Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.

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Argonaute 2 Controls Antiviral Activity against Sweet Potato Mild Mottle Virus in Nicotiana benthamiana

Plants ◽

10.3390/plants10050867 ◽

2021 ◽

Vol 10 (5) ◽

pp. 867

Author(s):

Erzsébet Kenesi ◽

Juan-Jose Lopez-Moya ◽

László Orosz ◽

József Burgyán ◽

Lóránt Lakatos

Keyword(s):

Sweet Potato ◽

Nicotiana Benthamiana ◽

Small Rnas ◽

Rna Silencing ◽

Mottle Virus ◽

Antiviral Defense ◽

Biological Processes ◽

Catalytic Subunits ◽

Argonaute 2

RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2−/− mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato.

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Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region

Journal of Virology ◽

10.1128/jvi.01566-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11603-11618 ◽

Cited By ~ 3

Author(s):

Maitreyi Chattopadhyay ◽

Vera A. Stupina ◽

Feng Gao ◽

Christine R. Szarko ◽

Micki M. Kuhlmann ◽

...

Keyword(s):

Small Rnas ◽

Rna Silencing ◽

Untranslated Region ◽

Higher Order ◽

Order Structure ◽

Silencing Suppressor ◽

Turnip Crinkle Virus ◽

Site Mutation ◽

Primary Mutation ◽

Positive Strand Rna

ABSTRACTTurnip crinkle virus (TCV) contains a structured 3′ region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3′ untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2′-hydroxylacylation analyzed byprimerextension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3′UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or indcl-2/dcl4orago1/ago2backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs.IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3′ UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3′ UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.

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Viroid-induced DNA methylation in plants

BioMolecular Concepts ◽

10.1515/bmc-2013-0030 ◽

2013 ◽

Vol 4 (6) ◽

pp. 557-565 ◽

Cited By ~ 13

Author(s):

Athanasios Dalakouras ◽

Elena Dadami ◽

Michael Wassenegger

Keyword(s):

Dna Methylation ◽

Small Rnas ◽

Rna Silencing ◽

De Novo ◽

Rna Degradation ◽

Double Stranded Rna ◽

Methyl Group ◽

Rna Molecules ◽

Cumulative Data

AbstractIn eukaryotes, DNA methylation refers to the addition of a methyl group to the fifth atom in the six-atom ring of cytosine residues. At least in plants, DNA regions that become de novo methylated can be defined by homologous RNA molecules in a process termed RNA-directed DNA methylation (RdDM). RdDM was first discovered in viroid-infected plants. Viroids are pathogenic circular, non-coding, single-stranded RNA molecules. Members of the Pospiviroidae family replicate in the nucleus through double-stranded RNA intermediates, attracting the host RNA silencing machinery. The recruitment of this machinery results in the production of viroid-derived small RNAs (vd-sRNAs) that mediate RNA degradation and DNA methylation of cognate sequences. Here, we provide an overview of the cumulative data on the field of viroid-induced RdDM and discuss three possible scenarios concerning the mechanistic details of its establishment.

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Apple chlorotic leaf spot virus 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana

Journal of General Virology ◽

10.1099/vir.0.82377-0 ◽

2007 ◽

Vol 88 (1) ◽

pp. 316-324 ◽

Cited By ~ 44

Author(s):

Hajime Yaegashi ◽

Tsubasa Takahashi ◽

Masamichi Isogai ◽

Takashi Kobori ◽

Satoshi Ohki ◽

...

Keyword(s):

Leaf Spot ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Fluorescent Protein ◽

Movement Protein ◽

Silencing Suppressor ◽

Spot Virus ◽

Double Stranded Rna ◽

Chlorotic Leaf ◽

Green Fluorescent

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.

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HC-Pro, a potyvirus RNA silencing suppressor, cancels cycling of Cucumber mosaic virus in Nicotiana benthamiana plants

Virus Genes ◽

10.1007/s11262-010-0460-0 ◽

2010 ◽

Vol 40 (3) ◽

pp. 440-446 ◽

Cited By ~ 22

Author(s):

Noriho Fukuzawa ◽

Noriko Itchoda ◽

Takeaki Ishihara ◽

Kazunori Goto ◽

Chikara Masuta ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Silencing Suppressor ◽

Rna Silencing Suppressor

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The role of RST1 and RIPR proteins in plant RNA quality control systems

Plant Molecular Biology ◽

10.1007/s11103-021-01145-9 ◽

2021 ◽

Author(s):

Mariann Auth ◽

Tünde Nyikó ◽

Andor Auber ◽

Dániel Silhavy

Keyword(s):

Quality Control ◽

Control Systems ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Rna Degradation ◽

Mrna Degradation ◽

Rna Quality ◽

Essential Components ◽

Rna Quality Control

AbstractTo keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3′-5′ and/or XRN4 5′-3′ exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome–mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex.

