Identification and characterization of genes associated with thermo-tolerance using virus induced gene silencing in Nicotiana benthamiana

Crop acclimatization to elevated temperature conditions often demands transcriptional reprogramming of an array of genes to evoke protection from heat stress. Previously, we addressed such question in potato and identified GLP1 (Germin-like protein 1), nsLTP (Non specific lipid transfer protein), PI-PLC (phosphoinositide-specific phospholipase-c), CHP (Conserved hypothetical protein) and RPL4 (60 S Ribosomal L4/L1 protein) as candidate thermo-tolerant genes. Herein, Nicotiana benthamiana knockdown lines were developed to study their functional relevance in high-temperature stress using virus induced gene silencing (VIGS). Among all the VIGS lines tested, silencing of GLP1 and CHP showed significant reduction (more than 75 %) in their endogenous transcript levels, whereas the expression of nsLTP, PI-PLC and RPL4 was reduced by 50 %. Thus suggesting the silencing of endogenous target gene was successful using VIGS. Among those genes tested, VIGS knockdown lines generated against GLP1 and nsLTP produced a very strong hypersensitive phenotype under gradual heat stress and sudden heat shock conditions. Knockdown of GLP1 and nsLTP also showed higher chlorophyll degradation and less cell viability upon stress compared with mock plants. Interestingly, down-regulation of PI-PLC plants showed thermo-sensitive to sudden heat shock, whereas CHP and RPL4 silenced plants showed relatively less sensitive phenotypes. Most importantly, silencing of GLP1 and nsLTP genes compromised acclimation induced H2O2 accumulation, which subsequently failed to activate reactive oxygen species (ROS) scavenging signaling pathways that controlling transcription of antioxidant and other heat stress related genes. These data suggest that GLP1 or nsLTP dependent H2O2 production is required for acquiring thermo-tolerance mediated via activation of ROS-related genes. These analyses will aid in an effort to engineer broad spectrum thermo-tolerance in economically important crop.