scholarly journals A new BSMV-based vector with modified β molecule allows simultaneous and stable silencing of two genes

2012 ◽  
Vol 17 (1) ◽  
Author(s):  
Adam Kawalek ◽  
Marta Dmochowska-Boguta ◽  
Anna Nadolska-Orczyk ◽  
Waclaw Orczyk
Keyword(s):  
Gene Silencing ◽  
Target Gene ◽  
Phytoene Desaturase ◽  
Gene Fragment ◽  
Virus Induced Gene Silencing ◽  
Reading Frame ◽  
Qrt Pcr ◽  
Bamhi Site ◽  
Functional Gene Analysis ◽  
The Stability

AbstractVirus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:β molecule was engineered with a unique BamHI site between the open reading frame of βc (ORF βc) and poly(A). The mixture of RNA particles α, βBamHI and γMCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to βBamHI and γMCS. Delivery of the target gene fragment in γMCS induced stronger silencing, while delivery in βBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of β enables simultaneous silencing of two genes. Quantifying the β and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than β, while the stability of the insert was higher in β compared with γ. The possible influence of the relative quantity of β and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.

Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis

2006 ◽  
Vol 33 (4) ◽  
pp. 347 ◽  
Author(s):  
Changchun Wang ◽  
Xinzhong Cai ◽  
Xuemin Wang ◽  
Zhong Zheng
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Function Analysis ◽  
Tobacco Rattle Virus ◽  
Phytoene Desaturase ◽  
Growth Stages ◽  
Virus Induced Gene Silencing ◽  
Gene Functions ◽  
Genes Encoding ◽  
Gene Function Analysis

Arabidopsis thaliana (L.) Heynh. is a model plant species in which to study plant gene functions. Recently developed virus-induced gene silencing (VIGS) offers a rapid and high-throughput technique platform for gene function analysis. In this paper we report optimisation of tobacco rattle virus (TRV)-induced gene silencing in Arabidopsis. The parameters potentially affecting the efficiency of VIGS in Arabidopsis were investigated. These included the concentration and pre-incubation of Agrobacterium inocula (agro-inocula), the concentration of acetosyringone included in agro-inocula, the Agrobacterium inoculation (agro-inoculation) method, the ecotypes and the growth stages of Arabidopsis plants for agro-inoculation, and the growth temperature of agro-inoculated plants. The optimised VIGS procedure involves preparing the agro-inocula with OD600 of 2.0, pre-incubating for 2 h in infiltration buffer containing 200 μm acetosyringone, agro-inoculating by vacuum infiltration, and growth of agro-inoculated plants at 22 −24°C. Following this procedure consistent and highly efficient VIGS was achieved for the genes encoding phytoene desaturase (PDS) and actin in Arabidopsis. The silencing phenotype lasts for at least 6 weeks, and is applicable in at least seven ecotypes, including Col-0, Cvi-0, Sd, Nd-1, Ws-0, Bay-0 and Ler. TRV-induced VIGS was expressed not only in leaves, but also in stems, inflorescences and siliques. However, VIGS was not transmissible through seed to the subsequent generation. The optimised procedure of the TRV-induced gene silencing should facilitate high-throughput functional analysis of genes in Arabidopsis.

Development of an efficient virus-induced gene silencing method in petunia using the pepper phytoene desaturase (PDS) gene

2019 ◽  
Vol 138 (3) ◽  
pp. 507-515 ◽  
Author(s):  
Aung Htay Naing ◽  
Hyun Young Song ◽  
Je Min Lee ◽  
Ki Byung Lim ◽  
Chang Kil Kim
Keyword(s):  
Gene Silencing ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Pds Gene

scholarly journals Stability of Barley stripe mosaic virus–Induced Gene Silencing in Barley

