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

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.

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 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.

scholarly journals Silencing of the Phytoene desaturase gene mitigates oxidative stress through the accumulation of free amino acids

2020 ◽  
Vol 49 (4) ◽  
pp. 927-935
Author(s):  
Md Sarafat Ali ◽  
Kwang-Hyun Baek
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Leaf Trait ◽  
High Concentrations ◽  
Rate Limiting ◽  
Pds Gene

Phytoene desaturase (PDS) is a rate-limiting enzyme involved in the biosynthesis of carotenoids, which converts phytoene to zeta-carotene in a two-step desaturation reaction. Transiently blocked carotenogenesis by silencing the PDS gene in Nicotiana benthamiana (NbPDS) using the virus-induced gene silencing (VIGS) technique was used. Silencing of NbPDS induced dwarfism and an albino-type leaf trait in N. benthamiana. The NbPDS-silenced leaves accumulated free amino acids in amounts 9.5-folds greater than those of the GFP-silenced control leaves, but contained only 59.6% of total soluble proteins. When treatment with 10 and 100 μM paraquat was carried out to induce oxidative stress, NbPDS-silenced N. benthamiana demonstrated more resistance at both concentrations compared to the control plants. These data strongly suggest that high concentrations of free amino acids occur because they are inadequately incorporated into proteins of the NbPDS-silenced plants, but reduce injury inflicted by oxidative stress even without the assistance of important antioxidants like carotenoids.

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.

scholarly journals Virus-induced Phytoene Desaturase (PDS) Gene Silencing Using Tobacco Rattle Virus in Lilium × formolongi

2019 ◽  
Vol 5 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Hua Xu ◽  
Leifeng Xu ◽  
Panpan Yang ◽  
Yuwei Cao ◽  
Yuchao Tang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Tobacco Rattle Virus ◽  
Phytoene Desaturase ◽  
Pds Gene

scholarly journals Knockdown of PHYTOENE DESATURASE in the field dodder, Cuscuta campestris, by Virus-Induced Gene Silencing

2021 ◽  
Author(s):  
Steven Dyer ◽  
Ryan Weir ◽  
Panagiotis Manesiotis ◽  
Johnathan J. Dalzell
Keyword(s):  
Gene Silencing ◽  
Reverse Genetics ◽  
Tobacco Rattle Virus ◽  
Beta Carotene ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Specific Sequence ◽  
Cuscuta Campestris ◽  
First Time ◽  
Globally Distributed

AbstractCuscuta campestris is a globally distributed obligate holoparasitic plant, and economically important crop pest. There is an urgent need for safe and effective new herbicides to control Cuscuta spp. PHYTOENE DESATURASE (PDS) is a biosynthetic enzyme within the carotenoid synthesis pathway, which is a target for several commercially available herbicides. The low transpiration rate of C. campestris results in sub-optimal translocation of PDS-targeting herbicides throughout the parasite, and resistance to these herbicides, and others, should be anticipated. Here we demonstrate that RNA interference (RNAi) can effectively reduce the expression of PDS in C. campestris. Virus Induced Gene Silencing (VIGS) is capable of inducing PDS knockdown in C. campestris, when Tobacco Rattle Virus (TRV) is used to deliver a PDS-specific sequence through the host plant Arabidopsis thaliana. This leads to a reduction in the accumulation of beta carotene, which is synthesised from phytoene, and significantly reduced growth of C. campestris. We hypothesise that secondary amplification and spread of PDS double-stranded RNA within C. campestris may circumvent the translocation limitations of other xylem and phloem-spread PDS-specific herbicides. These data demonstrate for the first time that VIGS can be used for reverse genetics interrogation of the C. campestris genome.

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