scholarly journals Multiple Functions of Rice Dwarf Phytoreovirus Pns10 in Suppressing Systemic RNA Silencing

2010 ◽  
Vol 84 (24) ◽  
pp. 12914-12923 ◽  
Author(s):  
Bo Ren ◽  
Yuanyuan Guo ◽  
Feng Gao ◽  
Peng Zhou ◽  
Feng Wu ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Stability ◽  
Antiviral Defense ◽  
Double Stranded Rna ◽  
Systemic Spread ◽  
Dsrna Viruses ◽  
Systemic Rna ◽  
Interfering Rna ◽  
Diverse Groups

ABSTRACT RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by Rice dwarf phytoreovirus (RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used Nicotiana benthamiana model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3′ overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.

scholarly journals Viral Class 1 RNase III Involved in Suppression of RNA Silencing

2005 ◽  
Vol 79 (11) ◽  
pp. 7227-7238 ◽  
Author(s):  
Jan F. Kreuze ◽  
Eugene I. Savenkov ◽  
Wilmer Cuellar ◽  
Xiangdong Li ◽  
Jari P. T. Valkonen
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Virus ◽  
Antiviral Defense ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Class 1 ◽  
Heterologous Virus ◽  
Silencing Suppression ◽  
Interfering Rna

ABSTRACT Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.

scholarly journals Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells

2004 ◽  
Vol 78 (6) ◽  
pp. 3149-3154 ◽  
Author(s):  
Eugene V. Ryabov ◽  
Rene van Wezel ◽  
John Walsh ◽  
Yiguo Hong
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Epidermal Cells ◽  
Turnip Crinkle Virus ◽  
Mechanical Inoculation ◽  
Long Distance ◽  
Systemic Spread ◽  
Systemic Rna ◽  
Green Fluorescent

ABSTRACT A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFPΔCP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFPΔCP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFPΔCP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFPΔCP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.

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

2008 ◽  
Vol 83 (5) ◽  
pp. 2188-2200 ◽  
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.

scholarly journals The Suppressor of Transgene RNA Silencing Encoded by Cucumber mosaic virus Interferes with Salicylic Acid-Mediated Virus Resistance

2001 ◽  
Vol 14 (6) ◽  
pp. 715-724 ◽  
Author(s):  
Liang-Hui Ji ◽  
Shou-Wei Ding
Keyword(s):  
Salicylic Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Virus Resistance ◽  
Rna Silencing ◽  
Defense Mechanism ◽  
Antiviral Defense ◽  
Oxidase Gene ◽  
Systemic Spread ◽  
Systemic Infections

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pre-treated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.

scholarly journals Antiviral Defense Involves AGO4 in an Arabidopsis–Potexvirus Interaction

2016 ◽  
Vol 29 (11) ◽  
pp. 878-888 ◽  
Author(s):  
Chantal Brosseau ◽  
Mohamed El Oirdi ◽  
Ayooluwa Adurogbangba ◽  
Xiaofang Ma ◽  
Peter Moffett
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Rna Silencing ◽  
Defense Mechanism ◽  
Rna Viruses ◽  
Antiviral Defense ◽  
Double Stranded Rna ◽  
Micro Rnas ◽  
Plantago Asiatica ◽  
Antiviral Activities

In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.

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

2007 ◽  
Vol 88 (1) ◽  
pp. 316-324 ◽  
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.

scholarly journals Dissection of Double-Stranded RNA Binding Protein B2 from Betanodavirus

2007 ◽  
Vol 81 (11) ◽  
pp. 5449-5459 ◽  
Author(s):  
Beau J. Fenner ◽  
Winnie Goh ◽  
Jimmy Kwang
Keyword(s):  
Rna Silencing ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Alanine Scanning Mutagenesis ◽  
Marine Aquaculture ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Fish Cells ◽  
Interfering Rna

ABSTRACT Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.

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

Plants ◽  
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.

Role of double-stranded RNA-binding proteins in RNA silencing and antiviral defense

2014 ◽  
pp. 1-16
Author(s):  
Jasleen Singh ◽  
Xiuchun Zhang ◽  
Lucy R. Stewart ◽  
Thomas Mitchell ◽  
Feng Qu
Keyword(s):  
Rna Silencing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Antiviral Defense ◽  
Double Stranded Rna

scholarly journals p24G1 Encoded by Grapevine Leafroll-Associated Virus 1 Suppresses RNA Silencing and Elicits Hypersensitive Response-Like Necrosis in Nicotiana Species

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1111
Author(s):  
Chen-Wei Zhang ◽  
Qing Liu ◽  
Qi Zeng ◽  
Wen-Ting Huang ◽  
Qi Wang ◽  
...  
Keyword(s):  
Hypersensitive Response ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Potato Virus X ◽  
Mobility Shift ◽  
Nicotiana Species ◽  
P24 Protein ◽  
Systemic Rna ◽  
Interfering Rna ◽  
Electrophoretic Mobility Shift Assays

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.

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