scholarly journals Identification of an RNA-silencing suppressor in the genome of Grapevine virus A

2006 ◽  
Vol 87 (8) ◽  
pp. 2387-2395 ◽  
Author(s):  
Z. Sh. Zhou ◽  
M. Dell'Orco ◽  
P. Saldarelli ◽  
C. Turturo ◽  
A. Minafra ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Higher Plants ◽  
Ectopic Expression ◽  
Rna Degradation ◽  
Plant Viruses ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Grapevine Virus ◽  
Transient Expression Assay ◽  
Grapevine Virus A

Higher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.

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

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. 

scholarly journals Effects of the Crinivirus Coat Protein–Interacting Plant Protein SAHH on Post-Transcriptional RNA Silencing and Its Suppression

2013 ◽  
Vol 26 (9) ◽  
pp. 1004-1015 ◽  
Author(s):  
M. Carmen Cañizares ◽  
Rosa Lozano-Durán ◽  
Tomás Canto ◽  
Eduardo R. Bejarano ◽  
David M. Bisaro ◽  
...  
Keyword(s):  
Coat Protein ◽  
Rna Silencing ◽  
Rna Degradation ◽  
Plant Viruses ◽  
Plant Protein ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Secondary Sirnas ◽  
Tomato Chlorosis Virus

In plants, post-transcriptional gene silencing (PTGS) is a sequence-specific mechanism of RNA degradation induced by double-stranded RNA (dsRNA), which is processed into small interfering RNAs (siRNAs). siRNAs are methylated and, thereby, stabilized by the activity of the S-adenosylmethionine-dependent RNA methyltransferase HEN1. PTGS is amplified by host-encoded RNA-dependent RNA polymerases (RDR), which generate dsRNA that is processed into secondary siRNAs. To counteract this RNA silencing-mediated response of the host, plant viruses express proteins with silencing suppression activity. Here, we report that the coat protein (CP) of crinivirus (family Closteroviridae, genus Crinivirus) Tomato chlorosis virus, a known suppressor of silencing, interacts with S-adenosylhomocysteine hydrolase (SAHH), a plant protein essential for sustaining the methyl cycle and S-adenosylmethionine-dependent methyltransferase activity. Our results show that, by contributing to an increased accumulation of secondary siRNAs generated by the action of RDR6, SAHH enhances local RNA silencing. Although downregulation of SAHH prevents local silencing, it enhances the spread of systemic silencing. Our results also show that SAHH is important in the suppression of local RNA silencing not only by the crinivirus Tomato chlorosis virus CP but also by the multifunctional helper component-proteinase of the potyvirus Potato virus Y.

scholarly journals FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chenjiang You ◽  
Wenrong He ◽  
Runlai Hang ◽  
Cuiju Zhang ◽  
Xiaofeng Cao ◽  
...  
Keyword(s):  
Rna Degradation ◽  
Transcriptional Gene Silencing ◽  
Rrna Processing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Processing Defects ◽  
And Function ◽  
Mirna Abundance ◽  
Unknown Link

Abstract Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5′−3′ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5′−3′ exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.

scholarly journals RNA Silencing against Geminivirus: Complementary Action of Posttranscriptional Gene Silencing and Transcriptional Gene Silencing in Host Recovery

2008 ◽  
Vol 83 (3) ◽  
pp. 1332-1340 ◽  
Author(s):  
Edgar A. Rodríguez-Negrete ◽  
Jimena Carrillo-Tripp ◽  
Rafael F. Rivera-Bustamante
Keyword(s):  
Gene Silencing ◽  
Intergenic Region ◽  
Rna Silencing ◽  
Methylation Status ◽  
Inverse Correlation ◽  
Plant Viruses ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Coding Region ◽  
Natural Defense

