Cross-Species Insights into PR Proteins: A Comprehensive Study of Arabidopsis thaliana, Solanum lycopersicum, and Solanum tuberosum

Abstract

This study provides a comprehensive analysis of pathogenesis-related (PR) proteins, focusing on PR1, PR5, and PR10, in three plant species: Arabidopsis thaliana (At), Solanum lycopersicum (Sl), and Solanum tuberosum (St). We investigated various physico-chemical properties, including protein length, molecular weight, isoelectric point (pI), hydrophobicity, and structural characteristics, such as RMSD, using state-of-the-art tools like AlphaFold and PyMOL. Our analysis found that the SlPR10-StPR10 protein pair had the highest sequence identity (80.00%), lowest RMSD value (0.307 Å), and a high number of overlapping residues (160) among all other protein pairs, indicating their remarkable similarity. Additionally, we used bioinformatics tools such as Cello, Euk-mPLoc 2.0, and Wolfpsort to predict subcellular localization, with AtPR1, AtPR5, and SlPR5 proteins predicted to be located in the extracellular space in both Arabidopsis and S. lycopersicum, while AtPR10 was predicted to be located in the cytoplasm. This comprehensive analysis, including the use of cutting-edge structural prediction and subcellular localization tools, enhances our understanding of the structural, functional, and localization aspects of PR proteins, shedding light on their roles in plant defense mechanisms across different plant species.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01343-1.

Introduction

Plant defense mechanisms are essential for the survival and growth of various plant species [1]. These mechanisms rely on a complex network of proteins, including pathogenesis-related (PR) proteins, which play pivotal roles in responding to biotic stresses [2]. PR proteins are involved in various aspects of plant defense [3], and understanding their structural [4], functional, and subcellular characteristics is crucial for unraveling the intricacies of plant defense mechanisms [5]. In this research, we delve into a comprehensive analysis of PR proteins, with a specific focus on PR1, PR5, and PR10, across three distinct plant species: Arabidopsis thaliana (At), Solanum lycopersicum (Sl), and Solanum tuberosum (St). By employing cutting-edge tools and methodologies, our study sheds light on the structural and functional aspects of these PR proteins, contributing to a deeper understanding of their roles in plant defense mechanisms across diverse plant species.

Despite the crucial roles of PR proteins in plant defense, several gaps in knowledge persist. Existing research has primarily focused on individual PR proteins associated with specific species [6], while comparative studies of PR proteins [7] are relatively limited. Notably, the comparative analysis of PR proteins across multiple plant species, as well as the exploration of their structural and subcellular characteristics, remains relatively unexplored [7]. To address these gaps, our study aims to comprehensively analyze PR1, PR5, and PR10 proteins in A. thaliana (At), S. lycopersicum (Sl), and S. tuberosum (St). Through a diverse array of bioinformatics analyses, we seek to unravel the physicochemical properties, subcellular localizations, topological features, secondary structures, functional annotations, sequence similarities, and protein–protein interactions, all within the context of At, Sl, and St.

Characterizing the unique physicochemical traits of PR proteins aids in identifying species-specific adaptations crucial for pathogen recognition [8], while predicting their subcellular localization provides insights into spatial distribution and defense strategy variations among plant species [9]. Analysis of transmembrane domains and secondary structures informs about PR protein architecture, guiding our understanding of their functional properties and interactions [10]. Furthermore, annotating conserved domains and constructing phylogenetic trees elucidates evolutionary relationships, shedding light on the molecular mechanisms underlying plant–pathogen interactions [11]. Using advanced structural prediction tools like AlphaFold 2 enables accurate modeling, facilitating the study of PR protein folding and stability [12]. Exploring protein–protein interaction through STRING [13] network unveils PR protein’s complex regulatory pathways governing plant defense, identifying potential targets for genetic manipulation to enhance plant immunity [14]. By bridging existing knowledge gaps and advancing our understanding of the roles played by PR proteins in the intricate defense mechanisms of plants, our study provides a comprehensive framework for unraveling their significance and functional implications. Ultimately, this study aims to enrich the field of plant biology by offering a nuanced perspective on the significance of PR proteins in plant defense, with potential implications for crop improvement and sustainable agriculture.

