Tobacco plant virus

A study demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in the perennial tree peony Xie et al.

Nishii et al. Moreover, several non-model plants, such as important commercial and medicinal plants, including cassava, flax, Papaver, Antirrhinum , mint, sweet basil, Ashwagandha, and olive tree, were also successfully explored with the TRV-VIGS system.

Two detailed Agrobacterium -mediated infection protocols have also been described in flax L. The systemic impact on the gene transcript levels in the stem demonstrated that the VIGS system can be applied for the functional study of cell wall genes in flax Chantreau and Neutelings, Chen et al.

Cruz et al. Tan et al. As a highly recalcitrant plant for genetic transformation, Ashwagandha Withania somnifera , an important Indian medicinal plant, has an established procedure to carry out VIGS for gene function studies Bomzan et al.

TRV-mediated VIGS through agroinoculation of olive plantlets has been successfully performed for functional genomic analyses in the olive tree Oleaceae Koudounas et al.

Not only can the TRV-VIGS system be used in most dicotyledonous species, it has also been adopted for gene-functional analysis of some monocotyledonous plants that are susceptible to TRV, such as wheat, corn, barley, rice, and Brachypodium sylvaticum in Gramineae and ornamental plants, such as orchids, a flowering plant of Orchidaceae. Virus-induced gene silencing has been successfully applied for functional characterization of genes involved in leaf rust resistance of wheat Scofield et al.

These results proved that this system is suitable for functional analysis of genes involved in seed germination and early plant development stages Zhang et al. Virus-induced gene silencing has also been adapted for functional validation of genes involved in floral growth and development of orchids Hsieh et al. Virus-induced gene silencing is a promising genetic tool for functional genomic studies in plants. First, the timing of VIGS appearance, as well as gene silencing duration, is usually species-specific.

So researchers should continue to prolong the gene silencing period of VIGS in the future. Another challenge of this technique is its varying penetrance of the phenotype in vegetative and reproductive tissue that requires a larger number of plants to be screened for phenotypes.

So far, studies have shown that silencing efficacy is often found regionally, dividing the whole plant or restricted to plant gene silenced regions without a few consecutive nodes Wege et al. Thus, proper positive control selection and a combination of different inoculation methods are necessary for an efficient VIGS system in diverse tissues at different developmental stages of crops.

However, most known positive controls are always visual phenotype of photo-bleaching on leaves, without any marker genes in other silenced regions. Hence, more positive control genes in different tissues should be screened in future VIGS system. Additionally, the stability of the environmental control and operation techniques is also worth noting in future VIGS studies, because the environment can cause changes in the silencing effect.

Since VIGS assay involves viruses that can be easily transmitted to other plants in a field environment, VIGS vector carrying inocula, seeds and other plant materials should be disposed under suitable biosafety regulation Senthil-Kumar and Mysore, a. Therefore, it is one of the important content for future researches to develop new VIGS vectors and also to modify the existing vectors to increase its host range and silencing efficiency Senthil-Kumar et al. In the past two decades, many VIGS vectors have been developed Supplementary Table 1 , and TRV was preferentially used for VIGS assays in most dicots and some monocots, due to the high susceptibility of a wide range of hosts with mild viral symptoms after infection Chen et al.

The increase in high-quality genome or transcriptome EST data has provided an excellent foundation and sufficient homologous sequence information for VIGS assays to target a specific gene, gene family, gene class, or miRNA mimic Senthil-Kumar and Mysore, a ; Figure 1. As viral vector construction of TRV is an important factor of an efficient VIGS system, many modifications have been done based on the original TRV vector to obtain better silencing efficiency in different species Figure 2.

Multiple inoculation methods for introducing the viral constructs into plants have also been developed to extend the application range of genes that can be studied by this system Figure 3. With the development of VIGS studies, scientists have found that the silencing efficiency of the VIGS system is strongly affected by the selection of Agrobacterium strain, inoculum concentration, environmental factors plant care , and positive control; thus, many established VIGS protocols have been optimized.

In total, all improvements of VIGS technology will speed up the application of this tool for identifying candidate genes involved in various aspects of plant biology Figure 4 , including plant-environment interactions, plant growth and development, metabolic processes, and other cellular processes in planta Senthil-Kumar et al. A deeper understanding of the VIGS mechanism has provided researchers with the option of combining VIGS with other functional genomic approaches conventional or molecular breeding and next-generation technology for crop breeding studies Cheng et al.

VIGS can also be performed in a stable plant that overexpresses, downregulates RNAi , or knocks out mutant an unrelated gene compared with the gene targeted by VIGS to characterize the function of a genetic pathway Baena-Gonzalez et al.