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A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

Journal of Virology ◽

10.1128/jvi.02339-06 ◽

2007 ◽

Vol 81 (6) ◽

pp. 2980-2994 ◽

Cited By ~ 133

Author(s):

Asuka Itaya ◽

Xuehua Zhong ◽

Ralf Bundschuh ◽

Yijun Qi ◽

Ying Wang ◽

...

Keyword(s):

Secondary Structure ◽

Small Rnas ◽

Rna Silencing ◽

Effective Means ◽

Critical Role ◽

Potato Spindle Tuber Viroid ◽

Biologically Active ◽

Antiviral Defense ◽

Silencing Suppressor ◽

Rna Structures

ABSTRACT RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

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Diverse and Newly Recognized Effects Associated with Short Interfering RNA Binding Site Modifications on the Tomato Bushy Stunt Virus P19 Silencing Suppressor

Journal of Virology ◽

10.1128/jvi.02186-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2188-2200 ◽

Cited By ~ 27

Author(s):

Yi-Cheng Hsieh ◽

Rustem T. Omarov ◽

Herman B. Scholthof

Keyword(s):

Symptom Severity ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Rna Binding ◽

Tomato Bushy Stunt Virus ◽

Silencing Suppressor ◽

Virus Spread ◽

Systemic Spread ◽

Specific Activities ◽

Interfering Rna

ABSTRACT The Tomato bushy stunt virus-encoded P19 forms dimers that bind duplex short interfering RNAs (siRNAs) to suppress RNA silencing. P19 is also involved in multiple host-specific activities, including the elicitation of symptoms, and in local and/or systemic spread. To study the correlation between those various roles and the siRNA binding by P19, predicted siRNA-interacting sites were modified. Twenty-two mutants were generated and inoculated onto Nicotiana benthamiana plants, to reveal that (i) they were all infectious, (ii) symptom differences did not correlate strictly with mutation-associated variation in P19 accumulation, and (iii) substitutions affecting a central domain of P19 generally exhibited symptoms more severe than for mutations affecting peripheral regions. Three mutants selected to represent separate phenotypic categories all displayed a substantially reduced ability to sequester siRNA. Consequently, these three mutants were compromised for systemic virus spread in P19-dependent hosts but had differential plant species-dependent effects on the symptom severity. One mutant in particular caused relatively exacerbated symptoms, exemplified by extensive morphological leaf deformations in N. benthamiana; this was especially remarkable because P19 was undetectable. Another striking feature of this mutant was that only within a few days after infection, viral RNA was cleared by silencing. One more original property was that host RNAs and proteins (notably, the P19-interactive Hin19 protein) were also susceptible to degradation in these infected N. benthamiana plants but not in spinach. In conclusion, even though siRNA binding by P19 is a key functional property, compromised siRNA sequestration can result in novel and diverse host-dependent properties.

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Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

Journal of Virology ◽

10.1128/jvi.00727-07 ◽

2007 ◽

Vol 81 (19) ◽

pp. 10379-10388 ◽

Cited By ~ 82

Author(s):

Hannes Vogler ◽

Rashid Akbergenov ◽

Padubidri V. Shivaprasad ◽

Vy Dang ◽

Monika Fasler ◽

...

Keyword(s):

Small Rnas ◽

Rna Silencing ◽

Fluorescent Protein ◽

Plant Viruses ◽

Micro Rna ◽

Silencing Suppressor ◽

Small Interfering Rnas ◽

Suppressor Activity ◽

Virus Family ◽

Green Fluorescent

ABSTRACT Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

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Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-3-0294 ◽

2010 ◽

Vol 23 (3) ◽

pp. 294-303 ◽

Cited By ~ 125

Author(s):

Inmaculada González ◽

Llucia Martínez ◽

Daria V. Rakitina ◽

Mathew G. Lewsey ◽

Félix A. Atencio ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Small Rnas ◽

Rna Silencing ◽

Silencing Suppressor ◽

Suppressor Activity ◽

Rna Silencing Suppressor ◽

2B Protein

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

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