High nitrogen fertilization suppresses the NtLRR‐RK4 defense pathway to enhance susceptibility to Alternaria alternata in flue‐cured tobacco

BACKGROUND: Tobacco brown spot disease, caused by the fungus Alternaria alternata, is a significant threat to global crop production, including in China. Our previous research identified NaLRR-RK4 as a key gene conferring resistance to this disease in wild tobacco (Nicotiana attenuata). In agricultural practice, the incidence of tobacco brown spot disease varies significantly with nitrogen levels, with plants under high nitrogen conditions exhibiting more severe symptoms. However, the relationship between nitrogen nutrition and the function of the homologous gene (NtLRR-RK4) in cultivated tobacco remains unknown. RESULTS: This study investigated the interaction between nitrogen levels and disease resistance in two cultivated tobacco varieties (N. tabacum), K326 and Hongda (HD). Tobacco plants were subjected to low, medium, and high nitrogen fertilizer levels and inoculated with A. alternata. Using RNA silencing and nitrogen treatments, we found that during infection, expression of the NtLRR-RK4 gene, along with its downstream targets NtERF109, NtDEF19, and the key scopoletin phytoalexin synthase gene NtF6'H1, was significantly higher in low nitrogen plants than in high nitrogen plants. Furthermore, under identical nitrogen conditions, gene expression was consistently higher in the K326 variety than in HD. Subsequent analysis revealed that nitrogen levels directly influence the NtLRR-RK4-mediated defense response. Specifically, high nitrogen input suppressed the activity of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) and reduced flavonoid biosynthesis. This suppression was more pronounced in HD, resulting in greater susceptibility to A. alternata compared with K326. CONCLUSION: In summary, we demonstrate for the first time that the disease resistance conferred by NtLRR-RK4 is modulated by nitrogen availability and host genetic factors. Overall, these results provide new insights into the molecular mechanisms of brown spot resistance in tobacco, informing the development of effective control strategies. © 2026 Society of Chemical Industry.