Antiviral RNA Silencing Research Articles

Cucumber mosaic virus coat protein modulates the accumulation of 2b protein and antiviral silencing that causes symptom recovery in planta.

Shoot apical meristems (SAM) are resistant to most plant viruses due to RNA silencing, which is restrained by viral suppressors of RNA silencing (VSRs) to facilitate transient viral invasion of the SAM. In many cases chronic symptoms and long-term virus recovery occur, but the underlying mechanisms are poorly understood. Here, we found that wild-type Cucumber mosaic virus (CMVWT) invaded the SAM transiently, but was subsequently eliminated from the meristems. Unexpectedly, a CMV mutant, designated CMVRA that harbors an alanine substitution in the N-terminal arginine-rich region of the coat protein (CP) persistently invaded the SAM and resulted in visible reductions in apical dominance. Notably, the CMVWT virus elicited more potent antiviral silencing than CMVRA in newly emerging leaves of infected plants. However, both viruses caused severe symptoms with minimal antiviral silencing effects in the Arabidopsis mutants lacking host RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) or SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that CMVWT induced host RDR6/SGS3-dependent antiviral silencing. We also showed that reduced accumulation of the 2b protein is elicited in the CMVWT infection and consequently rescues potent antiviral RNA silencing. Indeed, co-infiltration assays showed that the suppression of posttranscriptional gene silencing mediated by 2b is more severely compromised by co-expression of CPWT than by CPRA. We further demonstrated that CPWT had high RNA binding activity leading to translation inhibition in wheat germ systems, and CPWT was associated with SGS3 into punctate granules in vivo. Thus, we propose that the RNAs bound and protected by CPWT possibly serve as templates of RDR6/SGS3 complexes for siRNA amplification. Together, these findings suggest that the CMV CP acts as a central hub that modulates antiviral silencing and VSRs activity, and mediates viral self-attenuation and long-term symptom recovery.

CaRDR1, an RNA-Dependent RNA Polymerase Plays a Positive Role in Pepper Resistance against TMV

RNA silencing functions as a major natural antiviral defense mechanism in plants. RNA-dependent RNA polymerases (RDRs) that catalyze the synthesis of double-stranded RNAs, are considered as a fundamental element in RNA silencing pathways. In Arabidopsis thaliana, RDR1, 2 and 6 play important roles in anti-viral RNA silencing. Expression of RDR1 can be elevated following plant treatment with defense hormones and virus infection. RDR1 has been studied in several crop species, but not in pepper (Capsicum annuum L.). Here, a RDR1 gene was isolated from Capsicum annuum L., designated as CaRDR1. The full-length cDNA of CaRDR1 was 3,351 bp, encoding a 1,116-amino acid protein, which contains conserved regions, such as the most remarkable motif DLDGD. The transcripts of CaRDR1 could be induced by salicylic acid (SA), abscisic acid (ABA), H2O2, and tobacco mosaic virus (TMV). Silencing of CaRDR1 in pepper resulted in increased susceptibility to TMV as evident by severe symptom, increased of TMV-CP transcript, higher malondialdehyde (MDA) content and lower antioxidant enzymes activities compared with that of control plants. CaRDR1-overexpressing in Nicotiana benthamiana showed mild disease symptom and reduced TMV-CP transcripts than that of empty vector (EV) following TMV inoculation. The RNA silencing related genes, including NbAGO2, NbDCL2, NbDCL3, and NbDCL4 elevated expression in overexpressed plants. Alternative oxidase (AOX), the terminal oxidase of the cyanide (CN)-resistant alternative respiratory pathway, catalyze oxygen-dependent oxidation of ubiquinol in plants. It has an important function in plant defense against TMV. In addition, CaRDR1 overexpression promoted the expression of NbAOX1a and NbAOX1b. In conclusion, these results suggest that CaRDR1 plays a positive role in TMV resistance by regulating antioxidant enzymes activities and RNA silencing-related genes expression to suppress the replication and movement of TMV.

Interfering Satellite RNAs of Bamboo mosaic virus.

