Overexpression of NrCN improved TMV resistance in selection marker-free tobacco generated by Gene-Deletor system

Two expression cassettes, the Nicotiana tabacum Ubi.U4 promoter-driven tobacco mosaic virus (TMV)-resistant gene NrCN element and Arabidopsis thaliana senescence-expressive gene SAG12 promoter-driven LoxP/FRT site-specific recombinase gene FLP expression fragment, were both introduced into the TMV-sensitive tobacco variety K326 via Agrobacterium tumefaciens-mediated genetic transformation. In T0 transgenic plants inoculated TMV, the hypersensitive response (HR) and systemic HR (SHR) occurred at the inoculated leaf and the upper non-inoculated leaf, respectively. The chlorophyll, H2O2 and salicylic acid (SA) content but malonyldialdehyde (MDA) content in transgenic inoculation plants was significantly higher than non-transgenic plants. Meanwhile, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were exactly significantly higher than non-transgenic ones at the early stage after TMV inoculation, but the CAT activity in the former declined and was significantly lower than the latter at the later stage. In addition, the gene relative expression analysis of TMV-resistance-related protein components showed that the expressions of NrCN, NTF6, and PR-1a in genetically modified tobacco (GMT) plants were significantly upregulated after inoculation compared with wild-type tobaccos. Analysis of foreign gene deletion efficiency in transgenic plants indicated the foreign genes (e.g., recombinase gene FLP and screening reporter gene Bar::GUS) deletion events occurred in determinated GMT plant leaf with the development and maturity of the leaves. As a result, the transgenic plants became sensitive to herbicides. Therefore, the introduction of a plant expression vector (Gene-Deleter system with site-specific sequences LoxP/FRT and NrCN-contianing) into TMV-sensitive tobacco variety K326 enhanced the TMV resistance of sensitive tobacco plants. This study provides a basis for screening and acquisition of new varieties of marker-free and safe transgenic antiviral tobacco.