## 1 Introduction  
Plants are constantly attacked by various pathogens, including fungi, bacteria, and viruses, which can cause significant damage to plant health and reduce crop yields [1]. Consequently, plants have evolved multilayered defense mechanisms, including physical barriers (e.g., bark, waxy cuticles, and cell walls), chemical defenses via secondary metabolites, and inducible responses, such as producing antimicrobial proteins and enzymes [2,3]. These effective pathogen defenses are crucial for the survival and productivity of plants, particularly in agricultural settings where crop losses can have substantial economic and food security implications [4, 5, 6, 7]. _Nicotiana tabacum_ L., commonly known as cultivated tobacco, is an herbaceous annual plant in the genus _Nicotiana_. It is the most widely cultivated species in the genus and is commercially harvested for its leaves, which are processed into tobacco [8,9]. Due to genetic tractability and a well-characterized genome, it is an effective model organism for elucidating plant defense mechanisms. Beyond its economic value, _N. tabacum_ is known to produce a variety of phytochemicals--such as nicotine, polyphenols, and solanesol--with demonstrated antimicrobial and antioxidant properties that contribute to pathogen resistance [10,11]. Numerous studies of _N. tabacum_ extracts have reported antibacterial activity against _Bacillus subtilis_, _Corynebacterium pyogenes_, and _Pseudomonas_[12, 13, 14], antiparasitic properties against parasitic nematodes and other pathogens [15], analgesic and antifungal properties [16]. The phytochemicals in _N. tabacum_ have potential pharmaceutical value in treating neurodegenerative diseases (e.g., Alzheimer's and Parkinson's), inflammatory diseases (e.g., colitis, arthritis, sepsis, multiple sclerosis, and myocarditis), and metabolic syndrome (e.g., obesity and fatty liver) [16,17]. As a well-studied and genetically tractable plant, _N. tabacum_ is often a model organism in plant biology research, including studies on plant-pathogen interactions and defense mechanisms [18]. Understanding the defense mechanisms and bioactive compounds of _N. tabacum_ can inform the development of more disease-resistant and pest-tolerant crop varieties, which is crucial for sustainable agriculture [10,18,19]. As a commercially important crop, insights into its pathogen interactions can lead to the development of disease-resistant crop varieties, ultimately benefiting sustainable agriculture. It exhibits versatile defense mechanisms, including systemic and local resistance, providing broader insights into plant defense. Its significance lies in modeling plant defense mechanisms, showing resistance to various pathogens, and mounting systemic resistance, which is vital for understanding plant coordination against pathogens [20, 21, 22]. Studies on its interactions with pathogens, such as tobacco mosaic virus and _Pseudomonas tabaci_, reveal complex inductions of defense genes and the production of phytochemicals, including nicotine, which possess antimicrobial properties. _N. tabacum_'s genetic tractability, well-studied genome, agricultural relevance, and versatile defense make it invaluable for understanding plant defense principles and developing effective disease management strategies [10,18,19]. Computational methods enable the systematic analysis of plant proteins through sequence-based, expression-based, and interaction-based approaches. Sequence-based methods encompass alignment, domain analysis, and phylogenetic profiling, whereas expression-based approaches utilize transcriptomics and co-expression data. Interaction-based methods predict protein-protein interactions and reconstruct pathways. These tools facilitate high-throughput analysis, functional annotation, and integrative studies, advancing research in plant biology, agriculture, and biotechnology [23, 24, 25, 26, 27, 28, 29]. This study aimed to systematically identify and characterize pathogen defense proteins within the _N.