induced during PTI. (b) PTI pathway induced by bacterial flagellin. Sensing of flagellin or the derived elicitor peptide flg22 by the FLS2-BAK1/SERK3 receptor complex leads to activation of the RLCK BIK1 or its homolog PBL1, which induces phosphorylation of protein targets leading to Ca²⁺ influx and ROS production, and induction of PTI-responsive genes via activation of a MAPK cascade. Flagellin also induces callose deposition at PD, which depends on CML41 and the activation of CalS by a complex consisting of PDLP1, PDLP5, and NHL3. Consistent with CML41 being a Ca²⁺-binding protein, flg22-induced callose deposition is inhibited by the Ca²⁺ chelator EGTA. Bacterial effectors interfere with PRR function and PTI signaling. Several have been shown to regulate PD and move between cells, a likely function to support host susceptibility in recipient cells. (c) PTI pathway induced by viruses. dsRNA produced during viral replication in the VRC is recognized by a receptor complex consisting of an unknown PRR and SERK1. Recognition may occur at the PM, as depicted, or inside cells after internalization of the receptor complex. dsRNA perception leads to phosphorylation of the RLCK BIK1 in a SERK1-dependent manner and also causes deposition of callose at PD, which is dependent on PDLP1, PDLP2, and PDLP3 (likely acting redundantly) as well as on CML41. Consistent with CML41 being a Ca²⁺-binding protein, dsRNA-induced callose deposition is inhibited by EGTA, suggesting that BIK1 may activate a Ca²⁺ channel. dsRNA also leads to MAPK activation and induction of PTI-responsive genes. Viral infection also induces BAK1/SERK3-dependent responses, but the causal viral PAMP is unknown. Tobamoviral MP interferes with dsRNA-induced callose deposition by a yet unknown mechanism. Several potential targets in the cell or in the apoplastic space are suggested. Geminivirus-derived nucleic acids (not necessarily dsRNA) are sensed by the PM RLK NIK1, present as a homodimer or as a dimer with another PRR. Activation leads to phosphorylation of the downstream RPL10, which translocates to the nucleus to interact with the transcriptional repressor LYMYB to repress transcription of ribosomal protein genes, resulting in global translational silencing. The viral NSP interferes with this host defense response by interacting with the NIK kinase domain to prevent NIK1 phosphorylation and activation, thereby allowing viral protein translation and creating a more favorable environment for infection.
Abbreviations: ADR, ACTIVATED DISEASE RESISTANCE 1; BAK1, BRI1-ASSOCATED RECEPTOR KINASE 1; BG, β-1,3-glucanase; BIK1, BOTRYTIS-INDUCED KINASE 1; CalS, callose synthase; CC, coiled-coil; CML, CALMODULIN-LIKE; CNL, coiled-coil domain-containing NLR (nucleotide-binding-leucine-rich-repeat receptor); CPK, CALCIUM-DEPENDENT PROTEIN KINASE; DAMP, danger-associated molecular pattern; dsRNA, double-stranded RNA; EDS1, ENHANCED DISEASE SUSCEPTIBILITY 1; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N''-tetraacetic acid; ETI, effector-triggered immunity; FLS2, FLAGELLIN-SENSING 2; iDAMP, inducible DAMP; LYMYB, L10-INTERACTING MYB-DOMAIN-CONTAINING PROTEIN; MAPK, mitogen-activated protein kinase; MP, movement protein; NRG1, N REQUIRED GENE 1; NHL, NON-RACE SPECIFIC DISEASES RESISTANCE/HIN1 (HAIRPIN-INDUCED)-LIKE protein; NIK, NSP-INDUCED KINASE; NLR, nucleotide-binding leucine-rich repeat receptor; NSP, nuclear shuttle protein; P, phosphorylation; PAD4, PHOTOALEXIN-DEFICIENT 4; PAMP, pathogen-associated pattern; PBL1, PBS1-LIKE 1; PD, plasmodesmata; PDLP, PD-located protein; PM, plasma membrane; PRR, pathogen-recognition receptor; PTI, pattern-triggered immunity; RBOH, RESPIRATORY BURST OXIDASE HOMOLOG; RLCK, receptor-like cytoplasmic kinase; RLK, receptor-like kinase; ROS, reactive oxygen species; RP genes, ribosomal protein genes; RPL10, ribosomal protein L10; SAG101, SENESCENCE-ASSOCIATED GENE 101; SERK1, SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1; TIR, Toll/interleukin-1 receptor and resistance; TNL, Toll-interleukin 1-like receptor (TIR) NLR; VRC, viral replication complex; vRNP, viral RNP.

ROS: reactive oxygen species

perception of brassinosteroids (BRs), flagellin, and EF-Tu, respectively (44–46). Upon ligand binding, the ligand-induced complexes trigger overlapping downstream signaling events involving the phosphorylation of receptor-like cytoplasmic kinases (RLCKs) such as BOTRYTIS-INDUCED KINASE 1 (BIK1) and related AVRPPHB SUSCEPTIBLE 1 (PBS1)-like (PBL) proteins, activation of mitogen-activated protein kinase (MAPK) cascades, increased cytosolic Ca²⁺ levels, production of extracellular reactive oxygen species (ROS) and ethylene, transcriptional
reprogramming of defense-associated genes, and callose reinforcement of cell walls (47). Unlike RLKs, RLPs lack a kinase domain and rely on association with the regulatory LRR-RLK SUPPRESSOR OF BIR1-1 (SOBIR1) to activate cytoplasmic signaling pathways (48).

To enhance their virulence, pathogens produce effectors capable of suppressing PTI (49). Plants, in turn, have evolved a second layer of defense mediated by intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize the presence of effectors and activate ETI, which involves amplification of defense gene activation, often cell death at the site of infection, and systemic immunity known as systemic acquired resistance. Upon recognition of pathogen effectors, NLRs form oligomeric resistosomes that produce secondary messengers to activate downstream signaling responses, as exemplified by the Toll/interleukin-1 receptor (TIR) domain-containing NLR resistance proteins ROQ1 and RPP1 (50, 51), or lead to robust Ca²⁺ influx into cells by forming Ca²⁺-permeable channels in the PM, as shown by HOPZ-ACTIVATED RESITANCE 1 (ZAR1) (52) or wheat STEM RUST RESISTANCE 35 (Sr35) (53). PRR-mediated PTI and NLR-mediated ETI were initially thought to act independently. However, recent studies have shown that PTI and ETI are interdependent components of plant immunity, with PTI being necessary for priming the more potent ETI response and NLRs not only playing a role in ETI but also significantly enhancing PTI by upregulating PTI signaling components (54–59). To coordinate defense against multiple pathogens, PTI and ETI often rely on signaling cascades with extracellular ROS and calcium ion fluxes as secondary messengers, as well as the orchestration of transcription factors and hormone signaling pathways (60).

