The Triple Helix @ UChicago

Spring 2016

"Virus Induced Gene Silencing" by Clara Sava-Segal

 

At its most simple level, genetic research aims to identify the role of genes. Why? The most pertinent implication lies in a capability to diagnose and treat diseases. Genes code for proteins, which determine all fundamental characteristics and functions of life. With the uncovering of a “simple” gene, one can begin to fathom not only its role in said particular organism, but also begin to piece together certain imperative evolutionary comparisons. Various methods are employed to isolate the role of respective genes in organisms that are dependent on the interactions of the millions of gene-encoded proteins. Each method has its benefits and lapses, but their development is crucial. 

Oftentimes, it is easiest to do this by getting rid of a gene (“silencing”/”knocking out”) a gene and seeing the effect this has on the resulting organism. In some cases, silencing a gene is both more time consuming and economically efficient than screening through all of the genes of an organism, as is done more commonly. 

Let’s take an example at the floral level. Generally, the Aquilegia coerulea flower has the distinct rich reddish-pink seen in Figure 1. However, we can see the same flower depicted in Figure 2, without its pigment. This flower has a “silenced” Anthocyanidin Synthase (ANS) gene. Seeing that the flower is now white, we can extrapolate that the ANS gene is involved in pigmentation. The technique employed to “silence” this gene is termed “Virus Induced Gene Silencing,” a mode of reverse genetics.

 
 Figure 1

Figure 1

 
 Figure 2

Figure 2

 

It is crucial to understand the properties of general reverse genetics before looking at its subsets. As a whole, reverse genetics [“getting rid of a gene”] can be understood only by comparison to “regular” (“forward”) genetics. Within the latter, an organism with a naturally found or induced mutant phenotype is observed in order to identify which gene encoded that phenotype. That gene is correlated to DNA and amino acid sequences.[1] In contrast, reverse genetics does not start with the phenotype, but rather commences at the level of the DNA or protein sequence. This DNA is manipulated to create a specific mutant gene, and that gene codes for a mutant phenotype. The visible changed phenotype generally determines the function of the gene, as seen within the ANS mutant. More simply, as identified by Figure 3, the two processes are complete opposites and mirror one another. Of course, this only works when the genes code for a physical attribute.

 
 Figure 3

Figure 3

As previously identified, it is generally imperative to have a mutant phenotype or gene within the process. Having a mutant reveals function; take, for example, a Drosophila in which “knocking out” one gene or mutating it can have a drastic influence on the phenotype of the Drosophila. For instance, if the gene that codes for red eyes is mutated, the eyes will appear white. Thus, if the intention were to determine which region of the fly genome coded for eye color, various regions would be mutated until the resulting fly had the white-eye phenotype. This thought process, though, makes the assumption that a specific gene codes for a particular function. However, it is not always that simple. As the organisms become more complex, the genome has more and more regulatory mechanisms and genes that function in collaboration, making the selection of mutants much more intricate. 

For example, since Drosophila are vertebrates, the eye gene color is actually dependent on many regulatory elements including promoters, or regions on the DNA that initiate transcription. More simply, these promoter regions “let” the genes encode for certain phenotypes, and they themselves need to be “turned on” to do this. The promoter regions are “allowing” multiple genes to work. Therefore, reverse genetic techniques cannot simply mutate a gene, but also have to look at the mechanisms acting on that gene. From one perspective, having a promoter sequence could be easier: these could be blocked, and the gene would not be expressed independent of whether or not it is mutated. However, working with these regulatory mechanisms is much more complicated. The promoter could not only be regulating the eye color gene, but other genes, as well. Then, the resulting phenotype would have multiple changes and it would be impossible to record which change came from which gene. Very simply, reverse genetics only functions successfully when there is one unknown change being made at a time. There is too much interdependency for function and regulation that occurs in organisms otherwise. 

With all this being said, VIGS- the process used to make the changes in Figure 2- provides a more interesting, less invasive and simpler method. In part, this is due to its role in the plant genome, which has different regulatory mechanisms, but mostly the reason for the success of VIGS is its employment of the organism’s own immunodefense pathway. Employing the plant’s own system means that researchers do not need to take into account the various regulatory mechanisms that have the potential to disturb because the organism is “self-disturbing.” Unlike other methods of reverse genetics, the “mutation” is actually a “self-silencing.” How exactly does this occur? 

