Tomato and Tobacco Phytoene Desaturase Gene Silencing by Virus-Induced Gene Silencing (VIGS) Technique

Abstract

Background and Aims: Virus-Induced Gene Silencing (VIGS) is a virus vector technology that exploits antiviral defense mechanism. By infecting plants with recombinant viruses containing host genes inserted in the viral genome, VIGS achieves the RNA silencing process. The purpose of this study was to investigate the possibility of tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana benthamiana) phytoene desaturase (PDS) gene silencing, using VIGS technique by VIGS vector containing tomato PDS. Methods: PDS gene encodes one of the important enzymes in the carotenoid biosynthesis pathway. In this method Tobacco rattle tobravirus (TRV) vectors including pTRV 1 ،pTRV 2 and pTRV 2 PDS were used. The pTRV 2 PDS vector carried inserts derived from tomato PDS gene. Vectors were transformed into Escherichia coli strain DH 5 α. Agrobacterium tumefaciens strain GV3101 cells were transformed by the vectors containing specific genes. Results: Colony PCR with specific primers proved the presence of the PDS and RNAdependent RNA polymerase genes in selected agrobacterium random colonies. Agrobactrium containing pTRV 1 ،pTRV 2 and pTRV 2 PDS was inoculated into induction media culture. Finally, acetosyringone was added to pTRV 1 culture and then pTRV 1 and pTRV 2 PDS cultures mixed in a 1:1 ratio. Mixed culture was infiltrated into the test plant seedlings using 1ml needless syringe. Conclusion: Results showed the gene silencing in the form of photobleaching and mosaic occurred in tomato and tobacco plant leaves, respectively. However, plant infiltration with pTRV 1 and pTRV 2 (without PDS gene as control) mixed culture did not show any photobleaching.

Tomato (Solanum lycopersicum L.) in the service of biotechnology

“…An important breakthrough in the fast reverse genetics was achieved by using a powerful tool-virus induced gene silencing (VIGS) technology Fernandez-Moreno et al 2013). Jaberolansar et al (2010) and Romero et al (2011) successfully demonstrated that the Tobacco Rattle Virus (TRV)-based VIGS vector could be used in tomato to silence genes. On the other hand, Zhou et al (2012) applied Potato Virus X as a tool for virus-induced gene complementation for revealing a transcription factor network in the modulation of tomato fruit ripening.…”

Tomato tolerance to abiotic stress: a review of most often engineered target sequences

“…Since VIGS represents a useful tool for the identification of gene function, in the other approach different types of viruses (e.g. TRV, Tobacco Rattle Virus) were successfully used as the VIGS vectors and, among them, the TRV vector gave the most robust results in terms of ease of application, efficiency, and absence of disease symptoms (Jaberolansar et al 2010;Romero et al 2011;Wang et al 2015). Moreover, Wang et al (2015) demonstrated that this technology enabled achieving up to 100% VIGS efficiency in different tomato organs (leaves, flowers and fruits).…”