## Pattern-Triggered Immunity Against Viruses

Viruses are structurally diverse, encode different sets of genes, and are characterized by a high mutation rate. Therefore, for a long time it may have seemed unlikely that viruses could be recognized by plants through a highly conserved structural PAMP. (See the sidebar titled A New Field of Research.) This has changed with the discovery that *Arabidopsis* plants deficient in BAK1/SERK3 are hypersensitive to infection by tobacco mosaic virus (TMV), oilseed rape mosaic virus (ORMV), and turnip crinkle virus (TCV). Furthermore, treatment of plants with extracts from TCV-infected plants rapidly induced several PTI marker responses (e.g., MAPK activation, ethylene production, seedling growth inhibition) in a BAK1/SERK3-dependent manner. Seedlings treated with such an extract in the medium showed strong growth retardation, whereas medium containing purified virions had no such effect, suggesting that viruses induce PTI by PAMPs that are not part of the virion particle but are produced in the infected cell (61). Other studies showed that TCV-infected plants were also hypersensitive when BAK1-LIKE KINASE1 (BKK1)/SERK4 was deficient (62). Hypersusceptibility to plum pox virus (PPV) was observed in *Arabidopsis* plants deficient in both BAK1/SERK3 and BKK1/SERK4 (63). (See the sidebar titled The Role of BRI1-ASSOCIATED RECEPTOR KINASE 1 in Controlling Autoimmunity.) Furthermore, RLK NUCLEAR-SHUTTLE PROTEIN (NSP)-INDUCED KINASE 1 (NIK1), which belongs to the same LRR-RLK subfamily as the SERK clade (64), has been shown to play a role in plant defense against geminiviruses (DNA viruses with circular single-stranded genomes) (65). The involvement of different SERK proteins in defense against a given virus may indicate that a virus-infected cell produces multiple PAMPs that are recognized by different PRR complexes, or that a specific PRR complex can recruit different SERKs, as has previously been shown for the antimicrobial EFR and FLS2 immune receptor complexes (45). Recognized viral PAMPs could include viral protein(s) or nucleic acid(s) produced in the infected cell, or infection-induced host-derived iDAMP(s). However, among the many PAMPs and iDAMPs that can be produced by a virus-infected cell, dsRNA, a hallmark of viral replication, was identified as

nucleotide-binding
leucine-rich repeat
receptor
## A NEW FIELD OF RESEARCH

For a long time, it was thought that pattern-triggered immunity (PTI) against viruses was specific to animals and that plants, unlike vertebrates and other chordates, maintained RNA silencing as a major RNA-triggered defense system (2). Furthermore, research on PTI in plants was focused exclusively on fungal and bacterial microbes. The discovery of a double-stranded RNA (dsRNA)-induced, antiviral PTI response in plants has significantly changed the view of plant-pathogen interactions and opened a new field of research to explore the mechanism of dsRNA perception, the mode of action of dsRNA-induced PTI, how viruses overcome this defense mechanism, and how virus-induced PTI can lead to or be enhanced by effector-triggered immunity.

## THE ROLE OF BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 IN CONTROLLING AUTOIMMUNITY

The role of BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) in antiviral defense is supported by strongly enhanced susceptibility for tobacco rattle virus in plants deficient for BAK1-INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1), which binds to and controls BAK1. This also shows that BIR1, which during infection by itself is controlled for homeostasis through virus-activated small interfering RNA production from its transcript, acts as an important negative regulator of antiviral defense, protecting plants against the negative effects of autoimmunity (164).

a potent PTI elicitor. Treatment of *Arabidopsis* plants with viral dsRNA but also with unrelated dsRNA or the synthetic dsRNA analog polyinosinic–polycytidylic acid [poly(I:C)] elicited typical PTI responses and caused antiviral resistance, indicating that dsRNA-induced PTI is nucleotide sequence independent (66). These observations are consistent with the known role of viral RNA as a PAMP in animals (67, 68). In addition, bacterial RNA and DNA molecules act as PAMPs (69, 70). Interestingly, dsRNA-induced PTI responses in *Arabidopsis* are dependent on SERK1 and not on BAK1/SERK3 or BKK1/SERK4, which may therefore be involved in the recognition of other virus-induced PAMPs present in infected cells (66). The collective evidence for the roles of SERK1, BAK1/SERK3, BKK1/SERK4, and NIK1 in virus-induced PTI suggests that dsRNA and other virus-induced PAMPs or iDAMPs are sensed by PM-localized receptor complexes.

## Double-Stranded RNA-Induced Pattern-Triggered Immunity Signaling to Plasmodesmata

In the search for a mechanism by which PTI acts against viral infection, it was found that dsRNA-induced antiviral resistance does not affect virus replication but limits the efficiency of virus movement (66, 71) by rapidly inducing callose (β-1,3-glucan) deposition at PD, thereby limiting their permeability (71). The structure, function, and regulation of PD have been reviewed (72). To explain the effect of callose deposition, it has been proposed that the additional callose in the cell walls around PD pushes the PM toward the centrally located strand of the endoplasmic reticulum (ER) (the desmotubule), thus limiting the size of the cytoplasmic space between the two membranes available for molecular transport. The extent of callose deposition is dynamically determined by the activity of callose synthases (CalSs) or glucan synthases (GSLs) and β-1,3-glucanases (BGs), which synthesize and degrade callose, respectively (73–75). (See the sidebar titled The Role of MULTIPLE C2 DOMAINS AND TRANSMEMBRANE REGION

<footnote>poly(I:C)
polyinosinic–
polycytidylic acid</footnote>
THE ROLE OF MULTIPLE C2 DOMAINS AND TRANSMEMBRANE REGION PROTEINS IN REGULATING FLUX THROUGH PLASMODESMATA

Apart from callose deposition, cytoplasmic flux through plasmodesmata (PD) is also regulated by PD-localized MULTIPLE C2 DOMAINS AND TRANSMEMBRANE REGION PROTEINS (MCTPs), which are anchored to the desmotubule by their C-terminal transmembrane region and bind the desmotubule to the plasma membrane (PM). The binding of MCTPs to the PM occurs through the interaction of their C2 domains with the anionic lipid phosphatidylinositol 4-phosphate (PI4P). Therefore, cytoplasmic flux through the channels is regulated by the level of PI4P and thus the level of MCTP-membrane docking. *mctp* mutants are insensitive to callose levels and are unable to control cytoplasmic flow. Thus, regulation of cytoplasmic sleeve size by callose deposition may be mediated through effects on MCTP-membrane docking (165, 166).

