Virus-Induced Gene Silencing: Methods and Protocols
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But for the key genes controlling flower color, flower fragrance, and other ornamental characters in the plants that were the subject of the study, even if the change of characters is observed in transgenic model plants, it cannot be equated with the characters of the plants themselves as the object of study. This is also a major restriction on the use of transgenic technology to verify gene function.
In addition to using transgenic technology to study the function of genes, virus-induced gene silencing VIGS is also an efficient genetic tool for functional genomics in plants. Usually, VIGS needs to construct a recombinant virus carrying a fragment of the target gene to silence a specific endogenous gene [ 4 ].
The host defense machinery, such as post-transcriptional gene silencing PTGS , will be activated when the virus invades the host plant. In general, VIGS has become a simple and fast method to study gene function because of its simplicity and efficiency. However, VIGS technology also has its limitations. Due to the limitation of host range, different host plants may need to select corresponding virus vectors, and different virus vectors may have different requirements for the length of inserted exogenous gene fragments [ 8 ].
In addition, the sensitivity of plant materials to virus vectors also determines the method of introducing virus vectors suitable for use in plant materials. And the optimal plant culture temperature, the circadian growth rhythm and the optimum propagation temperature of the virus carrier determine the light and temperature conditions of the whole experimental system [ 9 ]. These uncertainties directly lead to the possibility that different genera and even different species of the same genus may need an independent VIGS operating system, but cannot use the same set of universal VIGS system simply to achieve the purpose of efficient silencing function gene.
For the selection of viral vectors, the most widely used vector is TRV virus. It is applicable for many plants because the virus has a broad host range [ 10 ]. The modified TRV-GFP vectors are also widely used because of its small molecular weight, wide host range, high infection rate, low pathogenicity and green fluorescence visualization after infection.
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At present, there are many inoculation methods for VIGS, and the following are commonly used: vacuum infiltration method, high pressure spray gun method leaf surface direct spraying method , root absorption method root irrigation method , back leaf injection method, etc. Generally speaking, the vacuum infiltration method has the highest silencing efficiency, but it has a high demand for utensils and cannot be used under the condition of the field. Although the high pressure spray gun method can be operated in a large area, because the leaves of Rosa are smaller and fluffier in the inoculation period, the contact area between the liquid and the blade is small and cannot penetrate the leaf surface.
The root absorption method is simple, but the species limitation is higher, so it is not suitable for Rosa.
Virus-induced gene silencing in plant roots.
In addition, high pressure spray gun method and root absorption method need to consume a large amount of infection liquid, which is also not suitable for operation under field conditions. Although the operation of leaf back injection is relatively simple, it cannot be injected with syringe because of the shallow leaves of Rosa. In order to solve the technical problems mentioned above, in this study, we provided a method for establishing VIGS system based on perennial Rosa plants as experimental objects under field conditions. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems were solved one by one.
And the successful establishment of VIGS system was confirmed by determining some biochemical indexes. For Rosa, R. We collected the samples leaves and petals in the forenoon on sunny days from 20 April to 10 May The integrity of RNA was detected by gel electrophoresis with 1. In order to specifically silence the RrGT1 gene in R. Two restriction endonuclease sites XbaI and XhoI were designed at the two ends of the fragment.
After double enzyme digestion, the digestion products were ligated with DNA ligase and transformed into Agrobacterium tumefaciens. The recombinant viral vector was confirmed by PCR and sequencing. Before the infection, the A. Then the mixed bacteria solution was kept at room temperature for 4 h with darkness. The experimental materials for gene silencing induced by virus were the perennial Rosa plants grown naturally in the field, and the experimental treatment time was about one month before Rosa flowering.
Because the leaves and twigs are difficult to be injected with syringes and vacuum infiltration cannot be used in the field, we have adopted the method of infecting leaves and twigs with A. The specific operation was to completely immerse the scratched leaves and branchlets in the 5 ml centrifuge tube containing the infective fluid for infection.
In order to improve the infection efficiency of A. Each gene was assessed with three biological replications. All samples 0. After centrifugation, each extract was passed through a membrane filter 0. The aqueous phase was used to determine the absorbance at nm and nm. Qualitative and quantitative analyses of anthocyanins were performed via HPLC. Three independent biological replicates were measured for each sample.