2007 ◽  
Vol 20 (11) ◽  
pp. 1323-1331 ◽  
Author(s):  
Marianne Bruun-Rasmussen ◽  
Christian Toft Madsen ◽  
Stine Jessing ◽  
Merete Albrechtsen
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Barley Stripe Mosaic Virus ◽  
Mrna Levels ◽  
Virus Induced Gene Silencing ◽  
Qrt Pcr ◽  
Transient Nature ◽  
Polymerase Chain ◽  
Insert Length ◽  
Pds Gene

Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting photobleaching in infected barley plants was used as a reporter for silencing. In addition, downregulation of PDS mRNA was measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Using fragments of PDS ranging from 128 to 584 nucleotides in BSMV, we observed that insert length influenced stability but not efficiency of VIGS. Silencing was transient in most cases; however, the decrease in PDS mRNA levels measured by qRT-PCR began earlier and lasted longer than the photobleaching. Occasionally, silencing persisted and could be transmitted through seed as well as via mechanical inoculation, although large parts of the insert had been lost from the virus vector. The instability of the insert, observed consistently throughout our experiments, offers an explanation for the transient nature of silencing when using BSMV as a VIGS vector.

scholarly journals Rose Phytoene Desaturase Gene Silencing by Apple Latent Spherical Virus Vectors

HortScience ◽  
2012 ◽  
Vol 47 (9) ◽  
pp. 1278-1282 ◽  
Author(s):  
Hiroaki Ito ◽  
Masaki Ochiai ◽  
Hiroaki Kato ◽  
Katsuhiro Shiratake ◽  
Daigo Takemoto ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Molecular Breeding ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Desaturase Gene ◽  
Apple Latent Spherical Virus ◽  
The Rose ◽  
Highly Uniform ◽  
Secondary Shoot

We have succeeded in establishing a virus-induced gene silencing (VIGS) of rose using Apple latent spherical virus (ALSV) vectors. An ALSV infection on rose did not cause any symptoms like those observed on other plant species and grew healthy. We have cloned and sequenced the phytoene desaturase (PDS) gene in wild rose, then used its fragment for silencing the rose internal PDS gene. The silencing phenotypes such as the highly uniform photo-bleached phenotype with PDS inhibitions were observed on the upper leaves of primary shoots and on a secondary shoot of R. rugosa for more than 5 months. ALSV vectors seemed useful for analyzing gene function and for the molecular breeding of rose.

scholarly journals A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

2019 ◽  
Author(s):  
Mei Liu ◽  
Zhiling Liang ◽  
Miguel A. Aranda ◽  
Ni Hong ◽  
Liming Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Nicotiana Benthamiana ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Economic Significance ◽  
Potential Alternative ◽  
Low Efficiency

AbstractCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiencies. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits. Here, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69-300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged. These results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function in cucurbits.One sentence summaryA CGMMV-based vector enables gene function studies in cucurbits, an extremely low efficiency species for genetic transformation.

scholarly journals Silencing of the phytoene desaturase (PDS) gene affects the expression of fruit-ripening genes in tomatoes

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Aung Htay Naing ◽  
Swum Yi Kyu ◽  
Phyo Phyo Win Pe ◽  
Kyeung Il Park ◽  
Je Min Lee ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Fruit Ripening ◽  
Tomato Fruit ◽  
Carotenoid Biosynthesis ◽  
Past Research ◽  
Ethylene Biosynthesis ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Pale Yellow ◽  
Ripening Genes