PROTEINS in Regulating Flux Through Plasmodesmata.) Proteomic studies in *Arabidopsis* and other plant species have revealed lists of additional PD-associated proteins, including a family of PD-located proteins (PDLPs) (76) that have been implicated in promoting callose synthesis, for example, by binding and stabilizing callose (77, 78) or by blocking the activity of BG, as recently shown for PDLP7 (78). PD closure is also induced by the perception of microbial elicitors, such as bacterial *flg22* and fungal chitin (79), and by the defense hormone salicylic acid (SA) (80). Consistently, PD contain numerous membrane-anchored receptors, including receptors that bind *flg22* (79) and chitin (81), as well as other RLKs and RLPs with unknown ligands (82, 83). Thus, by closing in response to PAMPs, PD serve as important physical barriers against invading pathogens, including viruses (84, 85).

The signaling pathway induced by dsRNA perception leading to callose deposition and PD closure shares common elements with the signaling pathways induced by microbial elicitors but also shows important differences (Figure 3b,c). Chitin and *flg22* induce PD closure by phosphorylating the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), production of ROS, and activation of callose synthesis by CalS1 through signaling by a complex formed by PDLP1, PDLP5, and NDR1 (NON-RACE SPECIFIC DISEASES RESISTANCE)/HIN1 (HAIRPIN-INDUCED)-LIKE protein 3 (NHL3) (86) (Figure 3b). PDLP1, PDLP5, and CalS1 are also required for callose deposition in response to the defense hormone SA (74, 80, 86). In addition, *flg22*-triggered but not chitin-triggered plasmodesmal closure depends on the PD-localized $Ca^{2+}$-binding protein CALMODULIN-LIKE 41 (CML41) (86, 87). The dsRNA-induced signaling pathway leading to callose deposition at PD depends on SERK1, BIK1, and/or its homolog PBL1, as well as CML41 and PDLP1, PDLP2, and PDLP3, whereas PDLP5 is dispensable (71) (Figure 3c). Genetic studies have shown that BIK1/PBL1 and CML41 are required for plant antiviral defense (71), and this important function remains to be tested for the PDLPs. BIK1 is an important component of PTI signaling by integrating signals from multiple PRRs, as shown by its direct interaction with PRR proteins (88–90) and its ability to phosphorylate and activate downstream targets such as the ROS ($H_2O_2$)-generating NADPH oxidase RBOHD (91) or the calcium channel proteins OSCA1.3 (92) and CYCLIC NUCLEOTIDE-GATED CHANNEL 2 (CNGC2) and CNGC4 (93). The presence of poly(I:C) induces phosphorylation of BIK1 in a SERK1-dependent manner, suggesting that BIK1 acts downstream of a SERK1-containing poly(I:C) receptor complex (71). However, unlike the bacterial and fungal elicitors *flg22* and chitin, poly(I:C) did not induce ROS ($H_2O_2$) production, suggesting that dsRNA-induced signaling to PD involves downstream BIK1 targets other than RBOHD (71). dsRNA-induced signaling also differs from *flg22*- and chitin-induced signaling

PDLP: PD-located protein
VRC: viral replication complex

in that it requires PDLP1, PDLP2, and PDLP3 rather than PDLP1 and PDLP5 (71). This suggests that the PDLP1-PDLP5-NHL3 node, which is required for chitin- and flg22-induced callose deposition (86), does not play a role in poly(I:C)-induced callose deposition. How dsRNA-induced signaling activates PDLP1, PDLP2, or PDLP3 [these PDLPs act redundantly (76, 94)] and how this leads to the execution of PD closure may be revealed by further studies. Poly(I:C)-induced signaling to PD is also independent of MAPK3 and MAPK6, although poly(I:C) induces MAPK3/6 phosphorylation (71). Similar to flg22, poly(I:C) also triggers the activation of PTI defense genes (66, 71). It is therefore conceivable that poly(I:C) is perceived by different receptors with different downstream signaling responses, i.e., MAPK-independent signaling toward PD- and MAPK-dependent signaling toward defense gene activation (Figure 3c). (See the sidebar titled Chitin Signaling Shows That an Elicitor Can Be Sensed by Different Receptors Causing Different Downstream Signaling Responses.) Recent studies suggest a role for PDLP7 in regulating callose deposition and virus movement through interaction with BG (78), confirming the critical involvement of PDLPs in plant-virus interactions and suggesting that PDLP7 may serve as an additional target of dsRNA-induced signaling, which may be demonstrated by further studies.

## Perception of Double-Stranded RNA

The requirement of SERK1 for poly(I:C)-induced activation of PTI, including callose deposition at PD, suggests that dsRNA is sensed by a PM-associated receptor complex. Given that plant viruses replicate intracellularly, this raises the interesting question of how viral dsRNA might be perceived in the extracellular space. It is conceivable that viral dsRNA is exported from the cytoplasmic viral replication complexes (VRCs) into the apoplastic space (Figure 3c). Extracellular RNA is a common feature of plants (95), and plants exploit the trafficking of small and long RNAs across cell walls in cross-kingdom RNA interference (RNAi) (96, 97). In addition, viral particles, viral proteins, and viral RNA have been found in the apoplast of infected plants (98, 99). However, alternative scenarios can also be considered. For example, considering that plant receptor complexes can signal from endosomes (100), viral dsRNA could be sensed upon internalization of SERK1-containing receptor complexes from the PM into endosomes and possible recruitment into membrane-associated VRCs through membrane fusion events (101). The possibility that dsRNA sensing occurs through an endosomal receptor may also be supported by the known recognition of viral RNAs in animals by internalized Toll-like receptors (TLRs). TLR3, which is mainly localized to endolysosomes, is activated by dsRNAs released by viruses during

