## 5 Conclusions  
This study identifies and characterizes pathogen defense proteins in _N. tabacum_ through computational approaches, focusing on an uncharacterized pathogen defense VLP. Traditionally known for their roles in reproduction, VLPs are now recognized for their immune-relevant and antioxidant activities in animals and plants. The structural and functional analysis of the _N. tabacum_ VLP revealed significant potential in plant defense, with strong binding affinity and stability towards \(\beta\)-glucan, a fungal PAMP. This study highlighted the dynamic interactions between VLPs and PAMP (PGN and \(\beta\)-glucan) in _N. tabacum_. MDS results showed that PGN exhibits more substantial binding stability and more extensive interactions with VLP than \(\beta\)-glucan. Our findings suggested that _N. tabacum_ VLPs could act as PRRs, recognizing and responding to pathogens by activating immune signaling pathways and exhibiting antimicrobial activity. Future research should focus on experimentally validating the predicted interactions and functional roles of VLPs in plant defense. Investigating the mechanistic pathways of VLPs in vivo will enhance our understanding of their role in immune modulation and pathogen neutralization. Additionally, exploring the evolutionary conservation and structural features of VLPs across different species within the Solanaceae family could provide insights into their universal functions and potential biotechnological applications. By leveraging the structural insights from this study, future work could aim to engineer VLP-based strategies for developing crops with enhanced resistance to a wide range of pathogens, contributing to sustainable agriculture and food security.  
The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app15084463/s1](https://www.mdpi.com/article/10.3390/app15084463/s1)]([https://www.mdpi.com/article/10.3390/app15084463/s1](https://www.mdpi.com/article/10.3390/app15084463/s1)). Table S1. Bioinformatics tools employed for structural, evolutionary, and molecular interaction analyses of _Nicotiana tabacum_ VLP, including software functions, access links, and critical parameters. Table S2. Accession Numbers, Protein Names, and Plant Species Corresponding to Hypothetical and Uncharacterized Proteins Used in the Phylogenetic Analysis of VLPs from _N. tabacum_ and Its Homologs. The sequences were retrieved from the NCBI Clustered Non-Redundant (nr) database via BLASTp to construct a phylogenetic tree to study the evolutionary relationships among VLPs in Nicotiana tabacum and its homologs. The accession numbers listed provide transparency and reproducibility of the analysis.  
Conceptualization, A.E.; methodology, A.E. and H.M.; validation, A.E., H.M. and A.Y.R.; formal analysis, A.E.; data curation, H.M. and A.Y.R.; writing--original draft preparation, A.E. and H.M.; writing--review and editing, H.M. and A.Y.R.; visualization, A.E., H.M. and A.Y.R.; supervision, A.E. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
Not applicable.  
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author. Meanwhile, the refined 3D structure of Pathogen Defense Vitellogenin-like Protein in _Nicotiana tabacum_ modeled by RoseTTAFold can be accessed via [[https://www.modelarchive.org/doi/10.5452/ma-wajr2](https://www.modelarchive.org/doi/10.5452/ma-wajr2)]([https://www.modelarchive.org/doi/10.5452/ma-wajr2](https://www.modelarchive.org/doi/10.5452/ma-wajr2)) (accessed on 13 April 2025).  
While preparing this work, the author(s) used ChatGPT v3.5 (OpenAI) to refine grammar and language. After utilizing this tool, the author(s) thoroughly review and edit the content as needed and take full responsibility for the accuracy and integrity of the publication.  
The authors declare no conflicts of interest.