Abstract Background Past research has shown that virus-induced phytoene desaturase (PDS) gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a pale-yellow fruit phenotype. Although the PDS gene is often used as a marker for gene silencing in tomatoes, little is known about the role of PDS in fruit ripening. In this study, we investigated whether the pepper PDS gene silenced endogenous PDS genes in the fruit of two tomato cultivars, Dotaerang Plus and Legend Summer. Results We found that the pepper PDS gene successfully silenced endogenous PDS in tomato fruit at a silencing frequency of 100% for both cultivars. A pale-yellow silenced area was observed over virtually the entire surface of individual fruit due to the transcriptional reduction in phytoene desaturase (PDS), zeta-carotene (ZDS), prolycopene isomerase (CrtlSO), and beta-carotene hydroxylase (CrtR-b2), which are the carotenoid biosynthesis genes responsible for the red coloration in tomatoes. PDS silencing also affected the expression levels of the fruit-ripening genes Tomato AGAMOUS-LIKE1 (TAGL1), RIPENING INHIBITOR (RIN), pectin esterase gene (PE), lipoxygenase (LOX), FRUITFULL1/FRUITFUL2 (FUL1/FUL2), and the ethylene biosynthesis and response genes 1-aminocyclopropane-1-carboxylate oxidase 1 and 3 (ACO1 and ACO3) and ethylene-responsive genes (E4 and E8). Conclusion These results suggest that PDS is a positive regulator of ripening in tomato fruit, which must be considered when using it as a marker for virus-induced gene silencing (VIGS) experiments in order to avoid fruit-ripening side effects.

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.

Human Norovirus RNA Persists in Seawater under Simulated Winter Conditions but Does Not Bioaccumulate Efficiently in Pacific Oysters (Crassostrea gigas)

2010 ◽  
Vol 73 (11) ◽  
pp. 2123-2127 ◽  
Author(s):  
D. DANCER ◽  
R. E. RANGDALE ◽  
J. A. LOWTHER ◽  
D. N. LEES
Keyword(s):  
Crassostrea Gigas ◽  
Limit Of Detection ◽  
Reading Frame ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Pacific Oysters ◽  
The United Kingdom ◽  
Winter Conditions ◽  
Rna Fragments ◽  
The Stability

Norovirus (NoV) is the principal agent of bivalve molluscan shellfish–associated gastroenteric illness worldwide. Currently, noncultivable human NoVs can be detected in bivalve molluscan shellfish by using molecular methods such as real-time reverse transcription PCR assays (qRT-PCR). In addition to infectious viruses, this methodology may also detect noninfectious NoV, including fragments of the NoV genome. This study addresses, in part, the implications of qRT-PCR results for the detection of NoV in shellfish in the absence of an infectivity assay. To evaluate environmental persistence, the stability of a short fragment of the NoV genome, spanning the qRT-PCR target in the open reading frame 1/2 junction, was assessed in seawater under artificial environmental conditions simulating winter in the United Kingdom (1 mW/cm2 UV irradiation, 8°C) during a 4-week period. Detectable RNA levels decreased exponentially (T90 of approximately 141 h); however, sequences were still detectable for up to 2 weeks. The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate NoV particles (from human feces) and RNA fragments was also compared using qRT-PCR. Oysters exposed to NoV particles subsequently were positive for NoV by qRTPCR at levels several orders of magnitude in excess of the theoretical limit of detection, whereas oysters exposed to similar quantities of NoV RNA were either negative or positive at significantly lower levels. Therefore, although noninfectious fragments of NoV RNA may persist in the environment under winter conditions, this type of material will not be efficiently bioaccumulated by Pacific oysters and should not significantly contribute to positive qRT-PCR results.

scholarly journals A Methodological Advance of Tobacco Rattle Virus-Induced Gene Silencing for Functional Genomics in Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Gongyao Shi ◽  
Mengyuan Hao ◽  
Baoming Tian ◽  
Gangqiang Cao ◽  
Fang Wei ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Viral Vector ◽  
Tobacco Rattle Virus ◽  
Influential Factors ◽  
Virus Induced Gene Silencing ◽  
Vector Construction ◽  
Functional Gene Analysis ◽  
Methodological Advance ◽  
Diverse Plant Species ◽  
Diverse Plant

As a promising high-throughput reverse genetic tool in plants, virus-induced gene silencing (VIGS) has already begun to fulfill some of this promise in diverse aspects. However, review of the technological advancements about widely used VIGS system, tobacco rattle virus (TRV)-mediated gene silencing, needs timely updates. Hence, this article mainly reviews viral vector construction, inoculation method advances, important influential factors, and summarizes the recent applications in diverse plant species, thus providing a better understanding and advice for functional gene analysis related to crop improvements.

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