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.

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 Tobacco mosaic virus 126-kDa Protein Increases the Susceptibility of Nicotiana tabacum to Other Viruses and Its Dosage Affects Virus-Induced Gene Silencing

2008 ◽  
Vol 21 (12) ◽  
pp. 1539-1548 ◽  
Author(s):  
Phillip A. Harries ◽  
Karuppaiah Palanichelvam ◽  
Sumana Bhat ◽  
Richard S. Nelson
Keyword(s):  
Nicotiana Tabacum ◽  
Gene Silencing ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Brome Mosaic Virus ◽  
Virus Induced Gene Silencing ◽  
Suppressor Activity

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein–green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

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.

Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek

2019 ◽  
Vol 99 (6) ◽  
pp. 917-926 ◽  
Author(s):  
Champa Wijekoon ◽  
Stacy D. Singer ◽  
Randall J. Weselake ◽  
Udaya Subedi ◽  
Surya N. Acharya
Keyword(s):  
Gene Silencing ◽  
Plant Species ◽  
Reverse Genetics ◽  
Functional Characterization ◽  
Homologous Gene ◽  
Virus Induced Gene Silencing ◽  
Forage Legumes ◽  
Crop Species ◽  
Barrel Medic ◽  
Trigonella Foenum Graecum L

Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene functions. However, while VIGS has been utilized successfully in the diploid model leguminous species Medicago truncatula (Gaertn.) (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10–15 d through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek. The introduction of a highly homologous gene fragment from a heterologous plant species to target endogenous genes for transient VIGS-based silencing in a range of species of interest represents a potentially useful strategy for the rapid functional characterization of candidate genes in forages.

scholarly journals A Ligation-Independent Cloning Tobacco Rattle Virus Vector for High-Throughput Virus-Induced Gene Silencing Identifies Roles for NbMADS4-1 and -2 in Floral Development

2007 ◽  
Vol 145 (4) ◽  
pp. 1161-1170 ◽  
Author(s):  
Yiyu Dong ◽  
Tessa M. Burch-Smith ◽  
Yule Liu ◽  
Padmavathi Mamillapalli ◽  
Savithramma P. Dinesh-Kumar
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Floral Development ◽  
Tobacco Rattle Virus ◽  
Virus Vector ◽  
Virus Induced Gene Silencing ◽  
Ligation Independent Cloning

scholarly journals An E3 ubiquitin ligase fromNicotiana benthamianatargets the replicase ofBamboo mosaic virusand restricts its replication

2018 ◽  
Author(s):  
I-Hsuan Chen ◽  
Jui-En Chang ◽  
Chen-Yu Wu ◽  
Ying-Ping Huang ◽  
Yau-Huei Hsu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Tobacco Rattle Virus ◽  
E3 Ubiquitin Ligase ◽  
Infection Process ◽  
Ring Domain ◽  
Virus Induced Gene Silencing ◽  
Ubiquitin E3 Ligase ◽  
Rapid Amplification

AbstractOne upregulated host gene identified previously was found involved in the infection process ofBamboo mosaic virus(BaMV). The full-length cDNA of this gene was cloned by 5′- and 3′-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved RING-domain and a transmembrane domain. The gene might function as an E3 ubiquitin ligase. We designated this protein inNicotiana benthamianaas ubiquitin E3 ligase containing RING-domain 1 (NbUbE3R1). Further characterization by usingTobacco rattle virus-based virus-induced gene silencing revealed an increased BaMV accumulation in both knockdown plants and protoplasts. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed inN. benthamianaplants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting BaMV accumulation. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an upregulated gene, NbUbE3R1, that plays a role in BaMV replication.

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 Tobacco rattle virus-induced PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH) gene silencing in Solanum pseudocapsicum L.

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4424 ◽  
Author(s):  
Hua Xu ◽  
Leifeng Xu ◽  
Panpan Yang ◽  
Yuwei Cao ◽  
Yuchao Tang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Tobacco Rattle Virus ◽  
Phytoene Desaturase ◽  
Vacuum Infiltration ◽  
Suitable Reference Gene ◽  
Marker Genes ◽  
Agrobacterium Strain ◽  
Suitable Reference ◽  
Mg Chelatase

Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. The present study constitutes the first application of VIGS in S. pseudocapsicum, which has great ornamental and pharmaceutical value, using tobacco rattle virus (TRV) vectors. Two marker genes, PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH), were used to test the VIGS system in S. pseudocapsicum. The photobleaching and yellow-leaf phenotypes of the silenced plants were shown to significantly correlate with the down-regulation of endogenous SpPDS and SpChlH, respectively (P ≤ 0.05). Moreover, the parameters potentially affecting the efficiency of VIGS in S. pseudocapsicum, including the Agrobacterium strain and the inoculation method (leaf syringe-infiltration, sprout vacuum-infiltration and seed vacuum-infiltration), were compared. The optimized VIGS parameters were the leaf syringe-infiltration method, the Agrobacterium strain GV3101 and the growth of agro-inoculated plants at 25°. With these parameters, the silencing efficiency of SpPDS and SpChlH could reach approximately 50% in S. pseudocapsicum. Additionally, the suitability of various reference genes was screened by RT-qPCR using three candidate genes, and the results demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) can serve as a suitable reference for assessing the gene expression levels of VIGS systems in S. pseudocapsicum. The proven application of VIGS in S. pseudocapsicum and the characterization of a suitable reference gene in the present work will expedite the functional characterization of novel genes in S. pseudocapsicum.

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