Comparison of Post-transcriptional Gene Silencing (PTGS) Strategies for Developing Transgenic Plants Resistant to Tomato Leaf Curl Virus (ToLCV)

Abstract

Tomato (Solanum lycopersicum L.), an economically important crop is host to many whitefly transmitted geminiviruses including tomato leaf curl virus (ToLCV). Genetically engineering resistance of pathogen through Post-transcriptional gene silencing (PTGS/RNAi) is a powerful strategy that can provide alternative to existing methods of producing virus resistant plants. We cloned and characterized ToLCV-replicase (TRP) gene from a local Dharwad, Karnataka, India ToLCV isolate for development of transgenic tomato plants. Plant expression vectors carrying viral replicase (rep) gene in sense, antisense, ihp (intron spaced hairpin) and HUTR (inverted repeats of heterologous 3΄-untranslated region) were constructed. Transgenic tomato plants carrying rep gene in different strategies when challenged with whiteflies carrying ToLCV showed varied degrees of resistance. Such plants were confirmed through PCR, GUS, Dot blot, Southern blot and semiquantitative PCR analysis. High degree of resistance was observed in the construct carrying both sense and antisense strand interrupted by intron (ihp). Our results demonstrate that, transgenic plants with simultaneous expression of sense and antisense strands are more efficient in gene silencing of ToLCV than those expressing either sense or antisense strand alone.