Tobacco Rattle Virus (Trv) Based Virus Induced Gene Silencing (Vigs) in Tomato (Solanum Lycopersicum)

Abstract

Virus-Induced Gene Silencing (VIGS) is a technology to test the function of genes that are involved in leaf and floral senescence. VIGS is a powerful tool for assaying the effects of gene silencing in plants. Efficient silencing requires the fragments that are more than 90% homologous to the target gene. Here, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and therefore allows to isolate a fragment of the homologous PDS gene from the species of interest i.e. tomato. We typically use silencing of phytoenedesaturase (PDS), involved in carotene biosynthesis which results in a characteristic photobleaching phenotype in the leaves. The purpose of this study was to investigate the possibility of tomato (Solanamlycopersicum) phytoenedesaturase (PDS) gene silencing, using VIGS technique by Tobacco rattle virus (TRV) based VIGS vector containing tomato PDS. The pTRV2:PDS vector carried inserts derived from tomato PDS gene. Vectors were transformed into DH5α Escherichia coli strain. GV3101 Agrobacterium tumefaciens strain cells were transformed by the vectors containing PDS genes. The construct was validated for physical integrity (through PCR and restriction digestion analysis) as well as for biological functionality (through transient expression of transgene protein i.e. PDS by agroinfiltration method in the experimental host plant).The construct shows gene silencing results in photobleaching phenotype occurred in tomato plant leaves. However, plant infiltration with pTRV1 and pTRV2 (without PDS gene as control) mixed culture did not show any photobleaching. This construct found structurally as well as functionally active under plant system and can be further modified into transgene construct to be used to study the function of other genes through VIGS in tomato.