scholarly journals Virus-Induced Gene Silencing, a Post Transcriptional Gene Silencing Method

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Turgay Unver ◽  
Hikmet Budak
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Viral Vectors ◽  
Large Scale ◽  
Reverse Genetics ◽  
Transcriptional Gene Silencing ◽  
Virus Induced Gene Silencing ◽  
Post Transcriptional Gene Silencing ◽  
Dicotyledonous Plants

Virus-induced gene silencing (VIGS) is one of the reverse genetics tools for analysis of gene function that uses viral vectors carrying a target gene fragment to produce dsRNA which trigger RNA-mediated gene silencing. There are a number of viruses which have been modified to silence the gene of interest effectively with a sequence-specific manner. Therefore, different types of methodologies have been advanced and modified for VIGS approach. Virus-derived inoculations are performed on host plants using different methods such as agro-infiltration and in vitro transcriptions. VIGS has many advantages compared to other loss-of-gene function approaches. The approach provides the generation of rapid phenotype and no need for plant transformation. The cost of VIGS experiment is relatively low, and large-scale analysis of screening studies can be achieved by the VIGS. However, there are still limitations of VIGS to be overcome. Nowadays, many virus-derived vectors are optimized to silence more than one host plant such as TRV-derived viral vectors which are used for Arabidopsis and Nicothiana benthamiana. By development of viral silencing systems monocot plants can also be targeted as silencing host in addition to dicotyledonous plants. For instance, Barley stripe mosaic virus (BSMV)-mediated VIGS allows silencing of barley and wheat genes. Here we summarize current protocols and recent modified viral systems to lead silencing of genes in different host species.

scholarly journals Characterization of a Brome mosaic virus Strain and Its Use as a Vector for Gene Silencing in Monocotyledonous Hosts

2006 ◽  
Vol 19 (11) ◽  
pp. 1229-1239 ◽  
Author(s):  
Xin Shun Ding ◽  
William L. Schneider ◽  
Srinivasa Rao Chaluvadi ◽  
M. A. Rouf Mian ◽  
Richard S. Nelson
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Festuca Arundinacea ◽  
Systemic Infection ◽  
Brome Mosaic Virus ◽  
Expression Vectors ◽  
Virus Induced Gene Silencing ◽  
Dicotyledonous Plants ◽  
Virus Expression

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMVA/G (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

scholarly journals Virus-Induced Gene Silencing in Transgenic Plants Expressing the Minor Capsid Protein of Beet western yellows virus

2002 ◽  
Vol 15 (8) ◽  
pp. 799-807 ◽  
Author(s):  
V. Brault ◽  
S. Pfeffer ◽  
M. Erdinger ◽  
J. Mutterer ◽  
V. Ziegler-Graff
Keyword(s):  
Coat Protein ◽  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Transcriptional Gene Silencing ◽  
Virus Induced Gene Silencing ◽  
Minor Capsid Protein ◽  
Beet Western Yellows Virus ◽  
Post Transcriptional Gene Silencing ◽  
Low Levels ◽  
Minor Coat Protein

Transgenic Nicotiana benthamiana expressing the minor coat protein P74 of the phloem-limited Beet western yellows virus (BWYV) exhibited an unusual spatial pattern of post-transcriptional gene silencing (PTGS) when infected with BWYV or related viruses. Following infection, transgenic P74 and its mRNA accumulated to only low levels, 21 to 23 nucleotide RNAs homologous to the transgene appeared, and the transgene DNA underwent methylation. The infecting viral RNA, however, was not subject to significant silencing but multiplied readily and produced P74 in the phloem tissues, although the P74 encoded by the transgene disappeared from the phloem as well as the nonvascular tissues.

scholarly journals Sequence-, Tissue-, and Delivery-Specific Targeting of RNA During Post-Transcriptional Gene Silencing

2002 ◽  
Vol 15 (8) ◽  
pp. 753-763 ◽  
Author(s):  
Ezequiel Balmori-Melian ◽  
Robin M. MacDiarmid ◽  
David L. Beck ◽  
Richard C. Gardner ◽  
Richard L. S. Forster
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Viral Vectors ◽  
Transcriptional Gene Silencing ◽  
Wild Type ◽  
Long Distance ◽  
Post Transcriptional Gene Silencing ◽  
In The Wild ◽  
Long Distance Movement ◽  
White Clover Mosaic Virus

Transgenic Nicotiana benthamiana plants expressing an untranslatable version of the coat protein (CP) gene from the Tamarillo mosaic virus (TaMV) were either resistant to TaMV infection or recovered from infection. These phenotypes were the result of a post-transcriptional gene silencing (PTGS) mechanism that targeted TaMV-CP sequences for degradation. The TaMV-CP sequences were degraded when present in the wild-type TaMV potyvirus, in transgene mRNA, or in chimeric viral vectors based on White clover mosaic virus. The more efficiently targeted region was mapped to a 134-nt segment. Differences were observed in the efficiency of targeting during cell-to-cell and long-distance movement of the chimeric viruses. However, the TaMV-CP sequences do not appear to be targeted for degradation when delivered by biolistics.

