Utilization of a Tobacco Rattle Virus Vector to Clone a Nicotiana benthamiana cDNA Library for VIGS

Virus–induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS vector to contain the Gateway system for high throughput cloning (Ko et al., J. Fac.Agr.,Kyushu Univ., 60(1), 139–149 (2015)), and utilized this system to express a library of N. benthamiana cDNA. Random c.300 bp N. benthamiana cDNA fragments were generated by ultrasonication and inserted into the TRV VIGS vector by Gateway cloning. N. benthamiana were agroinfiltrated with randomly selected TRV cDNA constructs in Agrobacterium tumefaciens GV 2260. Distinct visible phenotypes were identified in three sets of the inoculated N. benthamiana plants. The three distinguished phenotypes showed leaf malformation and necrosis. The three expressed gene inserts were homologous to EST fragments identified as CK290013.1, CK296346.1, and AM8112161.1, and presumably these genes are related to TRV pathogenesis in N. benthamiana. Identification of the selected genes by VIGS will aid further analysis to determine the relationship between VIGS phenotype and gene function.