Development Of Virus-induced Gene Silencing (VIGS)System For Disease Resistance Genes In Nicotiana

Most cloned plant resistance (R) genes encode nucleotide binding site-leucine rich repeat (NBS-LRR) domain. Plants with R genes recognize corresponding pathogens, and triggers hypersensitive response (HR) after infection, which limits the pathogen into the infected cells and consequently protects plants from systemic infection. Traditional methods for R gene cloning are expensive and time-consuming.11Nicotiana species were studied on the seedlings age, Agrobacterium concentration, Acetosyringone(AS) concentration and cultural temperature respectively in order to optimize the silencing system. The chosen system:seedlings with6leaves, Agrobacterium concentration OD600=0.6, AS concentration400mmol/L and culture conditions for22℃,16h light;20℃,8h dark. Another14Nicotiana species were studied on virus induced gene silencing(VIGS) in the chosen system.At the same time, this project tried to develop a strategy for rapid cloning of disease resistance genes. First, NBS-LRR encoding genes from tomato and potato genome were systemically analyzed and subfamilies were grouped. PCR primers were designed for each subfamily and PCR products were amplifed from Nicotian alata. The PCR products were cloned into a TRV-based virus induced gene silencing construct. A total of113VIGS constructs targeting resistance genes in species have been constructed, including all the R gene families known in Solanaceae. One of the constructs, the VIGS for the N gene, sucessfully silenced the resistance gene N in N. benthamiana, showing the potential of this system for future R gene cloning. The development of VIGS targeting disease resistance genes will be a critical step for rapid cloning of disease resistance genes from Nicotiana spp, which can be used in crops in the Solanaceae crops.