Knockdown of VvMYBA1 via virus-induced gene silencing decreases anthocyanin biosynthesis in grape berries

2020 ◽  
Vol 100 (2) ◽  
pp. 175-184
Author(s):  
Peng Fei Zhang ◽  
Yan Mei Dong ◽  
Hao Yu Wen ◽  
Chang Mei Liang ◽  
Tie Quan Niu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Viral Vectors ◽  
Anthocyanin Biosynthesis ◽  
Tobacco Rattle Virus ◽  
Fruit Trees ◽  
Virus Induced Gene Silencing ◽  
Wine Quality ◽  
Grape Berries ◽  
Total Anthocyanins

Anthocyanin in grapevines, regulated by structural and regulatory genes, determines the colour of grape berries and is a key factor in wine quality. The transgenic approach is a useful strategy for elucidating gene function. However, it is difficult to obtain transgenic fruit trees, including grapevines. Viral vectors offer an effective strategy for overcoming this challenge. In this study, we successfully knocked down the VvMYBA1 gene in ‘Red Globe’ and ‘JiZaoMi’ grape berries via virus-induced gene silencing (VIGS) by vacuum infiltration and silencing efficiencies of 73% and 93%, respectively, were obtained relative to the control. Total anthocyanins content was reduced 1.7- and 2.7-times relative to that in the control in ‘Red Globe’ and ‘JiZaoMi’ grape berries, respectively. The expression levels of VvUFGT and VvDFR were reduced by 80% and 8% and by 72% and 42% relative to the control in ‘Red Globe’ and ‘JiZaoMi’ grape berries, respectively, in the anthocyanin metabolic pathways. Tobacco rattle virus-mediated VIGS was, therefore, successfully established in grape berries. These findings provide direct evidence that VvMYBA1 encodes skin colour in red grape berries and suggest a possible application of VIGS for gene function studies in other fruits.

VIGS technology: an attractive tool for functional genomics studies in legumes

2013 ◽  
Vol 40 (12) ◽  
pp. 1234 ◽  
Author(s):  
Stéphanie Pflieger ◽  
Manon M. S. Richard ◽  
Sophie Blanchet ◽  
Chouaib Meziadi ◽  
Valérie Geffroy
Keyword(s):  
Gene Silencing ◽  
Functional Genomics ◽  
Target Gene ◽  
Reverse Genetics ◽  
Plant Viruses ◽  
Transcriptional Gene Silencing ◽  
Full Potential ◽  
Legume Species ◽  
Virus Induced Gene Silencing ◽  
Apple Latent Spherical Virus

Legume species are among the most important crops worldwide. In recent years, six legume genomes have been completely sequenced, and there is now an urgent need for reverse-genetics tools to validate genes affecting yield and product quality. As most legumes are recalcitrant to stable genetic transformation, virus-induced gene silencing (VIGS) appears to be a powerful alternative technology for determining the function of unknown genes. VIGS technology is based on the property of plant viruses to trigger a defence mechanism related to post-transcriptional gene silencing (PTGS). Infection by a recombinant virus carrying a fragment of a plant target gene will induce homology-dependent silencing of the endogenous target gene. Several VIGS systems have been developed for legume species since 2004, including those based on Bean pod mottle virus, Pea early browning virus, and Apple latent spherical virus, and used in reverse-genetics studies of a wide variety of plant biological processes. In this work, we give an overview of the VIGS systems available for legumes, and present their successful applications in functional genomics studies. We also discuss the limitations of these VIGS systems and the future challenges to be faced in order to use VIGS to its full potential in legume species.

scholarly journals RNA interference: advances and questions

2002 ◽  
Vol 357 (1417) ◽  
pp. 65-70 ◽  
Author(s):  
Elisabetta Ullu ◽  
Appolinaire Djikeng ◽  
Huafang Shi ◽  
Christian Tschudi
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Trypanosoma Brucei ◽  
Gene Function ◽  
Protozoan Parasite ◽  
Transcriptional Gene Silencing ◽  
Eukaryotic Lineage ◽  
Powerful Method ◽  
Double Stranded Rna ◽  
Post Transcriptional Gene Silencing

In animals and protozoa gene–specific double–stranded RNA triggers the degradation of homologous cellular RNAs, the phenomenon of RNA interference (RNAi). RNAi has been shown to represent a novel paradigm in eukaryotic biology and a powerful method for studying gene function. Here we discuss RNAi in terms of its mechanism, its relationship to other post–transcriptional gene silencing phenomena in plants and fungi, its connection to retroposon silencing and possibly to translation, and its biological role. Among the organisms where RNAi has been demonstrated the protozoan parasite Trypanosoma brucei represents the most ancient branch of the eukaryotic lineage. We provide a synopsis of what is currently known about RNAi in T. brucei and outline the recent advances that make RNAi the method of choice to disrupt gene function in these organisms.

scholarly journals Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing

2021 ◽  
Vol 49 (18) ◽  
pp. 10250-10264
Author(s):  
Hartmut Jahns ◽  
Rohan Degaonkar ◽  
Peter Podbevsek ◽  
Swati Gupta ◽  
Anna Bisbe ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Circular Rna ◽  
Antisense Strand ◽  
Rnai Pathway ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Sense Strand

Abstract In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

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