Evidence That RNA Silencing-Mediated Resistance to Beet necrotic yellow vein virus Is Less Effective in Roots Than in Leaves

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

Download Full-text

Recovery of Nicotiana benthamiana Plants from a Necrotic Response Induced by a Nepovirus Is Associated with RNA Silencing but Not with Reduced Virus Titer

Journal of Virology ◽

10.1128/jvi.01192-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12285-12297 ◽

Cited By ~ 49

Author(s):

Juan Jovel ◽

Melanie Walker ◽

Hélène Sanfaçon

Keyword(s):

Host Defense ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Defense Mechanism ◽

Fluorescent Protein ◽

Virus Titer ◽

Defense Responses ◽

Viral Rna ◽

Small Interfering Rnas ◽

Green Fluorescent

ABSTRACT Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed.

Download Full-text

The p27 open reading frame of tomato infectious chlorosis virus encodes a suppressor of RNA silencing

Journal of General Plant Pathology ◽

10.1007/s10327-019-00850-0 ◽

2019 ◽

Vol 85 (4) ◽

pp. 301-305 ◽

Cited By ~ 1

Author(s):

Takaaki Mashiko ◽

Wei-Qin Wang ◽

Sedyo Hartono ◽

Gede Suastica ◽

Yutaro Neriya ◽

...

Keyword(s):

Rna Silencing ◽

Open Reading Frame ◽

Reading Frame ◽

Tomato Infectious Chlorosis Virus ◽

Suppressor Of Rna Silencing

Download Full-text

Role of RNA Silencing Suppression in the Pathogenicity and Host Specificity of the Grapevine Virus A

10.32747/2010.7592114.bard ◽

2010 ◽

Author(s):

Munir Mawassi ◽

Valerian Dolja

Keyword(s):

Rna Silencing ◽

Defense Mechanism ◽

Plant Viruses ◽

Small Interfering Rnas ◽

Grapevine Virus ◽

Virus Transport ◽

Systematic Analysis ◽

Grapevine Virus A ◽

Silencing Suppression ◽

And Function

RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.

Download Full-text

Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication

Journal of Virology ◽

10.1128/jvi.79.8.5129-5141.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 5129-5141 ◽

Cited By ~ 30

Author(s):

Qingmei Jia ◽

Vasili Chernishof ◽

Eric Bortz ◽

Ian Mchardy ◽

Ting-Ting Wu ◽

...

Keyword(s):

Viral Replication ◽

Translation Termination ◽

Human Herpesvirus ◽

Open Reading Frame ◽

Viral Gene ◽

Small Interfering Rnas ◽

Reading Frame ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.

Download Full-text

RNA silencing machinery contributes to inability of BSBV to establish infection in Nicotiana benthamiana: evidence from characterization of agroinfectious clones of Beet soil-borne virus

Journal of General Virology ◽

10.1099/jgv.0.001530 ◽

2020 ◽

Author(s):

Mathieu Mahillon ◽

Alain Decroës ◽

Chloé Peduzzi ◽

Gustavo Romay ◽

Anne Legrève ◽

...

Keyword(s):

Sugar Beet ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Low Frequency ◽

Biological Properties ◽

Polymyxa Betae ◽

Local Infection ◽

Plant Parts ◽

Root Necrosis ◽

Viral Suppressors

Beet soil-borne virus (BSBV) is a sugar beet pomovirus frequently associated with Beet necrotic yellow veins virus, the causal agent of the rhizomania disease. BSBV has been detected in most of the major beet-growing regions worldwide, yet its impact on this crop remains unclear. With the aim to understand the life cycle of this virus and clarify its putative pathogenicity, agroinfectious clones have been engineered for each segment of its tripartite genome. The biological properties of these clones were then studied on different plant species. Local infection was obtained on agroinfiltrated leaves of Beta macrocarpa. On leaves of Nicotiana benthamiana, similar results were obtained, but only when heterologous viral suppressors of RNA silencing were co-expressed or in a transgenic line down regulated for both dicer-like protein 2 and 4. On sugar beet, local infection following agroinoculation was obtained on cotyledons, but not on other tested plant parts. Nevertheless, leaf symptoms were observed on this host via sap inoculation. Likewise, roots were efficiently mechanically infected, highlighting low frequency of root necrosis and constriction, and enabling the demonstration of transmission by the vector Polymyxa betae. Altogether, the entire viral cycle was reproduced, validating the constructed agroclones as efficient inoculation tools, paving the way for further studies on BSBV and its related pathosystem.

Download Full-text

Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity

Journal of Virology ◽

10.1128/jvi.79.11.7217-7226.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 7217-7226 ◽

Cited By ~ 88

Author(s):

Zsuzsanna Mérai ◽

Zoltán Kerényi ◽

Attila Molnár ◽

Endre Barta ◽

Anna Válóczi ◽

...

Keyword(s):

Rna Silencing ◽

Regulatory System ◽

Sequence Specificity ◽

Silencing Suppressor ◽

Small Interfering Rnas ◽

Reading Frame ◽

Dsrna Binding ◽

Silencing Suppression ◽

Orf 5

ABSTRACT RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.

Download Full-text

Inhibition of Gammaherpesvirus Replication by RNA Interference

Journal of Virology ◽

10.1128/jvi.77.5.3301-3306.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3301-3306 ◽

Cited By ~ 71

Author(s):

Qingmei Jia ◽

Ren Sun

Keyword(s):

Rna Interference ◽

Viral Gene Expression ◽

Open Reading Frame ◽

Viral Gene ◽

Specific Gene ◽

Small Interfering Rnas ◽

Viral Production ◽

Reading Frame ◽

Early Protein ◽

Expression Program

ABSTRACT RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.

Download Full-text

Chimeric cDNA Sequences from Citrus tristeza virus Confer RNA Silencing-Mediated Resistance in Transgenic Nicotiana benthamiana Plants

Phytopathology ◽

10.1094/phyto-96-0819 ◽

2006 ◽

Vol 96 (8) ◽

pp. 819-827 ◽

Cited By ~ 11

Author(s):

Gourgopal Roy ◽

Mysore R. Sudarshana ◽

Diane E. Ullman ◽

Shou-Wei Ding ◽

Abhaya M. Dandekar ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Rna Silencing ◽

Citrus Tristeza Virus ◽

Potato Virus X ◽

Northern Hybridization ◽

Transcriptional Gene Silencing ◽

Small Interfering Rnas ◽

Cdna Sequences ◽

Tristeza Virus ◽

Citrus Tristeza

RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3′ untranslated region (UTR); and pCTV2 contained CP, p23, and 3′ UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.

Download Full-text