VIGS employs the RNA interference (RNAi) pathway to induce transient gene silencing or knock down. The process depends on the use of viral c-DNA vectors that hold the designated gene. Plants are inoculated with Agrobacterium that contains the viral vectors. 4 These organisms then use their own innate antiviral defense mechanisms to fight off the viruses. [5] Since the host genes are within the viruses, the plants’ defense mechanisms end up killing off their own expression of the targeted genes. 

But, how exactly does the RNAi pathway work after the plant is exposed to the virus? Dicer enzymes chop up the viral vector double stranded DNA into short interfering RNA (siRNA). This siRNA combine with various proteins, which are species-dependent, to form an RNA-induced-silencing complex (RISC), that is then activated. This activation is dependent upon the unzipping of the siRNA that exposes the DNA strands. Normally, this exposure would permit for transcription into a messenger RNA that would be translated into proteins. These proteins would be the direct product of the gene expression and generate the encoded phenotype. 

However, since this is part of an immunodefense pathway, the exposure of the DNA preps it for destruction. The exposure of anticodons is crucial. These anticodons on the siRNA are complementary to messenger RNA that will come in, bind, and then be destroyed by the RISC. Therefore, translation will never occur and the gene product will not be produced. [6] The following figure (Figure 4) displays this entire process.

 
 Figure 4

Figure 4

 

Figure 4 shows a plasmid labeled “TRV2-AqANS.” This refers to “TRV,” standing for the Tobacco Rattle Virus, which is next to the Aquilegia plant ANS gene. Since the Aquilegia flower was treated with this plasmid, it fought off both the TRV and its own gene and thus lost its pigment. (Figure 2). 

To conclude, the process of VIGS is essential to understanding gene function within plants, and serves as a non-invasive mechanism of reverse genetics by using the organism’s internal immune system, thus avoiding many of the issues that arise with reverse genetics. With VIGS, researchers do not need to induce “mutations” directly, as the organism silences its own genes. Furthermore, this skips over the regulatory mechanisms existent in most organisms. A resulting phenotype can result from hundreds of the genes within the plant genome. We just need to select a gene and a plant will tell us its role. 

Having these kinds of techniques are instrumental to genetics research that can have great impact on not only establishing lineages in evolutionary biology, but also in medical research. Virus Induced Gene Silencing (VIGS) has so far been used exclusively with the plant genome. However, reverse genetics is a staple of modern day genetics research and has been used across hundreds of model organisms. The development of these advanced techniques is crucial for the development of procedures applicable to the human genome. Therefore, even though VIGS is not directly applicable to our everyday lives, each improvement in reverse genetics is imperative. For instance, since VIGS makes use of the innate immune system of the plant, it is not unrealistic that the human immune system, too, can be harnessed for our own genetic research. 

Furthermore, as initially mentioned, since so many genes are conserved across evolutionary development, understanding the scope of genes in certain vertebrates with reverse genetic techniques can help us identify genes that are influential to not only the development of the human species, but also the expansion of certain diseases. In the future, it may very well be possible to “silence” genes that predispose an individual for diseases.

References

[1] Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Reverse genetics. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21843/ 
[2] Steller, H., and V. Pirrotta. “Expression of the Drosophila White Gene under the Control of the hsp70 Heat Shock Promoter.” The EMBO Journal 4.13B (1985): 3765–3772. Print. 
[3] Gould, Bille, and Elena Kramer. "Plant Methods." Virus-induced Gene Silencing as a Tool for Functional Analyses in the Emerging Model Plant Aquilegia (columbine, Ranunculaceae). BioMed Central, 12 Apr. 2007. Web. 16 May 2016. 
[4] Lu, R., AM Martin-Hernandez, JR Peart, I. Malcuit, and DC Baulcombe. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 30 Aug. 2003. Web. 16 May 2016. 
[5] "RNA Interference." RNA Interference. N.p., n.d. Web. 16 May 2016.

 
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