## 4 Discussion  
This study uses computational approaches to identify and characterize pathogen defense proteins in _N. tabucum_. By analyzing the structural and functional characteristics of N. tabucum, we identified an uncharacterized pathogen defense VLP in its proteome. Vg and VLPs, traditionally known for their role in reproduction and egg development in oviparous animals, including insects, are now recognized for their functions beyond serving as nutrient reserves for developing embryos [82; 83]. Emerging evidence suggests these proteins exhibit immune-relevant and antioxidant properties in animals and plants. Vg and its derived yolk proteins, such as lipovitellin and phosvitin, possess potent antioxidant properties, scavenging free radicals and protecting cells from oxidative stress [84]. Additionally, VLPs can modulate the immune system by binding to and neutralizing pathogens and regulating the production of immune-related molecules. These properties suggest their involvement in plant pathogen defense mechanisms. For example, insect VLPs can suppress the oxidative burst response in plants, a key defense mechanism against pathogens, which may potentially facilitate pathogen infection and transmission. They may also bind to and neutralize pathogens, preventing disease and modulating plant immune signaling pathways [85; 86; 87]. The role of VLPs in plant pathogen defense is an emerging research area, with studies on insect-transmitted plant viruses providing initial insights. VLPs, identified in plants and increasingly recognized for their role in plant defense against pathogens such as bacteria and fungi, may act as PRRs. They can induce defense responses, exhibit antimicrobial activity, and interact directly with PAMPs, helping plants recognize and respond to pathogens. As PRRs, they may bind to PAMPs, activate immune signaling pathways, produce reactive oxygen species, and induce systemic acquired resistance. In plants such as rice and Ara  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline \multicolumn{2}{c}{**Free**} & & & & & & & & & & & & & & \\ \multicolumn{2}{c}{**Ener-**} & & & & & & & & & & & & & & \\ \multicolumn{2}{c}{**(gas/mol)**} & & & & & & & & & & & & & & & \\ \hline \multirow{9}{*}{0 ns} & VLP- & VLP- & VLP- & VLP- & VLP- & VLP \(\beta\)- & VLP- & VLP \(\beta\)- & VLP- & VLP \(\beta\)- & VLP- & VLP \(\beta\)- & VLP- & VLP \(\beta\)- \\  & PCN & glucan & PCN & glucan & PCN & glucan & PCN & glucan & PCN & glucan & PCN & glucan & PCN & glucan & PCN & glucan \\ \multicolumn{2}{c}{} \\ \multirow{9}{*}{0 ns} & \(-85.4146\) & \(-66.0888\) & \(-19.9471\) & \(-12.8384\) & \(10.039351\) & \(5.71073\) & \(-84.2806\) & \(-57.3172\) & \(-44.24888\) & \(-50.9057\) & \(-3.3445\) & \(-7.44361\) & \(55.42  
bidopsis, VLPs have been shown to recognize bacterial and fungal components, activate defense genes, and enhance resistance. These proteins emerge as essential players in plant immunity, potentially developing crops with improved disease resistance [88; 89]. The phylogenetic tree of _N. tabacum_ VLP protein sequence clusters closely with sequences from the genera Capsicum, Solanum, Datura, Lycium, and Anisodus, suggesting significant sequence homology among these VLP proteins, all of which belong to the Solanaceae family, indicating a common evolutionary origin and potential conservation because of shared functions or structural requirements (Figure 2). This clustering implies that these VLPs might perform similar roles, possibly related to plant defense mechanisms against viral infections, retaining crucial structural features. Evolutionary pressures, such as horizontal gene transfer, convergent evolution, or shared environmental factors, may have driven this conservation. Understanding the sequence similarity and structural conservation can inform biotechnological applications, such as vaccine development, by allowing data extrapolation across these related species.