## CHITIN SIGNALING SHOWS THAT AN ELICITOR CAN BE SENSED BY DIFFERENT RECEPTORS CAUSING DIFFERENT DOWNSTREAM SIGNALING RESPONSES

While chitin is normally perceived by the plasma membrane receptor CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1), the triggering of plasmodesmal closure by chitin depends on the plasmodesmata (PD)-localized receptor LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN 2 (LYM2) (81). The chitin response is also dependent on lysine motif receptor kinases, LysM-CONTAINING RECEPTOR-LIKE KINASE 4 (LYK4) and LYK5. Both LYM2 and LYK4 were localized to PD by fluorescent protein fusion microscopy, suggesting that chitin-triggered signaling at PD is mediated by an LYM2-LYK4 complex. The chitin response at PD mediated by LYM2-LYK4 is independent of mitogen-activated protein kinase (MAPK) activation induced by CERK1 (81). Therefore, signaling to PD may occur via a specific shortcut pathway that acts independently of signaling to the nucleus via MAPK cascades, allowing rapid closure of PD in the cell walls of infected cells.
infection (67). Importantly, poly(I:C), which induces plant immunity similarly to viral dsRNA (66), is one of the most potent immunoinducers of TLR3 activation (102). Alternatively, dsRNA perception in plants could occur via receptors located in the cytoplasm. Examples are cytoplasmic retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs), including RIG-I, MDA5 (melanoma differentiation-associated protein 5), and LGP2 (laboratory of genetics and physiology 2), which detect the presence of dsRNA in animals (103). Potential candidates for cytoplasmic receptors in plants are dsRNA-binding proteins (DRBs). *Arabidopsis* has five DRBs, of which DRB1/HYL1 and DRB4 are required for DCL1 and DCL4 activity, respectively (104). Notably, DRB2 accumulates in VRCs and plays a role in controlling virus accumulation independent of Dicer activity (105) or virus-induced necrosis (106), suggesting a defense role for DRB2 distinct from RNA silencing.

In addition to the DRBs, the DCL proteins could be cytosolic PRR candidates. As mentioned above, these proteins bind dsRNAs and process them into small RNAs during RNA silencing. Furthermore, they show close homology of their helicase domains with those of mammalian RLRs (107). However, *Arabidopsis* *dcl2 dcl4* double mutants, *dcl2 dcl3 dcl4* triple mutants, and *dcl1* mutants showed increased rather than decreased PTI responses upon poly(I:C) treatment, suggesting that DCL proteins do not play a direct role in dsRNA sensing toward PTI and that dsRNA-induced silencing and dsRNA-induced innate immunity are distinct antiviral responses requiring different protein machinery (66). Nevertheless, in DCL4-deficient plants, where DCL2 normally acts as a surrogate dicer in the processing of dsRNAs into siRNAs (108), DCL2 has the ability to activate intracellular NLRs and defense gene expression. This property of DCL2 depends on its ability to bind dsRNA but is independent of its dicer activity. Therefore, DCL2 may play an important role in activating immunity when dsRNA accumulates above a certain threshold and the RNAi mechanism fails (e.g., when DCL4 becomes saturated with dsRNA) (109). However, given the requirement for this threshold level of dsRNA, it seems unlikely that DCL2-mediated dsRNA sensing could play a role in triggering PTI at the viral infection front, where infection starts from zero and consequently viral dsRNA levels must be low. It seems possible that DCL2-induced immunity complements the lack of efficient RNA silencing during later stages of infection, when the infecting virus supports efficient replication and viral progeny production by expressing high levels of VSR to inhibit antiviral RNAi, likely leading to dsRNA accumulation.

## Specificity of Viral Double-Stranded RNA as a Pathogen-Associated Pattern

Activation of PTI responses in plants by dsRNA is independent of dsRNA length and nucleotide sequence (66, 71). Thus, PTI of infected cells may not necessarily be activated by viral dsRNA but could also be triggered by host-derived dsRNAs that accumulate during infection and may function as iDAMPs. Candidates are the dsRNA precursors of vasiRNAs, which originate from transcripts of host coding genes in an RDR-dependent manner (37–39, 110) (Figures 2e and 4). As is mentioned above, host dsRNA can also accumulate upon virus-induced disruption or overload of RNA decay pathways that normally eliminate aberrant transcripts resulting from RNA degradation, end cleavage, or end processing (Figure 2d). dsRNA molecules also occur in the absence of infection as widespread precursors of various siRNA species [e.g., heterochromatic siRNAs (hc-siRNAs), natural antisense siRNAs (nat-siRNAs), trans-acting siRNAs (ta-siRNAs), phased siRNA (phasiRNAs)] that function in transcriptional and post-transcriptional gene regulation (111). However, unlike virus-derived or virus-induced dsRNAs, these host-derived dsRNAs, which are also present in uninfected cells, are not sensed—i.e., unlike dsRNAs isolated from virus-infected plants, dsRNAs isolated from mock-treated plants did not induce PTI responses (66). This raises the question of how plants are able to distinguish housekeeping dsRNAs from foreign dsRNA PAMPs [viral dsRNA, in vitro-produced random dsRNA, poly(I:C)] and

phasiRNAs:
phased siRNAs
<figure><img src="image_10.png" /></figure>

(Caption for Figure 4 appears on following page)
<caption>**Figure 4** (Figure appears on preceding page)</caption>

