Viral Suppressors of Gene Silencing

2021

Electronic Journal of Biotechnology, 2004

Journal of Biosciences, 2020

Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism wherein transcripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will be subjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance and cross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might have evolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA) molecules of 21-23 nucleotides length act as homology guides for triggering the systemic degradation of transcripts homologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouring chalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has been achieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of gene silencing has led to the development of several alternative strategies for inducing gene silencing in a precise and controlled way. This has paved the way for using PTGS as one of the chief functional genomics tools in plants and has helped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides, opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencing the deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in a new era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined the basics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, as case studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenic male sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress the production of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistance in sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify the concept and utility of gene silencing in crop plants.

Nature Reviews Genetics, 2002

In the early days of molecular biology, genes were first defined through the description of their mutant phenotype. FORWARD GENETICS has the advantage that the phenotype of the mutant gives a clue to the function of the gene. But with the advent of large-scale genome sequencing the situation is different -literally thousands of genes have been identified, but we know nothing of their function. REVERSE GENETICS is now the most effective way to assess the function of a gene, but so far there has been no general method for reverse genetics other than gene targeting by homologous recombination, which is slow and costly. Antisense approaches, such as antisense oligonucleotide and RIBOZYME technologies, have been useful in reverse genetics, but only to a limited degree. By contrast, the promise of small interfering RNA (siRNA) technology to 'knock down' the expression of any gene in vertebrate cells is set to revolutionize reverse genetic approaches.

World Journal of Biology and Biotechnology, 2020

Ribonucleic acid (RNA) silencing, RNA interference (RNAi) or post-transcriptional gene silencing takes place in a variety of eukaryotes and it was discovered firstly in the plants. The RNA silencing process is activated by a trigger from dsRNA predecessor. A very important step in the silencing pathways the conversion of dsRNA into small duplexes of RNA of the representative length and arrangement. Then these small dsRNA monitor RNA silencing by different mechanisms. Post transcriptional gene silencing mechanisms were initially identified as an anti-viral process that give protection to the organisms from the viruses or which inhibit the unsystematic incorporation of transposable components. The basic aim of this review article is to study the mechanism of gene silencing by dsRNA and the roles of certain proteins in cellular post transcriptional RNA silencing machinery and finally we also discuss the RNA silencing as an anti-viral defense mechanism in the plants.

Proceedings of the …, 2011

Journal of Clinical Pharmacy and Therapeutics, 2004

Cell, 1999

Much of our current understanding of the PTGS mechanism comes from observations of its effects on viral replication (Vaucheret et al., 1998). Infection by many types of RNA or DNA viruses can stimulate PTGS. This was shown in several independent experiments using diverse viruses, all of which carried sequences from cloned plant genes, and a number of different plant genes were tested. In all cases, the transcripts of homol-It is now routine to construct trangenes with defined ogous plant genes became degraded following infection promoters and terminators and express them in a variety (reviewed in Bruening, 1998). PTGS also allows plants of organisms. However, some introduced transgenes to "cure themselves" of infecting viruses even if the are not expressed as they should be. For example, virus has no homology to the host genome. In several epigenetic gene silencing inactivates expression of examples (reviewed in Carrington and Whitham, 1998) transgenes in higher plants at frequencies of up to 30% viruses infect a plant and initially replicate, but as the of independent transformants. Although many examples infection spreads, the host plant begins degrading the of transgene silencing have been described in diverse viral transcripts until no virus particles can be detected, recipient species from plants to fungi to animals (rea process called "recovery." The "recovered" plant is viewed in Vaucheret et al., 1998), the gene regulation no longer susceptible to further infection by sequencemechanisms behind the process are poorly understood. related viruses. Not surprisingly, some plant viruses Gene silencing affects homologous sequences, for exhave evolved mechanisms to protect themselves against ample, repeats in transgenes or transgenes with se-PTGS in their hosts. For example, expression of particuquence homology to endogenous genes. Two types of lar gene products of several RNA viruses has recently gene silencing mechanisms have been observed in been shown to prevent PTGS of unrelated transgenes plants, transcriptional gene silencing (TGS) and postas well as to enhance replication of other viruses (Anantranscriptional gene silencing (PTGS). The promoters of dalakshmi et al., 1998; Brigneti et al., 1998; Kasschau TGS silenced genes are inactivated so that their tranand Carrington, 1998). scription is inhibited. In contrast, genes silenced by Why are both transgenes and viruses susceptible to PTGS continue to be transcribed, but polyadenylated PTGS? Dougherty and colleagues (Lindbo et al., 1993) transcripts are nearly undetectable. Both TGS and PTGS

International Journal of Bio-resource and Stress Management

Gene silencing is one of the most efficient and promising functional genomics tools which down regulates the expression of a gene in a very precise manner and has significant impact on crop improvement. Silencing of a target gene can be achieved at two levels; transcriptional and post-transcriptional stages. In broader sense, transcriptional gene silencing occurs through the repression of the process of transcription while the post transcriptional gene silencing can occur through the degradation of the mRNA. Small RNA molecules like siRNAs & miRNAs are key players which mediate the mechanism of gene silencing. These small RNA molecules are generated through Dicer digestion of the endogenous or exogenous dsRNA by which RISC (RNA induced gene silencing complex) is activated which inhibits expression of a gene. Apart from these small RNA molecules, viruses can also accomplish gene silencing which is known as VIGS (Virus induced gene silencing). VIGS consists of cloning and inserting plant endogenous gene sequences in recombinant viral vectors, which are then inoculated in plants, triggering RNA interference. Gene silencing as reverse genetic tool, has been exploited successfully to build up the resistant against biotic (insect/pest, nematodes, viruses etc.) and abiotic stresses (drought, salinity, frost etc.), and for the enhancement of yield and quality parameters. With the development of cutting edge biotechnological tools and techniques, it is now affordable to knock down/down regulate the expression of any undesirable gene. Ultimately this kind of technology can be important to global food security and sustainability.

International Journal of Plant Genomics, 2009

Virus-induced gene silencing (VIGS) is one of the reverse genetics tools for analysis of gene function that uses viral vectors carrying a target gene fragment to produce dsRNA which trigger RNA-mediated gene silencing. There are a number of viruses which have been modified to silence the gene of interest effectively with a sequence-specific manner. Therefore, different types of methodologies have been advanced and modified for VIGS approach. Virus-derived inoculations are performed on host plants using different methods such as agro-infiltration and in vitro transcriptions. VIGS has many advantages compared to other lossof-gene function approaches. The approach provides the generation of rapid phenotype and no need for plant transformation. The cost of VIGS experiment is relatively low, and large-scale analysis of screening studies can be achieved by the VIGS. However, there are still limitations of VIGS to be overcome. Nowadays, many virus-derived vectors are optimized to silence more than one host plant such as TRV-derived viral vectors which are used for Arabidopsis and Nicothiana benthamiana. By development of viral silencing systems monocot plants can also be targeted as silencing host in addition to dicotyledonous plants. For instance, Barley stripe mosaic virus (BSMV)-mediated VIGS allows silencing of barley and wheat genes. Here we summarize current protocols and recent modified viral systems to lead silencing of genes in different host species.