Materials and Methods

Retrieval of Protein Sequences

Three PR protein sequences viz. PR1, PR5, and PR10 of A. thaliana, S. lycopersicum, and S. tuberosum were downloaded from UniProt (http://www.uniprot.org) [15] in FASTA format for further analysis.

Analysis of Physicochemical Properties of PR’s

The physico-chemical properties of PR1, PR5, and PR10 of three species were calculated using the ExPASy ProtParam tool (https://web.expasy.org/protparam/) [16]. The cellular localization of PR proteins was predicted using web servers like CELLO v.2.5 (http://cello.life.nctu.edu.tw/) [17], Euk-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/) [18], Wolfpsort (https://wolfpsort.hgc.jp/) [19]. For detecting signal peptides, PrediSi (http://www.predisi.de/) [20] and SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) [21] were used.

Topological Analysis and Secondary Structure Prediction

The topological analysis of each PR protein was carried out using TMHMM—2.0, an online tool (http://www.cbs.dtu.dk/services/TMHMM/) [22]. Secondary structures of the PR proteins were predicted using the Expasy SOPMA tool (https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) [23]. This tool provides information about the different conformations of proteins, including the percentages of α-helices, β-sheets, turns, extended strands, and random coils.

Functional Annotation and Comparative Analysis

The functional annotation of PR1, PR5, and PR10 proteins from all three species were carried out using InterProScan (https://www.ebi.ac.uk/interpro/result/InterProScan/), tool for identifying functional domains and motifs in protein sequences [24]. This analysis allowed us to determine the conserved protein domains, families, and functional sites within these plant defense proteins. By comparing the results, we evaluated the differences and similarities in the functional annotations of PR proteins across the three plant species.

Sequence Alignment and Comparative Analysis

To determine the sequence similarity and alignment of PR proteins across three species, multiple sequence alignment was carried out using the MUSCLE tool [25] available at (https://www.ebi.ac.uk/Tools/msa/muscle/). Sequence similarity was calculated using the SIM—Alignment Tool for Protein Sequences (https://web.expasy.org/sim/). The aligned sequences were visualized and analyzed using the Jalview software [26] with a sequence identity threshold set at 50% to ensure robust alignment results. This computational approach aided in the identification of conserved regions and variations within these plant defense proteins. A phylogenetic tree was then constructed using the neighbor-joining method with the PAM 250 substitution matrix, commonly employed for protein sequence analysis [27], to provide insights into the evolutionary relationships among the analyzed protein sequences.

Structure Prediction

PR proteins were analyzed using AlphaFold 2 [8], a state-of-the-art deep learning-based method for structural biology. The protein sequences were analyzed using a Google Colab environment specifically designed for AlphaFold 2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb#scrollTo=kOblAo-xetgx). The resulting structural models were further analyzed and visualized using PyMOL [9]. To assess the structural similarity and identify homologous regions among PR proteins from different species, pairwise structural alignments were performed using PyMOL's built-in alignment tools. The RMSD values were calculated to quantify the structural deviation between the protein structures, providing insights into their overall structural similarity. Ramachandran plots were generated to evaluate the quality and stereochemistry of the predicted protein structures, aiding in the identification of regions with unfavourable backbone dihedral angles.

Protein–Protein Interactions

We used the STRING tool (https://string-db.org/) [13] to identify protein–protein interactions, our objective was to comprehensively characterize the interactome of these PR proteins, including their interacting partners, interaction strength via STRING-generated scores, and the functional implications of these associations. This approach combined diverse data sources, including experimental data, databases, and text mining, to predict and score protein interactions. We evaluated interaction scores to assess reliability and analyzed the functional annotations of interacting proteins to understand the biological significance of these interactions in pathogenesis-related responses. This method allowed us to not only identify specific interacting partners for PR proteins in the three plant species but also to compare their interaction networks.

Results

The results from studies on distinct PR proteins from A. thaliana, S. lycopersicum, and S. tuberosum, namely PR1, PR5, and PR10, are reported in terms of accession numbers, physicochemical properties, and cellular localization in S1 and Table 1.

Table 1.