Overview model of antiviral defense responses and their suppression by viral proteins. The upper part of the figure illustrates events during RNA virus infection. Early events that likely occur in cells at the infection front are shown in green, later events that depend on sustained viral replication and likely occur in cells behind the infection front are shown in dark red, and potentially even later events are shown in light red. Viral replication produces both dsRNA and positive-sense (+) replicated viral genomic RNA (Event 1). Tobamovirus MP forms a complex with replicated viral genomic (+)RNA (vRNP), which is targeted to PD to spread infection into adjacent cells (Event 2). Viral dsRNA molecules exported into the apoplasmic space (Event 3) are sensed by a SERK1-containing PRR complex (Event 4), which triggers PTI leading to Ca²⁺ influx, PD closure, and induction of immunity genes (Event 5). The MPs of tobamoviruses counteract this defense response and thereby keep the PD in an open state, which could occur through mechanisms at several levels (e.g., interference with the PRR complex, PTI signaling, or callose metabolism at the PD) (Event 6). Meanwhile, dsRNA triggers antiviral RNA silencing, which, together with RNA decay, controls viral RNA accumulation (Event 7). In this process, RNA decay limits antiviral RNA silencing by competing for RNA templates needed for secondary vsiRNA production. Infection causes several host coding genes to undergo PTGS and convert their mRNA transcripts into vasiRNAs with the potential to induce PTGS of other target genes (Event 8). The expression of NLR genes is controlled by the conversion of their transcripts into phasiRNAs (Event 9). Together with vasiRNAs and vsiRNAs, they can target host genes for PTGS (Event 10). In cells behind the infection front, VSR accumulates (Event 11) and blocks antiviral RNA silencing to allow increased production of viral progeny (Event 12). As an additional effect, the VSR can interfere with the normal miRNA-mediated regulation of developmentally important genes, leading to symptoms (Event 13). The VSR also initiates the expression of NLR proteins by suppressing the miRNA-mediated conversion of NLR transcripts into phasiRNAs (Event 14). Recognition of viral avirulence proteins by NLRs leads to resistosome formation and ETI (Event 15). High viral dsRNA accumulation during later stages of infection can saturate DCL4 activity and induce its surrogate dicer, DCL2, to activate NLR genes, thereby enhancing PTI and ETI (Event 16). Similarly, high viral RNA levels can lead to overloading of RNA decay, resulting in the conversion of RNAs into ct-siRNAs, which can target other host genes for PTGS under the control of the VSR (Event 17). Immediately after their production, those vsiRNAs, phasiRNAs, vasiRNAs, and ct-siRNAs that escape VSR activity are mobile and, as long as PD remain open by MP, can move between cells and systemically to regulate viral infection and gene expression in cells ahead of the viral infection front (Event 18). The lower part of the figure illustrates the antiviral defense response against geminiviruses, which is mediated by recognition of viral nucleic acid by the RLK NIK1 (Event 19), leading to translational repression (Event 20), and is inhibited by NSP (Event 21). In addition, geminiviruses and other DNA viruses are controlled by TGS (Event 22). Abbreviations: CPK, CALCIUM-DEPENDENT PROTEIN KINASE; ct-siRNA, coding transcript-derived siRNA; DCL, DICER-LIKE; ds, double-stranded; ETI, effector-triggered immunity; LYMYB, L10-INTERACTING MYB-DOMAIN-CONTAINING PROTEIN; MAPK, mitogen-activated protein kinase; miRNA, microRNA; MP, movement protein; mRNA, messenger RNA; NIK1, NSP-INDUCED KINASE 1; NLR, nucleotide-binding leucine-rich repeat receptor; NSP, nuclear shuttle protein; P, phosphorylation; PD, plasmodesmata; phasiRNA, phased siRNA; PRR, pathogen-recognition receptor; PTGS, post-transcriptional gene silencing; PTI, pattern-triggered immunity; RLK, receptor-like kinase; RP genes, ribosomal protein genes; RPL10, ribosomal protein L10; SERK1, SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1; siRNA, small interfering RNA; ss, single-stranded; TGS, transcriptional gene silencing; vasiRNA, virus-activated siRNA; vRNP, viral RNP; vsiRNA, viral siRNA; VSR, viral suppressor of RNA silencing.

virus-induced host dsRNA iDAMPs. Recognition of animal dsRNAs by the cytosolic receptors RIG-1 and MDA5 is length dependent (112), with RIG-1 recognizing sequences as short as 70 bp and MDA5 binding to dsRNAs longer than 100 bp. Viral dsRNAs in plants can be several kilobases in length. Consistently, poly(I:C) of 350–850 bp and in vitro-produced dsRNAs (700 bp) as well as long bacteriophage Phi6 dsRNAs (several kilobases) have been shown to induce PTI in plants (66, 71). However, single-stranded polyinosinic acid and 21-nt short dsRNAs did not induce an immune response (66). The membrane-associated TLR3 discriminates between self and nonself RNAs by compartmentalization. It recognizes nucleic acids by their presence in endosomes and lysosomes, i.e., nucleic acids derived from or associated with internalized cargo (112). Similarly, plant virus-derived dsRNAs could be distinguished as nonself by their specific compartmentalization within VRCs and recognized upon membrane fusion with endosomes associated with the dsRNA-receiving PRR complex. Viral dsRNAs could also be distinguished from cellular dsRNAs by structural features. The animal RIG-1 protein has a specific affinity for the 5'-triphosphate moiety of viral RNA (113), which is normally lost during maturation and capping of cellular RNA. Specific structural features of tobamoviruses include several kilobase-long dsRNA replication intermediates or transfer RNA (tRNA)-like structures at the 3' ends of the viral genomic and subgenomic RNAs. In addition, the tobamovirus positive-sense RNA genome, but not the
negative-sense RNA replication intermediate, has a 5' cap, and the viral genomic and subgenomic RNAs lack a 3' poly(A) tail. The genomic RNA of potyviruses carries a 3' poly(A) tail, but its 5' end carries the virus protein, genome linked (VPg) protein instead of a 5' cap (114). Poly(I:C) and in vitro synthesized dsRNA, which can induce PTI responses, also differ from normal RNA molecules in that they are both noncapped and lack a poly(A) tail.