Satellite RNAs (satRNAs) are sub-viral agents that may interact with their cognate helper virus (HV) and host plant synergistically and/or antagonistically. SatRNAs totally depend on the HV for replication, so satRNAs and HV usually evolve similar secondary or tertiary RNA structures that are recognized by a replication complex, although satRNAs and HV do not share an appreciable sequence homology. The satRNAs of Bamboo mosaic virus (satBaMV), the only satRNAs of the genus Potexvirus, have become one of the models of how satRNAs can modulate HV replication and virus-induced symptoms. In this review, we summarize the molecular mechanisms underlying the interaction of interfering satBaMV and BaMV. Like other satRNAs, satBaMV mimics the secondary structures of 5′- and 3′-untranslated regions (UTRs) of BaMV as a molecular pretender. However, a conserved apical hairpin stem loop (AHSL) in the 5′-UTR of satBaMV was found as the key determinant for downregulating BaMV replication. In particular, two unique nucleotides (C60 and C83) in the AHSL of satBaMVs determine the satBaMV interference ability by competing for the replication machinery. Thus, transgenic plants expressing interfering satBaMV could confer resistance to BaMV, and interfering satBaMV could be used as biological-control agent. Unlike two major anti-viral mechanisms, RNA silencing and salicylic acid-mediated immunity, our findings in plants by in vivo competition assay and RNA deep sequencing suggested replication competition is involved in this transgenic satBaMV-mediated BaMV interference. We propose how a single nucleotide of satBaMV can make a great change in BaMV pathogenicity and the underlying mechanism.

Crispr/Cas9 Mediated Inactivation of Argonaute 2 Reveals its Differential Involvement in Antiviral Responses

RNA silencing constitutes an important antiviral mechanism in plants. Small RNA guided Argonaute proteins fulfill essential role in this process by acting as executors of viral restriction. Plants encode multiple Argonaute proteins of which several exhibit antiviral activities. A recent addition to this group is AGO2. Its involvement in antiviral responses is established predominantly by studies employing mutants of Arabidopsis thaliana. In the virological model plant, Nicotiana benthamiana, the contribution of AGO2 to antiviral immunity is much less certain due to the lack of appropriate genetic mutants. Previous studies employed various RNAi based tools to down-regulate AGO2 expression. However, these techniques have several disadvantages, especially in the context of antiviral RNA silencing. Here, we have utilized the CRISPR/Cas9 technology to inactivate the AGO2 gene of N. benthamiana. The ago2 plants exhibit differential sensitivities towards various viruses. AGO2 is a critical component of the plants’ immune responses against PVX, TuMV and TCV. In contrast, AGO2 deficiency does not significantly influence the progression of tombusvirus and CMV infections. In summary, our work provides unequivocal proof for the virus-specific antiviral role of AGO2 in a plant species other than A. thaliana for the first time.

Identification and characterization of two RNA silencing suppressors encoded by ophioviruses

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K–25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MPCPsV and 24KCPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MPCPsV and 24KCPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway.

SAGA complex mediates the transcriptional up-regulation of antiviral RNA silencing

Pathogen recognition and transcriptional activation of defense-related genes are crucial steps in cellular defense responses. RNA silencing (RNAi) functions as an antiviral defense in eukaryotic organisms. Several RNAi-related genes are known to be transcriptionally up-regulated upon virus infection in some host organisms, but little is known about their induction mechanism. A phytopathogenic ascomycete, Cryphonectria parasitica (chestnut blight fungus), provides a particularly advantageous system to study RNAi activation, because its infection by certain RNA viruses induces the transcription of dicer-like 2 (dcl2) and argonaute-like 2 (agl2), two major RNAi players. To identify cellular factors governing activation of antiviral RNAi in C. parasitica, we developed a screening protocol entailing multiple transformations of the fungus with cDNA of a hypovirus mutant lacking the RNAi suppressor (CHV1-Δp69), a reporter construct with a GFP gene driven by the dcl2 promoter, and a random mutagenic construct. Screening for GFP-negative colonies allowed the identification of sgf73, a component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, a well-known transcriptional coactivator. Knockout of other SAGA components showed that the histone acetyltransferase module regulates transcriptional induction of dcl2 and agl2, whereas histone deubiquitinase mediates regulation of agl2 but not dcl2 Interestingly, full-scale induction of agl2 and dcl2 by CHV1-Δp69 required both DCL2 and AGL2, whereas that by another RNA virus, mycoreovirus 1, required only DCL2, uncovering additional roles for DCL2 and AGL2 in viral recognition and/or RNAi activation. Overall, these results provide insight into the mechanism of RNAi activation.

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing.

Phytoviruses encode viral suppressors of RNA silencing (VSRs) to counteract the plant antiviral silencing response, which relies on virus-derived small interfering (si)RNAs processed by Dicer RNaseIII enzymes and subsequently loaded into ARGONAUTE (AGO) effector proteins. Here, a tobacco cell-free system was engineered to recapitulate the key steps of antiviral RNA silencing and, in particular, the most upstream double-stranded (ds)RNA processing reaction, not kinetically investigated thus far in the context of plant VSR studies. Comparative biochemical analyses of distinct VSRs in the reconstituted assay showed that in all cases tested, VSR interactions with siRNA duplexes inhibited the loading, but not the activity, of antiviral AGO1 and AGO2. Turnip crinkle virus P38 displayed the additional and unique property to bind both synthetic and RNA-dependent-RNA-polymerase-generated long dsRNAs, and inhibited the processing into siRNAs. Single amino acid substitutions in P38 could dissociate dsRNA-processing from AGO-loading inhibition in vitro and in vivo, illustrating dual-inhibitory strategies discriminatively deployed within a single viral protein, which, we further show, are bona fide suppressor functions that evolved independently of the conserved coat protein function of P38.