Subcellular localization of PR proteins predicted by different tools

Tool PR	Cello	Euk-mPLoc 2.0	Wolfpsort
PR1
At	Extracellular	Extracell. Vacuole	Chloroplast
Sl	PlasmaMembrane	Cell membrane. Extracell	Chloroplast
St	Extracellular	Extracell. Vacuole	Chloroplast
PR5
At	Extracellular	Extracellular	Extracellular
Sl	Extracellular	Vacuole	Vacuolar
St	Vacuole	Vacuole	Vacuole
PR10
At	Cytoplasmic	Vacuole	Cytoplasmic
Sl	Cytoplasmic	Cytoplasmic	Cytoplasmic
St	Cytoplasmic	Cytoplasmic	Cytoplasmic

Physico-chemical Properties

Computed values for physico-chemical properties including protein length, molecular weight, isoelectric point (pI), number of negatively and positively charged residues, extinction coefficient, instability index, aliphatic index, and grand average of hydropathicity (GRAVY) for PRs were obtained using the ExPASy ProtParam tool (S1). Among all PRs, the minimum length was observed in StPR1 (119) while the maximum length was observed for PR5 (239 in AtPR5 and 227 in StPR5). The minimum molecular weight was observed for StPR1 (13,631.14) while the maximum molecular weight was observed for AtPR5 (25,252.31) and StPR5 (25,009.12). Four PR proteins, including AtPR1, SlPR1, SlPR5, and StPR1, were found to be basic in nature while the remaining five PRs, including AtPR5, AtPR10, SlPR10, StPRP5, and StPR10, were acidic. The AtPRs had EC values ranging from 38,765 (AtPR12) to 19,940 M⁻¹ cm⁻¹ (AtPR10), while SlPRs had values ranging from 49,345 (SlPR5) to 13,410 M⁻¹ cm⁻¹ (SlPR10) and StPRs had values ranging from 46,785 (StPR1) to 10,555 M⁻¹ cm⁻¹ (StPR10).

The instability index of all PRs in S. lycopersicum was less than 40, indicating stability, while AtPR5, AtPR10, SlPR10, and StPRP5 had values greater than 40. Aliphatic index values ranged from 92.45 (AtPR10) to 61.67 (AtPR5) for A. thaliana PRs, 88.25 (SlPR10) to 51.06 (SlPR5) for S. lycopersicum, and 90.75 (StPR10) to 45.13 (StPR1) for S. tuberosum PRs. All three PR proteins analyzed across the three species had negative GRAVY scores, with A. thaliana ranging from − 0.133 to − 0.288, S. lycopersicum from − 0.009 to − 0.426, and S. tuberosum from − 0.164 to − 0.782.

Three separate tools, Cello, Euk-mPLoc, and Wolfpsort, were used to determine the subcellular localization of various PR proteins. Results revealed that AtPR5 was localized extracellularly in all three tools, similarly, AtPR1 and StPR1 were localized extracellular in both Cello and Euk-mPLoc tools. RP10 of Sl and St was found to be localized in the cytoplasmic region, and StPR5 was localized in the vacuole region. The tools PrediSi and SignalP 4.1 predicted the presence of signal peptides in AtPR1 and Atpr5 PR proteins, but none of the other PR proteins possessed signal peptides.

Topological Analysis and Secondary Structure Prediction

To determine the topological features of PR proteins, including the presence or absence of transmembrane domains, we utilized the TMHMM-2.0 web tool. Our examination revealed the occurrence of transmembrane domains exclusively in AtPR5, while all other PR proteins exhibited a consistent absence of transmembrane domains across all three plant species. This information provides valuable insights into the structural diversity of these defense proteins among different taxa.

To determine the secondary structure composition of PR proteins, we utilized the SOPMA tool. The predominance of random coils across all PR proteins and species, with percentages ranging from 27.10% in AtPR10 to 60.59% in SlPR5. Interestingly, alpha helices exhibited their lowest occurrence in SlPR5 (4.71%) and their highest prevalence in StPR10 (39.38%). Furthermore, AtPR1 displayed the minimum proportion of extended strands (16.77%), while SlPR1 showed the maximum (30.48%) (S2). These findings provide insights into the distinct secondary structure profiles of PR proteins across diverse plant species.