Pattern-Triggered Immunity Suppression by Tobamovirus Movement Proteins

In the plant-pathogen arms race, pathogens evolve effectors that overcome host defenses by inhibiting specific steps in the host defense pathway or in the expression, synthesis, delivery, or subcellular localization of its components. For example, plant viruses have evolved VSR effector proteins to inhibit the host's antiviral RNA silencing response (10). In addition, tobamovirus movement proteins (MPs) act as effector proteins capable of inhibiting dsRNA-induced PTI, thereby disrupting callose deposition at PD and facilitating virus movement (71). Consistent with dsRNA-induced callose deposition at PD and its suppression by MP, the level of callose deposition is greatly reduced in cells at the viral infection front (71). These findings are consistent with a previous study showing that replication of a cell-autonomous TMV replicon lacking MP induced strong callose deposition at PD, whereas lower callose levels were observed in the presence of MP in association with virus movement (115). Based on classical microinjection studies, it was thought that PD have a relatively low size exclusion limit, allowing only small molecules of about 1 kD to pass, and that viruses evolved MPs to open the PD pores, which would otherwise be too small for macromolecular transport and passage of the viral complex (116, 117). The new findings suggest that MPs do not open PD pores but rather prevent their closure. However, current evidence for the ability of MPs to inhibit stress/dsRNA-induced PD closure is limited to the MPs of tobamoviruses. To establish a general model, it will be necessary to demonstrate that MPs from other viruses also possess this ability. Further studies are also needed to determine the mechanism by which tobamoviral MPs interfere with the PTI response. Because MPs are expressed in cytoplasmic VRCs, they may interfere with the internalized PRR complex or they may be secreted to interfere with the PRR complex at the PM (Figure 3c). This hypothesis is supported by evidence that the MPs of TMV, turnip vein-clearing virus (TVCV), and CaMV interact with the cell wall enzyme pectin methylesterase (PME) in Nicotiana tabacum (118, 119). An interaction between MPs and this enzyme may simply allow MPs to reach the apoplast, but, hypothetically, MPs may also use this enzyme to alter the structural state of cell wall pectins, which are enriched in microdomains surrounding PD channels (120), thereby increasing the PD size exclusion limit. The MPs may also alter the generation of pectic fragments from de-esterified pectin, which have been reported to act as signals regulating the FERONIA (FER)-mediated dynamic clustering of receptor complexes at the PM (121). The MPs may also interfere with the internalization of the dsRNA-receiving PRR complex. The MPs of TMV and several other viruses were found to interact with the synaptotagmin SYT1/SYTA, which is essential for the regulation of endocytosis and MP-directed cell-to-cell transport of several viruses, including TMV and TVCV (122, 123). In addition, the MPs may interfere with downstream components of the dsRNA-induced signaling pathway. Considering that the MPs localize to PD, the activity of PDLPs, CML41 or the CalSs, and BG could be modified by MPs. (See the sidebar titled Movement Protein Function at Plasmodesmata May Involve the Receptor-Like Kinase BARELY ANY MERISTEM 1.) However, although indirect interactions between MPs and BGs have been observed (124, 125), this activity would act downstream of elicitation and signaling and would likely reduce callose levels irrespective of the specific elicitor. This is in contrast to the observation that TMV MP interfered with dsRNA-induced callose deposition but not flg22-induced callose

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MOVEMENT PROTEIN FUNCTION AT PLASMODESMATA MAY INVOLVE
THE RECEPTOR-LIKE KINASE BARELY ANY MERISTEM 1

The movement protein (MP) of tobacco mosaic virus (TMV) was reported to undergo a functional interaction with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) at plasmodesmata. BAM1 overexpression enhanced TMV movement in *Arabidopsis* and *Nicotiana benthamiana*, whereas virus movement was strongly inhibited in an *Arabidopsis bam1-3* loss-of-function mutant or after knockdown of BAM1 expression in *N. benthamiana*. Binding of MP occurred to the transmembrane and kinase domains of BAM1, but not to its leucine-rich repeats. Furthermore, phosphorylation assays using coexpression of the two proteins in *Escherichia coli* led to the detection of BAM1 autophosphorylation but not phosphorylation of MP. Taken together, these findings suggest that MP is not a ligand of BAM1, but that BAM1 is involved in MP function (167).

BAM1 also plays a role in promoting the intercellular spread of RNA silencing and is a virulence target of geminivirus C4 proteins. It has been proposed that C4 supports viral infection by inhibiting the BAM1-supported non-cell-autonomous RNA interference against the virus (168, 169).

deposition, suggesting that MP interferes with a dsRNA-specific receptor or signaling component
(71).

The Role of Plasma Membrane Nanodomain Organization in Pattern-Triggered Immunity and Virus Movement

Recent studies have revealed a critical role for the regulation of PM nanodomains in plant signal-
ing. For example, the receptor proteins FLS2 and BRI1 are localized to distinct PM nanodomains
(126). Furthermore, the specific nanoscale organization of the PM domain associated with FLS2
and BAK1 is required for flg22-induced signaling and is regulated by FER and its ligand RALF 23
(121, 127). The PM subcompartment at PD is enriched in members of the REMORIN (REM)
family, which is a typical feature of PM nanodomains in plants (82). Group 1 and group 6 REMs
are involved in the regulation of the plasmodesmal aperture leading to the modification of viral
movement (128, 129). In particular, the *Solanum tuberosum* REM1.3 protein has been shown to
interfere with the ability of the potato virus X (PVX) triple gene block virus 1 (TGB1) movement
protein to increase PD permeability (129). The ability of REM1.3 to inhibit PVX movement
depends on its lateral organization into PM nanodomains as well as its specific phosphorylation
by membrane-bound *Arabidopsis* CALCIUM-DEPENDENT PROTEIN KINASE 3 (CPK3) in-
duced by perception of TGB1 or the viral coat protein (130). REM1.3 phosphorylation was also
induced by TMV MP, and REM1.3 interfered with the ability of TMV MP to increase PD per-
meability and TMV movement upon overexpression (129, 130). Based on these findings, Perraki
et al. (129) proposed that viral proteins such as PVX TGB1 or TMV MP induce the activation
of protein kinases, which in turn phosphorylate REM1.3 at its N-terminal domain. The phos-
phorylation status of REM1.3 regulates its organization at the PM and its association with PD,
whereby the latter leads to PD closure through the induction of callose deposition and restricts vi-
ral cell-to-cell movement (130). It will be interesting to see whether the proposed CPK3-mediated
pathway, induced upon recognition of viral proteins, is linked to the dsRNA-induced pathway, as
both pathways lead to callose deposition and PD closure. Because TMV MP promotes viral move-
ment by inhibiting dsRNA-induced, PTI-mediated closure of PD (71), it is tempting to speculate
that REM-dependent inhibition of MP activity may act as a PD-based second line of defense
against TMV movement.
Pattern-Triggered Immunity–Inducing and –Inhibiting Activities
of Other Viruses

The example of PVX shows that, in addition to TMV, other viruses trigger PTI or PTI-like signal-
ing leading to PD closure. Another example is hibiscus green spot virus (HGSV), which encodes
two MPs called binary movement block 1 (BMB1) and BMB2. While BMB1 is related to PVX
TGB1 and is thought to form a complex with viral RNA for movement, BMB2 is an ER-associated
protein that facilitates BMB1 movement across PD (131). Recently, it was shown that BMB1 in-
duces a plant defense response leading to callose deposition at the PD, which is suppressed in the
presence of BMB2. Consistently, the efficiency of BMB proteins in facilitating viral cell-to-cell
movement depends largely on the expression ratio between the two proteins, i.e., compared to a
1:1 ratio, a tenfold higher amount of BMB1 than BMB2 inhibited movement, whereas a tenfold
higher amount of BMB2 than BMB1 further enhanced movement (132). Although the detailed
mechanisms remain to be investigated, the observations confirm the emerging paradigm that viral
movement is linked to plant defense responses that are activated and suppressed during infection
and modulate the conductance of PD.

