Gene silencing in tomato plants to study Rhizoctonia solani pathogenesis
Rhizoctonia solani is a necrotrophic soil borne fungal pathogen, responsible for causing diseases to numerous plant species such as tomato, rice, sorghum, soybean, cauliflower and lettuce. It accounts for great loss in crop yield annually leading to negative impact on socio-economic development. Here, I am involved in a research project, which focuses on understanding tomato- R. solani interaction using gene silencing approaches. The present lab has identified few significant putative genes in tomato plants, which have been predicted to play a crucial role in R. solani pathogenesis. Therefore, in order to identify efficient approach for transient gene silencing in tomato, we conducted different inoculation methods to induce VIGS mediated gene silencing of endogenous reporter gene PDS which is responsible for carotenoid biosynthesis in plants. Of the different methods tried, stem node wounding and injection method was found to exhibit efficient photo-bleaching of phenotype due to PDS silencing in tomato plants. Along with this, my work is also focused on developing RNAi-constructs for target tomato genes to decipher and validate the role of predicted tomato genes associated with R. solani pathogenesis. In this context, target genes sense and antisense orientation of GST and LRR have been cloned in cloning vector pJET1.2 and further sub-cloning in RNAi vector pGEM-T Easy (containing intron) is in progress. Finally, sense-intron-antisense cassettes of both genes will be introduced in binary vector pCambia1301. In addition, VIGS mediated silencing of a few target genes, NR, DMR, GST and LRR was also repeated using agro-infiltration assay in tomato to validate their role in R. solani pathogenicity.
Keywords: Rhizoctonia solani, RNA interference, VIGS, Pathogenicity, Cloning.