Functional Annotation and Comparative Analysis

The protein PR1 analysis found AtPR1 to feature a signal peptide region, CAP domain, CRISP family signatures, and part of the Cysteine-rich secretory protein-related family. StPR1 shared similar features with AtPR1, including CRISP family signatures and Cysteine-rich secretory protein-related family membership, but lacked a signal peptide. Additionally, StPR1 contained a CAP domain from Pfam and matches to the Venom allergen 5 signature. However, SlPR1's data only provided its amino acid sequence length (210 amino acids) without further information, making direct comparisons challenging.

All three proteins, AtPR5, SlPR5, and StPR5, belong to the Thaumatin family and the Osmotin/thaumatin-like superfamily, sharing a common conserved Thaumatin-related site. AtPR5 features additional TMhelix domains, not found in SlPR5 and StPR5, signifying unique functional elements. Whereas both AtPR5 and SlPR5 are associated with various MetaCyc pathways, this information is absent for StPR5, suggesting diverse biological roles. Furthermore, AtPR5 and SlPR5 are linked to gene identifiers (OS03G0233200 PROTEIN), while StPR5's gene identifier remains unspecified, which is crucial for genetic and functional investigations. Additionally, AtPR5 includes details about the CAP domain and Venom allergen 5 signature, which are not present in SlPR5 and StPR5, and StPR5 possesses multiple instances of the Pathogenesis-related protein signature at different locations in its sequence, not mentioned for the other two proteins. These dissimilarities likely contribute to their unique functionalities.

The PR10 proteins from A. thaliana, S. lycopersicum, and S. tuberosum exhibit both commonalities and distinctions in their structural and functional characteristics. All three proteins share a conserved Bet v1-like domain, indicated by the presence of Major Pollen Allergen Bet V1 signatures in their sequences. This domain is crucial for their roles as allergens. Additionally, they possess a START-like domain superfamily, suggesting potential involvement in various metabolic pathways. Functionally, these proteins are associated with similar Gene Ontology terms related to allergenicity and stress responses. However, there are subtle differences among them. AtPR10 has an MLP-LIKE PROTEIN 423 domain, while SlPR10 and StPR10 lack this feature. Moreover, AtPR10 and StPR10 have PRINTS PR00634 signatures in regions different from those in SlPR10. The Pfam domain PF00407, associated with the Pathogenesis-related protein Bet v 1 family, is conserved in all three proteins, further emphasizing their roles in pathogenic responses. Despite these similarities, minor variations in domain locations and domain profiles indicate some distinctions between these PR10 proteins, possibly reflecting subtle functional differences in their respective species.

Sequence Alignment and Comparative Analysis

Multiple sequence alignment was performed using the MUSCLE tool, the conservation of amino acids across the sequences was evaluated. A 50% sequence identity threshold was set, and visualized in Jalview as shown in Fig. 1, where the color scheme was applied according to the percentage identity. The higher the identity, the darker the blue color of the amino acids. The phylogenetic tree generated for PR proteins showed that AtPR1 and StPR1 clustered closely together, indicating a close evolutionary relationship, while SlPR1 occupied a distinct clade. Similarly, AtPR5 and StPR5 displayed a close clustering, indicating their evolutionary proximity, while SlPR5 was positioned in a separate clade. In the case of PR10 proteins, SlPR10 and StPR10 formed a close-knit cluster, implying a shared evolutionary history, while AtPR10 was situated in a separate branch of the tree. The percentage identity obtained using SIM alignment tool in Table 2 showed, alignment score, residue overlap, and gap frequency between various protein pairs. Notable, the highest percentage identity was (80.00%) and a score of 682 for the Slpr10-Stpr10 protein pair, while Slpr1-Stpr1 exhibits lowest percentage identity (with 30.00%) score of 26.

Fig. 1.

Multiple sequence alignment indicated in (A), (C), (E) and phylogenetic analysis of PR proteins displayed in (B), (D), (F) representing closely aligned protein pairs

Table 2.