In conclusion, a good VIGS system should be established with proper viral vector construction, inoculation method, optimized inoculum type and concentration, proper positive controls with good plant care and plant vigor, thus eliciting high gene silencing efficiency with a uniform phenotype and simple operation procedure with lower equipment requirements and largely extending the scope of genes that can be studied Liu et al. Finally, a proper VIGS assay together with other genetic tools and related biochemical studies Lu et al.

GS, ZX, and MH researched data for the article, substantially contributed to discussion of the content, and wrote the manuscript. All authors have read and approved the manuscript to be published. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U. Front Plant Sci. Published online Jun 4. Author information Article notes Copyright and License information Disclaimer.

Omar, Zagazig University, Egypt. Zhengqing Xie, nc. Received Feb 23; Accepted May The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.

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Abstract As a promising high-throughput reverse genetic tool in plants, virus-induced gene silencing VIGS has already begun to fulfill some of this promise in diverse aspects. Introduction Virus-induced gene silencing VIGS is a high-throughput reverse genetics technique that exploits an RNA-mediated antiviral defense mechanism [post-transcriptional gene silencing PTGS ] for functional gene analysis Ratcliff et al. Open in a separate window. Methodology Progress of TRV-VIGS The delivery of the constructed recombinant viral vector carrying partial cDNA sequences of target genes into plants for gene silencing is always very important for the efficacy of VIGS; therefore, scientists have developed many ways to introduce the viral vectors into plants.

Leaf Injection Leaf injection syringe infiltration is the most common agroinfiltration method used for leaf or floral development-related gene studies in dicots with high silencing efficiency Figure 3.

Agrodrench Apart from leaf injection and spray inoculation, researchers also developed other methods to study early root development genes Figure 3. Fruit Agroinjection In addition to the previous infection methods carried out on leaves or roots, researchers also developed a method to study fruit genes.

Secondary Infiltration Scientists have also developed a more complex inoculation procedure that is applicable for high-throughput applications of VIGS Lu et al. Important Factors of TRV-VIGS As an experiment of TRV-VIGS carried out from viral vector selection, recombinant viral vector construction, Agrobacterium transformation in vivo transcription of virus genome , the delivery of inoculum into plants, and gene silencing in plants, each step may strongly affect the silencing efficiency of the target gene.

Viral Vector Selection Different viral vectors are suitable for VIGS research in diverse plants with different silencing efficiencies for details, see Supplementary Table 1.

Agrobacterium Strain The optimal A. Inoculum Concentration As the agroinoculation methods and characteristics of diverse infected plants are different, the concentration of the infection solution strongly affects the gene silencing efficiency of VIGS experiments. Environmental Factors Studies have shown that environmental factors directly affect plant growth status and also virus accumulation and spread in plants, which are closely related to gene silencing efficiency and gene silencing duration inheritance of TRV-VIGS Senthil-Kumar and Mysore, b.

Ambient Temperature Previous reports showed that temperature is a key player in influencing the gene silencing phenotype development in plants with VIGS Szittya et al.

Inoculation Method To realize the gene functions across different plant development stages, researchers have developed many VIGS inoculation methods Figure 3. Leguminosae Tobacco rattle virus-VIGS has also been used to study the functional roles of disease resistance genes in soybean.

Ornamental Plants A study demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in the perennial tree peony Xie et al. Other Commercial and Medicinal Plants Moreover, several non-model plants, such as important commercial and medicinal plants, including cassava, flax, Papaver, Antirrhinum , mint, sweet basil, Ashwagandha, and olive tree, were also successfully explored with the TRV-VIGS system.

Monocotyledon Not only can the TRV-VIGS system be used in most dicotyledonous species, it has also been adopted for gene-functional analysis of some monocotyledonous plants that are susceptible to TRV, such as wheat, corn, barley, rice, and Brachypodium sylvaticum in Gramineae and ornamental plants, such as orchids, a flowering plant of Orchidaceae.

Gramineae Virus-induced gene silencing has been successfully applied for functional characterization of genes involved in leaf rust resistance of wheat Scofield et al. Orchidaceae Virus-induced gene silencing has also been adapted for functional validation of genes involved in floral growth and development of orchids Hsieh et al. Limitations Virus-induced gene silencing is a promising genetic tool for functional genomic studies in plants.

Conclusion and Future Prospects In the past two decades, many VIGS vectors have been developed Supplementary Table 1 , and TRV was preferentially used for VIGS assays in most dicots and some monocots, due to the high susceptibility of a wide range of hosts with mild viral symptoms after infection Chen et al.

Author Contributions GS, ZX, and MH researched data for the article, substantially contributed to discussion of the content, and wrote the manuscript. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Footnotes Funding.

References Albert M. An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato. Plant J. A central integrator of transcription networks in plant stress and energy signaling. Nature — RNA silencing. VIGS-genomics goes functional. Trends Plant Sci. The potential of virus-induced gene silencing for speeding up functional characterization of plant genes.

