Mutation And Evolution Analysis Of TMV Resistance N Gene In Tobacco

The N gene in tobacco is the first cloned plant disease resistance gene encoding Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) domains. It confers resistance to tobacco mosaic virus (TMV) by mediating hypersensitive response (HR) at the infection site. A 50 kDa amino acid sequence from the C-terminal helicase motif of TMV repilicase was enough to trigger the HR in tobacco plants with the N gene. The p50 sequence of the TMV was transformed into a susceptible tobacco plant (the homozygote transgenic plant is called P50 in this study). The P50 plant was crossed with a N-containing tobacco cultivar SamsunNN. Therefore, the hybrid seeds have both the N gene and its corresponding avirulence gene P50. Systemic HR occurs in the F1 seedlings and the seedlings usually die 1-2 weeks after germination. Only when the N gene or p50 mutates and loses its function, can the hybrid seedlings survive.In this study,910,000 F1 seeds derived from crosses between SansunNN and transgenic P50 were produced and geminated. Only 350 seedlings survived 2 weeks after germination. Innoculation with TMV on detached leaf showed that 41 of these survivals had no HR response to TMV, indicating the loss of the N gene function. Therefore, the N gene has a loss of function mutation rate of approximately 1/22,000. PCR amplifications using a series of N-specific primers showed that 40 of the 41 mutants yielded no PCR products, suggesting deletions of the entire N gene. In contrast, the other mutant had PCR products using all N-specific primers. Sequencing the PCR products discovered a point mutation, which causes amino acid change from F(1008) to L(1008). We also obtained N homologes from tobacco cultivar Hicks broadleaf through database mining and PCR amplification. The N gene sequences from seven genotypes of Nicotiana gultinosa, were obtained by sequencing PCR products amplified using N-specific primers. No sequence exchanges were discovered between the N gene and its homologues, and N gene is highly conserved in N. glutinosa, showing a typical evolutionary pattern of TypeⅡdisease resistance genes.