Pathogenesis‐related proteins (PR), including osmotins, play a vital role in plant defense, being activated in response to diverse biotic and abiotic stresses. Despite their significance, the mechanistic insights into the role of osmotins in plant defense have not been extensively explored. The present study explores the cloning and characterization of the osmotin gene (WsOsm) from Withania somnifera, aiming to illuminate its role in plant defense mechanisms. Quantitative real‐time PCR analysis revealed significant induction of WsOsm in response to various phytohormones e.g. abscisic acid, salicylic acid, methyl jasmonate, brassinosteroids, and ethrel, as well as biotic and abiotic stresses like heat, cold, salt, and drought. To further elucidate WsOsm's functional role, we overexpressed the gene in Nicotiana tabacum, resulting in heightened resistance against the Alternaria solani pathogen. Additionally, we observed enhancements in shoot length, root length, and root biomass in the transgenic tobacco plants compared to wild plants. Notably, the WsOsm‐ overexpressing seedlings demonstrated improved salt and drought stress tolerance, particularly at the seedling stage. Confocal histological analysis of H2O2 and biochemical studies of antioxidant enzyme activities revealed higher levels in the WsOsm overexpressing lines, indicating enhanced antioxidant defense. Furthermore, a pull‐down assay and mass spectrometry analysis revealed a potential interaction between WsOsm and defensin, a known antifungal PR protein (WsDF). This suggests a novel role of WsOsm in mediating plant defense responses by interacting with other PR proteins. Overall, these findings pave the way for potential future applications of WsOsm in developing stress‐tolerant crops and improving plant defense strategies against pathogens.
