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.

scholarly journals California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jamilur Rahman ◽  
Ian T. Baldwin ◽  
Klaus Gase
Keyword(s):  
Gene Silencing ◽  
Host Plant ◽  
Target Genes ◽  
Elevated Temperatures ◽  
Tobacco Rattle Virus ◽  
Vector System ◽  
Nicotiana Attenuata ◽  
Cdna Clones ◽  
Virus Induced Gene Silencing ◽  
In Planta

Abstract Background Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant’s native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. Results Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. Conclusions The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

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.

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 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 Virus-induced gene silencing in the perennial woody Paeonia ostii

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7001
Author(s):  
Lihang Xie ◽  
Qingyu Zhang ◽  
Daoyang Sun ◽  
Weizong Yang ◽  
Jiayuan Hu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Functional Characterization ◽  
Tobacco Rattle Virus ◽  
Vacuum Infiltration ◽  
Genomic Research ◽  
Virus Induced Gene Silencing ◽  
Tree Peony ◽  
Medicinal Value

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii is presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized for Agrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more complete Agrobacterium penetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter gene PoPDS encoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenous PoPDS transcripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

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