scholarly journals Tobacco rattle virus -induced gene silencing in Hevea brasiliensis

2020 ◽  
Author(s):  
Hui-Liang Li ◽  
Dong Guo ◽  
Ying Wang ◽  
Jia-Hong Zhu ◽  
Long Qu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Hevea Brasiliensis ◽  
Rubber Tree ◽  
Tobacco Rattle Virus ◽  
Functional Gene ◽  
Small Interfering Rnas ◽  
Virus Induced Gene Silencing ◽  
Rubber Biosynthesis ◽  
Natural Rubber Biosynthesis ◽  
First Time

Abstract BackgroundSince it is very difficult to obtain gene knockouts in rubber tree (Hevea Brasiliensis) due to low genetic transformation efficiency. Virus-induced gene silencing (VIGS) is a powerful gene silencing tool that has been intensively applied in plant. Up to now, the application of VIGS in rubber tree has not yet been reported.ResultsHevea brasiliensis phytoene desaturase (HbPDS) was identified in H. brasiliensis genome. The prediction of small interfering RNAs (siRNAs) from HbPDS and the silencing gene fragment (SGF) were predicted and a length of 409 bp SGF was chosen to be tested. We show that the tobacco rattle virus (TRV) -VIGS is able to induce effective HbPDS silencing in rubber tree. The TRV-VIGS system has the potential for functional gene studies in rubber tree.ConclusionsThis is the first time to report VIGS in rubber tree. The present TRV-VIGS method could be further applied to produce gene silenced rubber tree plants, to advance functional gene of rubber tree. The applied TRV-VIGS method will achieve deeper underground into the natural rubber biosynthesis and regulation in this important rubber-producing plant.

scholarly journals Tobacco rattle virus–induced gene silencing in Hevea brasiliensis

2021 ◽  
Vol 85 (3) ◽  
pp. 562-567
Author(s):  
Hui-Liang Li ◽  
Dong Guo ◽  
Ying Wang ◽  
Jia-Hong Zhu ◽  
Long Qu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Hevea Brasiliensis ◽  
Rubber Tree ◽  
Tobacco Rattle Virus ◽  
Reverse Genetic ◽  
Small Interfering Rnas ◽  
Virus Induced Gene Silencing ◽  
Gene Encoding ◽  
Efficient Gene ◽  
Unknown Gene

ABSTRACT Virus-induced gene silencing (VIGS) is a powerful gene-silencing tool that has been intensively applied in plants. To data, the application of VIGS in rubber tree has not yet been reported. In this study, we described the efficient gene silencing in rubber tree by VIGS. The gene encoding Hevea brasiliensis phytoene desaturase (HbPDS) was identified in rubber tree genome. Small interfering RNAs from HbPDS and the silencing gene fragment were predicted and a length of 399 bp was selected to be tested. We showed that the tobacco rattle virus (TRV)-VIGS could induce effective HbPDS silencing in rubber tree. This study was the first to report VIGS in rubber tree. The present TRV-VIGS method could be used to perform reverse genetic approaches to identify unknown gene functions and might be further applied to produce gene silenced rubber tree plants, to advance functional gene of rubber tree.

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.

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 Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava

2019 ◽  
Vol 20 (16) ◽  
pp. 3976 ◽  
Author(s):  
Hongqiu Zeng ◽  
Yanwei Xie ◽  
Guoyin Liu ◽  
Yunxie Wei ◽  
Wei Hu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Functional Genomics ◽  
Transient Expression ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Mosaic Disease ◽  
African Cassava Mosaic Virus ◽  
Cassava Mosaic Disease ◽  
Virus Induced Gene Silencing ◽  
Transient Expression Assay

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

scholarly journals An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits

2021 ◽  
Vol 12 ◽  
Author(s):  
Yifan Wang ◽  
Ning Huang ◽  
Niu Ye ◽  
Lingyu Qiu ◽  
Yadong Li ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Function Analysis ◽  
Juglans Regia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tobacco Rattle Virus ◽  
Post Inoculation ◽  
Virus Induced Gene Silencing ◽  
Gene Function Analysis

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

scholarly journals Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors

2021 ◽  
Author(s):  
Verónica Aragonés ◽  
Flavio Aliaga ◽  
Fabio Pasin ◽  
José-Antonio Daròs
Keyword(s):  
Gene Silencing ◽  
Genome Editing ◽  
Recombinant Proteins ◽  
Viral Vectors ◽  
Tobacco Rattle Virus ◽  
Vector System ◽  
Efficient Production ◽  
Virus Induced Gene Silencing ◽  
Guide Rnas ◽  
One Step

Genome editing and gene expression engineering using CRISPR-Cas systems in plants usually rely on labor-intensive tissue culture approaches to generate stably transformed plants that express the components of the reaction. Viral vectors have demonstrated to be a quick and effective alternative to express multiple guide RNAs, DNA templates for homologous recombination, and even Cas nucleases. Here we have developed an improved vector system based on tobacco rattle virus (TRV) to simplify logistics in genome editing and gene silencing approaches. The new system consists in a single Agrobacterium tumefaciens clone co-transformed with two compatible mini binary vectors from which TRV RNA1 and an engineered version of TRV RNA2 are expressed. Sequences of recombinant proteins, gene fragments for virus-induced gene silencing (VIGS) or guide RNAs can be easily inserted by one-step digestion-ligation and homology-based cloning methods in the RNA2 plasmid to produce vectors with a size substantially smaller than usual. Using this new one-Agrobacterium TRV mini vector system, we show robust VIGS of an endogenous host gene after infiltration of bacterial suspensions at low optical densities, and efficient production of recombinant proteins in Nicotiana benthamiana. Most importantly, we also show highly efficient heritable genome editing in more than half of the seedling originating from inoculated N. benthamiana plants that express Cas9.

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