PhOBF1, a petunia ocs element binding factor, plays an important role in antiviral RNA silencing.

Virus-induced gene silencing (VIGS) is a common reverse genetics strategy for characterizing the function of genes in plants. The detailed mechanism governing RNA silencing efficiency triggered by viruses is largely unclear. Here, we reveal that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of the basic leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and a reporter gene, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or the white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of the silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside, and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or PhOBF1-overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by the SA biosynthesis pathway.

Dicer-Like 4 Is Involved in Restricting the Systemic Movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.

Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Ros1 systemic accumulation dramatically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.

The Potyvirus Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways.

Potyviruses represent the largest group of known plant viruses and cause significant losses of many agriculturally important crops in the world. In order to establish infection, potyviruses must overcome the host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection.

Modulation of host plant immunity by Tobamovirus proteins.

To establish successful infection, plant viruses produce profound alterations of host physiology, disturbing unrelated endogenous processes and contributing to the development of disease. In tobamoviruses, emerging evidence suggests that viral-encoded proteins display a great variety of functions beyond the canonical roles required for virus structure and replication. Among these, their modulation of host immunity appears to be relevant in infection progression. In this review, some recently described effects on host plant physiology of Tobacco mosaic virus (TMV)-encoded proteins, namely replicase, movement protein (MP) and coat protein (CP), are summarized. The discussion is focused on the effects of each viral component on the modulation of host defense responses, through mechanisms involving hormonal imbalance, innate immunity modulation and antiviral RNA silencing. These effects are described taking into consideration the differential spatial distribution and temporality of viral proteins during the dynamic process of replication and spread of the virus. In discussion of these mechanisms, it is shown that both individual and combined effects of viral-encoded proteins contribute to the development of the pathogenesis process, with the host plant's ability to control infection to some extent potentially advantageous to the invading virus.

Characterization of small interfering RNAs derived from Rice black streaked dwarf virus in infected maize plants by deep sequencing

Rice black streaked dwarf virus (RBSDV) is the casual agent of maize rough dwarf disease, which frequently causes severe yield loss in China. However, the interaction between RBSDV and maize plants is largely unknown. RNA silencing is a conserved mechanism against viruses in plants. To understand the antiviral RNA interfering response in RBSDV-infected plants, the profile of virus-derived small interfering RNAs (vsiRNAs) from RBSDV in infected maize plants was obtained by deep sequencing in this study. Our data showed that vsiRNAs, accumulated preferentially as 21- and 22-nucleotide (nt) species, were mapped against all 10 genomic RNA segments of RBSDV and derived almost equally overall from both positive and negative strands, while there were significant differences in the accumulation level of vsiRNAs from segments 2, 4, 6, 7 and 10. The vsiRNAs (21 and 22 nt) generated from each segment of RBSDV genome had a 5′-terminal nucleotide bias toward adenine and uracil. The single-nucleotide resolution maps showed that RBSDV-derived siRNAs preferentially distributed in the 5′- or 3′-terminal regions of several genomic segments. In addition, our results showed that the mRNA levels of some components involved in antiviral RNA silencing pathway were differentially modified during RBSDV infection. Among them, the accumulation levels of ZmDCL1, ZmDCL2, ZmDCL3a, ZmAGO1a, ZmAGO1b, ZmAGO2a, ZmAGO18a and ZmRDR6 mRNAs were significantly up-regulated, while those of ZmDCL3b, ZmDCL4 and ZmAGO1c mRNAs showed no obvious changes in RBSDV-infected maize plants.

Mechanisms, applications, and perspectives of antiviral RNA silencing in plants.

Viral diseases of plants cause important economic losses due to reduction in crop quality and quantity to the point of threatening food security in some countries. Given the reduced availability of natural sources, genetic resistance to viruses has been successfully engineered for some plant-virus combinations. A sound understanding of the basic mechanisms governing plant-virus interactions, including antiviral RNA silencing, is the foundation to design better management strategies and biotechnological approaches to engineer and implement antiviral resistance in plants. In this review, we present current molecular models to explain antiviral RNA silencing and its application in basic plant research, biotechnology and genetic engineering.

Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing.

Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2 Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.

Current advances and prospectus of viral resistance in horticultural crops

Abstract Viruses are a major threat causing massive yield loss and economical damage to crop production worldwide. Through complex evolutionary processes, plants encounter and overcome viral infection by developing effective resistance mechanisms. Over the past decade, remarkable progress has been made in understanding the nature of plant resistance to viruses at the molecular level. This review summarizes the major resistance strategies that plants use to prevent viral infection. Recent investigations suggest that antiviral RNA silencing is the most prevalent defense strategy in plants. Other forms of resistance include R gene-mediated resistance and host factor-related recessive resistance. Naturally occurring resistances arise and are maintained in numerous virus-plant pathosystems based mainly on arms-race relationships and the cost-efficiency of resistance acquisition. In addition to the current status of the known resistance mechanisms, this review discusses the future prospectus for the practical application of plant resistances that influence resistance durability in agricultural ecosystems. Such applications include molecular breeding strategies using advanced molecular marker systems and the utilization of trans- or cis- genetics via the acquisition of engineered disease resistances.

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases

Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

A Signaling Cascade from miR444 to RDR1 in Rice Antiviral RNA Silencing Pathway.

Plant RNA-DEPENDENT RNA POLYMERASE1 (RDR1) is a key component of the antiviral RNA-silencing pathway, contributing to the biogenesis of virus-derived small interfering RNAs. This enzyme also is responsible for producing virus-activated endogenous small interfering RNAs to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific microRNA, miR444, is a key factor in relaying the antiviral signaling from virus infection to OsRDR1 expression. The expression of miR444 is enhanced by infection with Rice stripe virus (RSV), and overexpression of miR444 improves rice (Oryza sativa) resistance against RSV infection accompanied by the up-regulation of OsRDR1 expression. We further show that three miR444 targets, the MIKC(C)-type MADS box proteins OsMADS23, OsMADS27a, and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, an increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 on OsRDR1 transcription, thus activating the OsRDR1-dependent antiviral RNA-silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection, and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results reveal a molecular cascade in the rice antiviral pathway in which miR444 and its MADS box targets directly control OsRDR1 transcription.

Salicylic acid treatment and expression of an RNA-dependent RNA polymerase 1 transgene inhibit lethal symptoms and meristem invasion during tobacco mosaic virus infection in Nicotiana benthamiana.

BackgroundHost RNA-dependent RNA polymerases (RDRs) 1 and 6 contribute to antiviral RNA silencing in plants. RDR6 is constitutively expressed and was previously shown to limit invasion of Nicotiana benthamiana meristem tissue by potato virus X and thereby inhibit disease development. RDR1 is inducible by salicylic acid (SA) and several other phytohormones. But although it contributes to basal resistance to tobacco mosaic virus (TMV) it is dispensable for SA-induced resistance in inoculated leaves. The laboratory accession of N. benthamiana is a natural rdr1 mutant and highly susceptible to TMV. However, TMV-induced symptoms are ameliorated in transgenic plants expressing Medicago truncatula RDR1.ResultsIn MtRDR1-transgenic N. benthamiana plants the spread of TMV expressing the green fluorescent protein (TMV.GFP) into upper, non-inoculated, leaves was not inhibited. However, in these plants exclusion of TMV.GFP from the apical meristem and adjacent stem tissue was greater than in control plants and this exclusion effect was enhanced by SA. TMV normally kills N. benthamiana plants but although MtRDR1-transgenic plants initially displayed virus-induced necrosis they subsequently recovered. Recovery from disease was markedly enhanced by SA treatment in MtRDR1-transgenic plants whereas in control plants SA delayed but did not prevent systemic necrosis and death. Following SA treatment of MtRDR1-transgenic plants, extractable RDR enzyme activity was increased and Western blot analysis of RDR extracts revealed a band cross-reacting with an antibody raised against MtRDR1. Expression of MtRDR1 in the transgenic N. benthamiana plants was driven by a constitutive 35S promoter derived from cauliflower mosaic virus, confirmed to be non-responsive to SA. This suggests that the effects of SA on MtRDR1 are exerted at a post-transcriptional level.ConclusionsMtRDR1 inhibits severe symptom development by limiting spread of virus into the growing tips of infected plants. Thus, RDR1 may act in a similar fashion to RDR6. MtRDR1 and SA acted additively to further promote recovery from disease symptoms in MtRDR1-transgenic plants. Thus it is possible that SA promotes MtRDR1 activity and/or stability through post-transcriptional effects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0705-8) contains supplementary material, which is available to authorized users.

Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.