The specific conditions of the anthocyanin analysis were the same as those in the report of Yang et al. The eluent was aqueous solution A 0. Then the PCR Figure 1 b was verified by specific primers Table 1 , and the base mutation and deletion were verified by double enzyme digestion Figure 1 c and sequencing. Figure 1. M: Marker; V: The plasmid of the empty virus vector was used as the control; V1: The empty virus vector was digested by double enzymes; R: The plasmid carrying the target gene fragment was digested by two enzymes. The red box indicates the portion that needs to be retained for subsequent operations.
Three repeated PCR assays were performed on the recombinant virus vector and the results showed positive. M: Marker; V: The empty virus vector was used as the control; A1, A2, A3 was the three repetitions which were larger than the empty virus vector due to the inserted RrGT1 fragment. M: Marker; V: The plasmid of the empty virus vector was used as the control; TR: The plasmid of the recombinant vector was digested into two correct parts. Table 1. Primers used in the present study.
A para-genetic approach, called virus-induced gene silencing VIGS , offers a rapid means of gaining insight into gene function in plants. VIGS system could be used to suppress endogenous gene expression by infecting plants with a recombinant virus vector VIGS vector carrying host-derived sequence. Nicotiana benthamiana, Nicotiana. Antibody Data Search Beta. Authors: S.
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Dinesh-Kumar 1 ,. Radhamani Anandalakshmi 1 ,. Rajendra Marathe 1 ,. Michael Schiff 1 ,. Yule Liu 1. Full text PDF Related articles. The regions corresponding to gene fragments inserted into the VIGS vector are highlighted in orange. The developed CMV 20 vector allows silencing of targeted genes of interest with high infection rate and mild disease symptoms.
In our study, we tested a number of methods to inoculate CMV 20 empty vector or CMV 20 vector with a targeted silence gene insert. While agro-inoculation systems have been widely adopted for virus delivery into plants 65 , 66 , 67 , inoculation of plant viruses with an agrobacterium-assisted system on Musa plants has not been successfully established prior to this study. Compared to inoculating CMV 20 on N.
Such an approach may allow minimal wounding on banana, and injecting an Agrobacterium mixture carrying CMV 20 to the center of banana pseudostem-rhizome juncture, likely allowed virus infection to take place on the meristem or yet-to-emerge leaves before the true banana leaf is developed. Interestingly, it has been previously reported that 2b protein-defective mutant is unable to invade the shoot apical meristem We speculate that the meristem tissue is vulnerable to transformation of agrobacterium, and serves as the initial infection site for CMV.
As no vascular system in the meristem is available for virus movement, virus infection is confined to the meristem, and VIGS phenotype is only visualized with the development of the leaves. It has been reported that lower temperature during inoculation may increase the transformation efficiency of Agrobacterium The temperature control allowed us to achieve a balance for plant growth and VIGS efficacy.
We demonstrated that the combination of the developed CMV 20 vector and inoculation strategy enabled silencing of the targeted gene of interest in banana with high infection rate and silencing efficacy. Although VIGS may be limited by the duration of silencing or inherently having to observe plant phenotype under virus infection, the availability of the system developed here may enable previously unavailable rapid functional analysis of genes in banana and expedite a more complete understanding of different biological questions in this important staple and cash crop.
The 4. An AfeI restriction enzyme site was added on the 2b ORF to generate a gene insertion site similar to the method previously described Total RNA extracted from N. For N. Mock plants were infiltrated with infiltration solution containing A. Systemic leaves were collected at 8 days post-inoculation dpi for further analysis. For bananas, preparation of the inoculum was similar to that previously described.
For detection and quantification of CMV20 in both N. Actin of N. For qRT-PCR assay of silencing experiments, gene-specific primers were designed to prime outside the region targeted for silencing. RNA derived from mock- or 35S control vector-inoculated plants, as specified in the figures, was set as 1 for relative quantitative analysis. For raw read processing, the FastX-Toolkit v.
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Next, the trimmed reads were mapped to the banana genome and cDNA sequences retrieved from the banana genome hub Musa acuminata DH-Pahang version 2; banana-genome-hub. De Langhe, C. Why bananas matter: an introduction to the history of banana domestication. Ethnobotany Research and Applications 7 , — Heslop-Harrison, J.
Domestication, genomics and the future for banana. Arias, P. The World Banana Economy, — The banana Musa acuminata genome and the evolution of monocotyledonous plants. Davey, M. A draft Musa balbisiana genome sequence for molecular genetics in polyploid, inter- and intra-specific Musa hybrids.