Nitric oxide (NO), cyclic GMP (cGMP), and cyclic ADP-ribose (cADPR) play critical roles in plant defense responses. In this study, it was shown that infection of resistant tobacco with tobacco mosaic virus (TMV) increased NO synthase (NOS) activity, while susceptible tobacco did not. Administration of NO donors or recombinant mammalian NOS to tobacco plants or suspension cells triggered the expression of defense-related genes, including those encoding pathogenesis-related 1 (PR-1) protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cGMP and cADPR, which are second messengers in mammalian NO signaling. NO treatment increased endogenous cGMP levels, and NO-induced activation of PAL was blocked by inhibitors of guanylate cyclase, suggesting both cGMP-dependent and -independent signaling pathways. NO was shown to induce PR-1 and PAL gene expression through SA-dependent and -independent pathways, respectively. cGMP and cADPR were found to activate PAL and PR-1 gene expression, with cADPR acting through Ca²⁺ mobilization. The study demonstrated that NO signaling in plants involves components similar to those in animals, including guanylate cyclase and cGMP. NO donors such as GSNO and SNAP, as well as the NO second messenger cGMP, induced PAL expression in tobacco suspension cells. cADPR also induced defense gene expression, with its effects mediated by Ca²⁺ release. The study highlights the role of NO in plant defense responses and suggests that NO signaling in plants involves multiple pathways, including cGMP and cADPR. The findings provide evidence for a mammalian-type NO-responsive defense gene activation pathway in tobacco, involving cGMP-dependent components.Nitric oxide (NO), cyclic GMP (cGMP), and cyclic ADP-ribose (cADPR) play critical roles in plant defense responses. In this study, it was shown that infection of resistant tobacco with tobacco mosaic virus (TMV) increased NO synthase (NOS) activity, while susceptible tobacco did not. Administration of NO donors or recombinant mammalian NOS to tobacco plants or suspension cells triggered the expression of defense-related genes, including those encoding pathogenesis-related 1 (PR-1) protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cGMP and cADPR, which are second messengers in mammalian NO signaling. NO treatment increased endogenous cGMP levels, and NO-induced activation of PAL was blocked by inhibitors of guanylate cyclase, suggesting both cGMP-dependent and -independent signaling pathways. NO was shown to induce PR-1 and PAL gene expression through SA-dependent and -independent pathways, respectively. cGMP and cADPR were found to activate PAL and PR-1 gene expression, with cADPR acting through Ca²⁺ mobilization. The study demonstrated that NO signaling in plants involves components similar to those in animals, including guanylate cyclase and cGMP. NO donors such as GSNO and SNAP, as well as the NO second messenger cGMP, induced PAL expression in tobacco suspension cells. cADPR also induced defense gene expression, with its effects mediated by Ca²⁺ release. The study highlights the role of NO in plant defense responses and suggests that NO signaling in plants involves multiple pathways, including cGMP and cADPR. The findings provide evidence for a mammalian-type NO-responsive defense gene activation pathway in tobacco, involving cGMP-dependent components.