ABSTRACT RNA silencing in plants is a natural defense system mechanism against invading nucleic acids such as viruses. Geminiviruses, a family of plant viruses characterized by a circular, single-stranded DNA genome, are thought to be both inducers and targets of RNA silencing. Some natural geminivirus-host interactions lead to symptom remission or host recovery, a process commonly associated with RNA silencing-mediated defense. Pepper golden mosaic virus (PepGMV)-infected pepper plants show a recovery phenotype, which has been associated with the presence of virus-derived small RNAs. The results presented here suggest that PepGMV is targeted by both posttranscriptional and transcriptional gene silencing mechanisms. Two types of virus-related small interfering RNAs (siRNAs) were detected: siRNAs of 21 to 22 nucleotides (nt) in size that are related to the coding regions (Rep, TrAP, REn, and movement protein genes) and a 24-nt population primarily associated to the intergenic regions. Methylation levels of the PepGMV A intergenic and coat protein (CP) coding region were measured by a bisulfite sequencing approach. An inverse correlation was observed between the methylation status of the intergenic region and the concentration of viral DNA and symptom severity. The intergenic region also showed a methylation profile conserved in all times analyzed. The CP region, on the other hand, did not show a defined profile, and its methylation density was significantly lower than the one found on the intergenic region. The participation of both PTGS and TGS mechanisms in host recovery is discussed.

Post-transcriptional gene silencing and virus resistance in Nicotiana benthamiana expressing a Grapevine virus A minireplicon

2008 ◽  
Vol 18 (3) ◽  
pp. 331-345 ◽  
Author(s):  
Marina Brumin ◽  
Svetlana Stukalov ◽  
Sabrina Haviv ◽  
Mookkan Muruganantham ◽  
Yoni Moskovitz ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Virus Resistance ◽  
Nicotiana Benthamiana ◽  
Transcriptional Gene Silencing ◽  
Grapevine Virus ◽  
Grapevine Virus A ◽  
Post Transcriptional Gene Silencing

scholarly journals Geminivirus AL2 and L2 Proteins Suppress Transcriptional Gene Silencing and Cause Genome-Wide Reductions in Cytosine Methylation

2009 ◽  
Vol 83 (10) ◽  
pp. 5005-5013 ◽  
Author(s):  
R. Cody Buchmann ◽  
Shaheen Asad ◽  
Jamie N. Wolf ◽  
Gireesha Mohannath ◽  
David M. Bisaro
Keyword(s):  
Gene Silencing ◽  
Host Defense ◽  
Transcriptional Activation ◽  
Cytosine Methylation ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Viral Proteins ◽  
Adenosine Kinase ◽  
Transcriptional Gene Silencing ◽  
Efficient Production

ABSTRACT Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS.

scholarly journals The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step

2003 ◽  
Vol 77 (1) ◽  
pp. 511-522 ◽  
Author(s):  
Feng Qu ◽  
Tao Ren ◽  
T. Jack Morris
Keyword(s):  
Coat Protein ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Rna Degradation ◽  
Plant Viruses ◽  
Early Initiation ◽  
Turnip Crinkle Virus ◽  
Small Interfering Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Initiation Step

ABSTRACT Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants.

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

2007 ◽  
Vol 81 (19) ◽  
pp. 10379-10388 ◽  
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.

scholarly journals Tomato ringspot virus Coat Protein Binds to ARGONAUTE 1 and Suppresses the Translation Repression of a Reporter Gene

2014 ◽  
Vol 27 (9) ◽  
pp. 933-943 ◽  
Author(s):  
Rajita A. Karran ◽  
Hélène Sanfaçon
Keyword(s):  
Coat Protein ◽  
Reporter Gene ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Ringspot Virus ◽  
Small Interfering Rnas ◽  
Gfp Expression ◽  
Translation Repression ◽  
Tomato Ringspot Virus

RNA silencing regulates plant gene expression and antiviral defenses and functions by cleaving target RNAs or repressing translation. As a counter defense, many plant viruses encode suppressor proteins that sequester small RNAs or inactivate Argonaute (AGO) proteins. All known plant virus silencing suppressor activities eventually inhibit the degradation of target mRNAs. Using a transiently expressed green fluorescent protein (GFP) reporter gene, we show that Tomato ringspot virus (ToRSV) coat protein (CP) is a suppressor of RNA silencing that enhances GFP expression but does not prevent the degradation of the GFP mRNA or the accumulation of GFP small interfering RNAs (siRNAs). Coexpression of the CP with GFP resulted in increased association of residual GFP mRNAs with polysome fractions and reduced association of GFP siRNAs with monosome fractions. AGO1 was co-immunoprecipitated with the CP and CP expression destabilized AGO1. A WG motif within the CP was critical for the enhanced GFP expression, AGO1 interaction, and AGO1 destabilization, suggesting that the ToRSV CP acts as an AGO-hook protein and competes for AGO binding with a plant cellular GW/WG protein involved in translation repression.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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