In further support of the general importance of antiviral PTI, several viruses were shown to en-
code proteins capable of suppressing PTI triggered by bacterial and fungal elicitors. These include
the MP of cucumber mosaic virus (CMV) (133), the coat protein of PPV (63), and the P6 protein
of CaMV (134). Further studies are needed to show whether these proteins suppress PTI induced
during plant infection by their respective viruses. Interference with PTI signaling has also been re-
ported for proteins encoded by geminiviruses, which have circular single-stranded DNA genomes.
For example, the βC1 protein of the tomato yellow leaf curl China virus (TYLCCNV) beta satel-
lite interferes with PTI by targeting components of the MAPK cascade activated by viral infection
(135). Furthermore, the NSP of several geminiviruses, including tomato golden mosaic virus
(TGMV), tomato crinkle leaf yellows virus (TCrLYV), and cabbage leaf curl virus (CaLCuV),
interacts with the PM-localized RLK NIK1 and prevents antiviral defense, in this case via antiviral
translational shutdown (65, 136, 137) (Figure 3c). This defense response is elicited by sensing
RNA or DNA isolated from geminivirus-infected *Arabidopsis thaliana* plants, but not by RNA or
DNA isolated from uninfected plants (136). Although NIK1 belongs to the same LRR-RLK sub-
family as the SERK clade (64), it elicits immune responses that are completely distinct from the
SERK1-dependent dsRNA-induced PTI response (66, 71) and the canonical SERK-dependent
PTI responses against microbial pathogens (64, 136, 137). Nevertheless, although the outcome of
translational repression is not directly related to viral movement, this is another important example
of viral suppression of an antiviral receptor-mediated immune response elicited by virus-derived or
virus-induced RNA/DNA. Further studies may clarify which specific nucleic acid ligand activates
NIK1 and whether this RLK actually functions as a viral RNA/DNA sensing receptor/coreceptor
or rather acts more downstream. Another geminiviral effector is the tomato yellow leaf curl virus
(TYLCV) C4 protein, which has the potential to interact with a wide range of RLKs, includ-
ing FLS2 and BRI1, suggesting that C4 may broadly interfere with PM transduced signaling
(138).

Regulation of Immunity by RNA Silencing

Given that dsRNA induces both RNA silencing and PTI, it is likely that the two defenses are ei-
ther spatially and temporally separated during infection or that they control each other in infected
cells. PTI inhibits virus movement and is therefore likely to be induced by low levels of dsRNA at
the virus front, whereas RNA silencing is more likely to be induced in the VSR-expressing cells
behind the infection front, which are involved in virus progeny production (71). Nevertheless,
the observation that dsRNA-induced PTI responses are enhanced in *dcl* mutants (66) may suggest that DCL proteins compete with dsRNA immune receptor(s) for dsRNA binding in the same cells. In addition, inhibition of silencing by VSRs may lead to increased cellular dsRNA levels, thereby triggering PTI, possibly via DCL2. However, the enhancement of dsRNA-induced PTI responses in *dcl* mutants could also reflect the role of DCLs, and RNA silencing in general, as negative regulators of NLR resistance genes involved in PTI and ETI. PTI and ETI resistance genes are under tight control as they would otherwise induce autoimmunity and inhibit plant growth. Consistent with the negative control of NLR genes by post-transcriptional silencing, *rdr6* mutants have been shown to exhibit constitutive PTI (139). The underlying mechanism involves initial cleavage of NLR transcripts under the control of a specific 22-nt-long microRNA (miRNA), which then recruits RDR6 to convert the cleaved transcripts into dsRNAs for subsequent stepwise processing into secondary siRNAs. These phasiRNAs are able to mediate the enhanced silencing of the specific NLR genes as well as related genes that are sequence unrelated to the miRNA trigger. Furthermore, their mobility as secondary siRNAs allows miRNA-mediated NLR repression to spread from the miRNA-expressing domain to neighboring tissues (140–143). However, while these mechanisms silence NLR genes in the absence of infection, the genes need to be activated in the presence of pathogens. This may occur through transcriptional activation upon pathogen sensing by PRRs (56) or through attenuation of miRNA/phasiRNA-mediated NLR silencing. Indeed, changes in the levels of miRNAs involved in NLR silencing altered flg22-induced immune responses, such as ROS production or MAPK activation (139, 144). Importantly, this system is likely capable of sensing viruses through the activity of their VSRs, as well as any other pathogens that encode silencing effectors, all of which can inactivate miRNA/phasiRNA-based regulation of NLR genes and thereby switch on the immune response (Figure 4). Thus, while the dsRNA-induced PTI response is independent of VSR activity (66), VSR accumulation in later stages of infection may reach levels capable of inactivating the negative regulation of NLRs, thereby enhancing the antiviral immune response.

VSRs can further enhance immune responses if they are themselves targets for specific recognition by NLRs (Figure 4). Examples of VSR proteins that induce ETI by specific recognition include P38 of TCV (145), P25 of PVX (146), NSs of tomato spotted wilt virus (147), and the 126k VSR (small replicase subunit) of TMV (148). The 126k VSRs of TMV and the related tomato mosaic virus are recognized by the tobacco N protein (149), whereas the tobamoviral MPs, which act as viral effectors to suppress antiviral dsRNA-induced PTI (71), are recognized by the tomato NLRs Tm-2 and TM-2² (150). Thus, plants employ sophisticated, multilayered mechanisms to sense invading viruses and respond to invasion by RNA silencing, RNA decay, and dsRNA-triggered PTI that restrict virus replication and movement, and may ultimately induce cell death in response to viral virulence effectors (Figure 4).

