Nitric oxide (NO) is involved not only in the regulation of plant growth, development, and stress responses but also in the regulation of plant-microbe interactions. Here, we demonstrate that Piriformospora indica can induce tobacco nitrate reductase to produce a NO signal in roots which enhances nitrogen uptake capacity by inducing the expression of ammonium and nitrate transporter genes and the development of lateral root and root hair, thereby promoting tobacco growth. In addition, the NO signal induced by P. indica is significantly different from that induced by the pathogen Phytophthora nicotianae. Inoculation with P. indica did not produce H₂O₂ and maintained high expression of Phytoglobin 1 in roots, resulting in a significantly lower NO level than in the roots inoculated with P. nicotianae. These findings suggest that an appropriate NO level is the likely basis of plant-P. indica symbiosis, which promotes the growth of host plants.

Tobacco (Nicotiana tabacum L.) is an important economic crop whose production is severely threatened by black shank and bacterial wilt, caused by Phytophthora nicotianae and Ralstonia solanacearum, respectively. Kunitz-type trypsin inhibitors (KTIs), as key components of plant innate immunity, play crucial roles in defense against biotic stress in various plant species. In this study, we systematically identified 22 KTI genes in N. tabacum genome and conducted a comprehensive analysis of their phylogenetic relationships, structural features, and expression patterns. By comparing transcriptomic profiles under colonization by the beneficial endophyte Piriformospora indica versus infection by the pathogenic fungus P. nicotianae, we identified a pathogen-specifically induced gene, NtKTI9. This gene was significantly activated upon infection by both P. nicotianae and R. solanacearum. Functional studies revealed that the NtKTI9 protein possesses trypsin inhibitory activity and exhibits antimicrobial capacity in vitro. Overexpression of NtKTI9 in tobacco activated the phenylpropanoid metabolism pathway, which promoted lignin deposition in the roots and enhanced plant resistance against both pathogens. Further mechanistic analysis showed that the expression of NtKTI9 is induced by jasmonic acid (JA) signaling, while its overexpression, in turn, strengthens JA signaling by regulating α-linolenic acid metabolism, suggesting a potential positive feedback loop that synergistically amplify plant immune responses. Based on gain-of-function evidence, this study indicates that NtKTI9 positively contributes to immune regulation in tobacco, which not only deepens our understanding of how KTI genes participate in plant immunity but also provides a novel candidate gene and theoretical foundation for molecular breeding of disease-resistant tobacco.