Virus-induced-gene-silencing is an approach of reverse genetics that has been successfully used for to study gene fucntion. It is employed at postranscriptional level by taking advantage of plant defence mechanism against parasite infection. Usually, after viral infection, plants produce double stranded RNA (dsRNA) to degrade RNA viruses. By simulating this approach, in VIGS , genes underlying pathogeneic effects on host plant in viral genome are removed. A sequence of the target gene is inserted into a VIGS vector such as Tobacco rattle virus, and delivered to the host plants thorugh Agrobacterium tumefaciens. After delivery of the recombinant viral genome into plant cells, the insert amplify and generate double stranded RNA (dsRNA). A Dicer like protein cleave the dsRNA into small interefrring RNA (siRNA) of about 21-24 nulceotides. The siRNA form a complex with RNA interfering silencing complex (RISC), which target homologues RNA for degradation(Lu, Peart, Malcuit, & Baulcombe, 2003; Ramegowda, Mysore, Senthil-kumar, & Willmann, 2014; Physiol, Bekele, Tesfaye, & Fikre, 2019)
Silencing of Phytoene desaturase (PDS) gene in Nicotiana benthamiana using Tobacco rattle virus (TRV) VIGS vector, and Agrobacterium tumefacien, stain GV2260. The main steps are involved in VIGS are agrobacterium preparation, infiltration, VIGS phenotyping, and silenced gene expression (Padmanabhan & Dinesh-kumar, 2009)
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- a) Innocualtion of Agrobacterium tumefaciens GV2260 with TRV1 and TRV2, allow the grothe of the culture overnnight.
- b) Cells collection by centrifugation at approximtively 3000 rpm.
Incubation of the culture at rom temperature for an average of 4 hours
Make a mixture of Agrobacterium containing TRV1 and TRV2 at 1:1 ratio
Innoculation of plants leaves using either Syringe, Spray or Vacuum infiltration approach.
- c) Allow plant growth at 25C
- d) VIGS Phenotyping 9 days after infiltration
- e) Transcritpome profiling of the silenced genes using nother blot or qPCR
The VIGS is a powerfull method that have been employed to investigate genes funcctions. Genes involve in biotic related-stresses have been studied in many hosts plants. The Phytoene desaturase (PDS) gene in Nicotiana benthamiana was the first to be silenced using Tobacco mosaic virus (TMV). Later, meristem gene was silenced in N. benthamiana using tomato golden mosaic DNA virus (TGMV)-VIGS vector.The function of wheat starch regulator 1 (TARSR1) was characterized through VIGS-derived Barley Stripe mosaic virus (BSMV) vector. It was observed that down-regulation of infection of wheat with BSMV-VIGS led to down regulation of TARSR1. Similarly, genes in ripped tomato and strawberry after detachment from the parental plant were silenced using VIGS techniques. Through Pepper huastero yellow vein virus (PHYVV), Comt, pAmt, and kas genes have been silenced. These three genes are involved in biosynthesis of capsaicinoids, repsobsible for pugent taste in pepper fruit (Physiol, Bekele, Tesfaye, & Fikre, 2019)
Furthermore, VIGS have also been used to study genes involved in abiotic stress-related response in plant. The tobacco rattle virus (TRV) was used to silence lea4 gene, which code for embryogenesis protein (LEA), yeidling to tomato susceptibility to drought. Likewise, enhancmenet of drought tolerance in wheat was observed when Era1 and Sal1 are down regulated due to silencing of genes thanks to BSMV-VIGS. It was shown that the down regulation of Era1 and Sal1 genes led to the decrease of ABA sensitivity.
Besides using VIGS to investigate biotic and drought stresses in plant, this technique was also employed in investigating salt stress. The function of SIGRX1 gene in enhancing salt tolerance in tomato was found after this gene was silenced using satellite DNA mβ-VIGS vector. This resulted in yellowing of tomato leaves compared to control treatments.
VIGS has been globally used for charactwriyation of genee function in a wide range of plants. Howver, it has been reported that the introduction of virus vector in host plants interfere with host metabolism, thus affecting plant-microbe aossociation. Moreover, irus multiplication can be hindered by cloning of genes into VIGS, leading many viruses to delete the cloned genes during amplification and spread in host plants. Besides problems related to plant metabolism and gene delivery, off target silencing is another major concern about VIGS. In off target silencing, the similarity between the dsRNA and some host mRNA sequences may result in the degradation of these mRNA, this can affect host gene expression patern.
References
- Lu, R., Peart, J. R., Malcuit, I., & Baulcombe, D. C. (2003). Virus-induced gene silencing in plants, 30, 296–303. https://doi.org/10.1016/S1046-2023(03)00037-9
- Padmanabhan, M., & Dinesh-kumar, S. P. (2009). Virus-Induced Gene Silencing as a Tool for Delivery of dsRNA into Plants, 4(2), 1–5. https://doi.org/10.1101/pdb.prot5139
- Physiol, J. P. B., Bekele, D., Tesfaye, K., & Fikre, A. (2019). Journal of Plant Biochemistry & Applications of Virus Induced Gene Silencing ( VIGS ) in Plant Functional Genomics Studies, 7(1), 1–7. https://doi.org/10.4172/2329-9029.1000229
- Ramegowda, V., Mysore, K. S., Senthil-kumar, M., & Willmann, M. R. (2014). Virus-induced gene silencing is a versatile tool for unraveling the functional relevance of multiple abiotic-stress-responsive genes in crop plants, 5(July), 1–12. https://doi.org/10.3389/fpls.2014.00323
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