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.

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 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.

Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana

2019 ◽  
Vol 74 (5-6) ◽  
pp. 151-159
Author(s):  
Feng Zhu ◽  
Yanping Che ◽  
Fei Xu ◽  
Yangkai Zhou ◽  
Kun Qian ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Target Genes ◽  
Function Analysis ◽  
Tobacco Rattle Virus ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Biotic Stresses ◽  
Overlap Extension

Abstract Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 (NbSABP2) and NbNAC1 in Nicotiana benthamiana to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length NbNAC1 gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of NbSABP2 and NbNAC1 was successfully amplified. Bacteria liquid PCR confirmed that the combination of NbSABP2 and NbNAC1 was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the NbSABP2 and NbNAC1 gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of NbSABP2 and NbNAC1 were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

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.

scholarly journals Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Meng Wang ◽  
Tingting Ren ◽  
Prince Marowa ◽  
Haina Du ◽  
Zongchang Xu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Normalize Gene Expression ◽  
Suaeda Glauca ◽  
Suitable Reference ◽  
Germinating Seeds ◽  
Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

scholarly journals Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madhab Kumar Sen ◽  
Kateřina Hamouzová ◽  
Pavlina Košnarová ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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