Virus-induced gene silencing for functional genomics in Withania somnifera , an important indian medicinal plant. Methods Mol. Stability of Barley stripe mosaic virus-Induced gene silencing in barley.

Plant Microbe Interact. Applications and advantages of virus-induced gene silencing for gene function studies in plants. Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. Development of a virus-induced gene silencing system for functional analysis of the RPS2-dependent resistance signaling pathways in Arabidopsis. Plant Mol. Using viral vectors to silence endogenous genes. Virus-induced gene silencing of cell wall genes in flax Linum usitatissimum.

Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence. Silencing a prohibitin alters plant development and senescence. Virus-induced gene silencing to investigate alkaloid biosynthesis in Opium Poppy. Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism.

BMC Plant Biol. A rapid and efficient method for uniform gene expression using the Barley stripe mosaic virus. Plant Methods 13 : A new barley stripe mosaic virus allows large protein overexpression for rapid function analysis. Sucking insects such as aphids do not spread TMV. Chewing insects such as grasshoppers and caterpillars occasionally spread the virus but are usually not important in spread.

Vegetative propagation perpetuates TMV and other virus diseases. Cuttings taken from an infected plant usually are infected even if no symptoms are immediately exhibited by the cutting. The virus particles are found in all parts of the plant except the few cells at the tips of the growing points. Infected stock plants should be discarded immediately. TMV can also survive outside the plant in sap that has dried on tools and other surfaces. The next person to open the door can pick up the TMV and spread it to any plant that they touch.

Tobacco products, particularly those containing air-cured tobacco, may carry TMV. Flue-cured tobacco, used in making cigarettes, is heated repeatedly during its processing, thereby inactivating most if not all TMV.

When tobacco products are handled or kept in pockets, hands and clothing can become contaminated with TMV and be a source of virus. TMV is not spread in the smoke of burning tobacco.

Symptoms vary with the species of plant infected and the environmental conditions. In some cases environmental conditions bring out symptoms while other conditions mask or hide symptoms. Symptoms associated with TMV infections:.

Some of the above symptoms can also be caused by high temperature, insect feeding, growth regulators, herbicides, mineral deficiencies, and mineral excesses. TMV diseases cannot be diagnosed on the basis of symptoms alone. Let's Stay Connected. Plants can be grown in climate-controlled, pharmaceutical-grade greenhouses that keep out bugs and pests but do not require sterile conditions.

The tray of plants is swiftly turned upside down and dunked into a metal basin of liquid containing millions of agrobacteria, a group of bacteria that naturally infect plants. A few seconds later, the vacuum is released, causing the leaves to re-expand and, like a sponge, soak up the liquid carrying the agrobacteria, which spread throughout the entire vascular structure of the plant. In a matter of minutes, the Nicotiana benthamiana plants have been transformed into mini bioreactors.

The agrobacteria transfer the viral DNA to the plant cells, which then make millions of copies of virus-like particles that serve as antigens but are not infectious. The plants are resettled in the greenhouse and after five or six days, the leaves are harvested, placed on a conveyor belt, chopped into tiny pieces, and soaked in an enzyme bath that breaks down the hard plant cell wall and releases millions of virus-like particles, which are purified and packaged, Ward explains.

The finished product is a plant-derived vaccine. For conventional vaccines, once the virus or viral particles are extracted from the cells that grew them and purified, they must be kept cold. But other plant-based vaccines skirt this problem by skipping the purification step altogether.

Genetically modified lettuce is also commonly used to make vaccines. Chloroplasts carry about copies of their genome—the genetic material that provides the cell with instructions to function and make copies of itself—unlike most other cells, which have only one copy. This means chloroplasts can produce up to times the amount of target antigen.

Once the viral gene has been inserted into the genome, the seed is grown under controlled but otherwise normal conditions at a farm or greenhouse and then harvested. But here, because lettuce is an edible plant, instead of purifying the virus-like particles by removing all the plant cells and debris, the chloroplasts containing the antigen are ground up into a powder that is then formulated into a pill or capsule, which would then be taken orally.

Several lettuce-based vaccines for humans and animals are under development, but none have advanced to clinical trials. The advantage to a pill form of a vaccine is that it can be stored at room temperature for long periods, thus eliminating the cold chain problem.

The bioreactor fermentation facilities are hundreds of millions of dollars, and then you need to purify and cold chain injection and so on. Emerging plant-based vaccine technology will not only help the world respond to the current and future pandemics but also offers an opportunity to expand vaccine production to developing countries, according to Hefferon.

Vaccines remain a cornerstone of public health, preventing some 4 to 5 million deaths each year. And yet many places around the world lack access to vaccines for measles, meningitis, and whooping cough. That means that some 1. All rights reserved. Plants as vaccine factories Plant-based vaccines eliminate the need for bioreactors because they themselves are the bioreactors. Share Tweet Email. Read This Next Did hallucinogenic booze fuel politics in ancient Peru?

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