## CONCLUDING REMARKS AND PERSPECTIVES

It is now clear that, in addition to RNA silencing, PTI plays a central role in plant defense against viruses, and it is expected that further research will provide important new insights into the different PTI pathways triggered by viral infection and the different mechanisms used by viruses to inhibit PTI.

In combination with corresponding viral effectors that inhibit these host responses to achieve effector-triggered susceptibility (ETS), these two plant defense pathways fit into the coevolutionary arms race model, where the host's fitness is increased if it can inhibit the pathogen, while the pathogen's fitness depends on its ability to counteract the host's immune defense system. This evolutionary arms race between the pathogen and its host is illustrated by the zigzag model originally

miRNA: microRNA
proposed by Jones & Dangl (151) and may involve multiple rounds of PTI-ETS interactions until NLR-mediated ETI finally leads to hypersensitive response (HR) and programmed cell death (PCD) to confine the pathogen to the initial sites of infection. However, while both RNA silencing and PTI apply to this arms race model, they are part of an interaction network in which these two pathways cooperate with each other and are also controlled by interactions with other components of the plant immune system. I have mentioned several interactions, and an overview is given in **Figure 4**. In addition, the immune system includes important roles of autophagy, protein degradation, and hormones that I could not mention here and that are reviewed elsewhere (152–154). Certainly, further detailed analysis of the antiviral plant immune system will allow the development of novel strategies against viral diseases in cultivated plants. Efforts to control plant diseases by expressing dominant resistance (NLR) genes (155) or using CRISPR/Cas9 to target viruses or engineer recessive resistance by knocking out genes required for virus susceptibility (156) have limitations as they are often virus specific and also exert selective pressure leading to the evolution of viruses that overcome resistance. To avoid these limitations, new strategies could include breeding for tolerance, which limits the selective pressure on the virus while maintaining plant fitness (157). Several studies have shown that silencing-based mechanisms and viral self-attenuation strategies play a role in tolerance (158, 159). A different yet highly promising approach to protect plants against pests and pathogens, including viruses, is to expose them to pest/virus genome-homologous dsRNA, which can be done by producing transgenic plants or by spraying [RNA-based plant protection (160, 161)]. Spraying avoids the use of transgenic plants but has the limitation that to be effective against viruses, the sprayed dsRNA must enter the cell through the cuticle and cell wall. However, once the technology is developed to overcome this important hurdle, formulated dsRNA agents could be used to protect plants against viruses by activating both antiviral RNA silencing and PTI. Mixtures of dsRNAs with homology against multiple viruses could be produced and applied on demand for broad and environmentally sustainable protection against viruses. However, to be successful, this strategy must ensure that the dsRNA-induced RNA silencing and PTI responses are sufficiently strong (or early) to overcome (or avoid) the counteraction of the virus-encoded effectors. In addition to uptake and efficiency, the stability of dsRNA molecules and the ability to synthesize dsRNA on a large scale and at an affordable price are just some of the parameters that need to be considered for the large-scale practical application of dsRNA for virus control.

## SUMMARY POINTS

1. Plants recognize viruses and use antiviral defense mechanisms to control their accumulation and cell-to-cell movement.

2. Double-stranded RNA (dsRNA) produced during virus replication induces antiviral RNA silencing and pattern-triggered immunity (PTI).

3. Antiviral RNA silencing is induced by recognition of viral dsRNA by DICER-LIKE proteins and interferes with virus accumulation by targeting the viral genome or viral transcripts for cleavage. Viruses control this host defense response with their viral suppressors of RNA silencing (VSRs).

4. Viral infection initiates the production of various host small interfering RNA (siRNA) species that, together with virus-derived siRNAs, have the potential to control host gene expression in cells at, and ahead of, the spreading viral infection front.
5. RNA virus infection induces PTI upon recognition of dsRNA by a SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1-containing receptor complex, which triggers signaling through BOTRYTIS-INDUCED KINASE 1 and other identified components, leading to the closure of plasmodesmata (PD), thereby interfering with the ability of the virus to spread infection from the infected cell to neighboring cells.

6. Tobamovirus movement proteins (MPs) interfere with dsRNA-induced PD closure, thereby facilitating virus movement.

7. Receptor-mediated recognition of geminivirus-derived nucleic acids leads to translational shutdown via a pathway distinct from the PTI pathway induced by microbes and RNA viruses, which is suppressed by the virus-encoded nuclear shuttle protein (NSP).

8. Virus-encoded proteins can be recognized by intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), leading to effector-triggered immunity (ETI). In the absence of viruses and their VSRs, NLR expression is controlled by post-transcriptional gene silencing.

## FUTURE ISSUES

1. Which pathogen-recognition receptor or receptors detect the presence of dsRNA?

2. Do plant cells secrete dsRNA and other viral products into the apoplastic space to allow recognition by receptor complexes at the PM, or does recognition depend on receptor internalization? Could there be a general role of the plant extracellular matrix in plant-virus interactions?

3. What is the mechanism by which tobamovirus MPs suppress dsRNA-induced closure of PD? Do MPs from other viruses also have this activity?

4. What other elements, in addition to those identified, distinguish the PTI response elicited by RNA viruses from that elicited by microbes?

5. Given that viruses propagate within cells, how are plants able to recognize viruses and virus-derived proteins and nucleic acids as nonself?

6. What other pathogen-associated patterns, danger-associated molecular patterns, or inducible danger-associated molecular patterns besides dsRNA are produced in the virus-infected plant cell, and how are the elicited host response pathways coordinated?

7. Which virus-induced siRNAs target host genes, and what role do they play in virus-host compatibility?

8. How can the different PTI responses induced by RNA viruses and geminiviruses be reconciled?

## DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS

The author apologizes to all the scientists whose research could not be appropriately cited owing
to space limitations. The author would like to thank Laura Elvira-Gonzalez, Ana Sede, Karl-
Heinz Kogel, and Todd Blevins for critical reading of the manuscript and providing thoughtful
comments. The author acknowledges current research funding by the Agence Nationale de la
Recherche for projects GreenTolerance (ANR grant ANR-21-CE20-0020-01) and BioProtect
(ANR-21-SUSC-0003-01). BioProtect is carried out under the second call of the ERA-NET
Cofund SusCrop, being part of the Joint Programming Initiative on Agriculture, Food Security
and Climate Change. SusCrop has received funding from the European Union's Horizon 2020
research and innovation program under grant agreement 771134.

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