Protein pair alignments—percentage identity, score, residue overlap, and gap frequency

PR–PR	Percentage identity (%)	Score	Residue overlap	Gap frequency (%)
Atpr1–Slpr1	37.00	39	27 residues	0.00
Atpr1–stpr1	60.90	379	115 residues	1.70
Slpr1–Stpr1	30.00	26	30 residues	0.00
Atpr5–Slpr5	43.30	268	141 residues	5.70
Atpr5–Stpr5	42.50	383	219 residues	8.20
Slpr5–stpr5	62.70	571	169 residues	3.60
Atpr10–Slpr10	30.90	101	94 residues	2.10
Atpr10–Stpr10	35.10	123	94 residues	2.10
Slpr10–Stpr10	80.00	682	160 residues	0.00

Structure Prediction and Homology

We analyzed the structural predictions of all three PR proteins using AlphaFold2 in Google Colab. Each protein had five structural predictions, along with predicted local distance difference test (pLDDT) scores, aligned error, and sequence coverage. We selected the top-ranked prediction based on the pLDDT scores and visualized it using PyMOL as shown in Figs. 2 and 3. To evaluate the interactions between PR proteins, we calculated RMSD values for each protein pairs and in Fig. 3A–C) analyzed the Ramachandran plot. The SlPR10–StPR10 interaction had the lowest RMSD (0.307 Å), indicating a high degree of structural alignment, while ATPR1-SLPR1 had the highest RMSD (21.254 Å), indicating greater structural divergence. The remaining protein pairs displayed structural similarity in the decreasing order ATPR5-SLPR5 (0.884 Å), ATPR5-STPR5 (0.743 Å), SLPR5-STPR5 (0.505 Å), and AtPR1-STPR1 (0.355 Å). These pairs had relatively closer structural similarity, suggesting potential functional similarities in their interactions.

Fig. 2.

PR1 and PR5 Protein structures predicted by AlphaFold2, and its PAE plot representing the predicted alignment error between each residue in the model, along with homology model of protein pairs generated using PyMOL, providing insights into its structural characteristics

Fig. 3.

PR10 Protein structures predicted by AlphaFold2, and its PAE plot representing the predicted alignment error between each residue in the model, along with homology model of protein pairs generated using PyMOL. The Ramachandran plot of PR1(A), PR5(B), and PR10(C) indicates the distribution of phi (ϕ) and psi (ψ) angles for each amino acid residue in the protein

Protein–Protein Interaction

PR1 Interactions

In the Fig. 4 examination of PR1 dataset results discloses that there are no common protein–protein interactions between PR1-2 in AtPR1 and M0ZJH6\_SOLTU in STPR1. In AtPR1, PR1-2 interacts with several proteins involved in plant defense, such as BG2, CHI-B, HEL, PR5, PAD4, ICS1, NPR1, PDF1.2A, and itself, but none of these interactions directly overlap with the interactions of M0ZJH6\_SOLTU in StPR1. Similarly, M1DTJ6\_SOLTU in StPR1 interacts with distinct proteins, such as RCR3 and M1AN72. This analysis indicates distinct protein interaction networks for these two datasets, suggesting unique functional roles for PR1-2 and M0ZJH6\_SOLTU in their respective contexts.

Fig. 4.

Protein protein interactions of PR1, PR5 and PR10 in At, Sl, and St indicating mutually interacting protein pairs

PR5 Interactions

In the Fig. 4 PR5 dataset indicated that AtPR5 interacts with a variety of proteins, including BG2, CHI-B, EDS1, HEL, NPR1, PAD4, PDF1.2A, PR1, PR1-2, and PRB1. These interactions imply a crucial role for AtPR5 in the defense against pathogens and acquired pathogen resistance in A. thaliana. Furthermore, in SlPR5, PR5 interacts with several proteins, including Phenylalanine Ammonia-Lyase, PAL2, PR-5, PR1B1, and NPR1. These interactions indicate a diverse range of functional connections, such as those related to phenolic compound synthesis, pathogenesis-related leaf proteins, and defense responses in S. lycopersicum. Additionally, PR5 in StPR5, represented by multiple isoforms (A0A3Q7EQU5, A0A3Q7FGA3, A0A3Q7HW47, A0A3Q7ID95, A0A3Q7IPA2, A0A3Q7HTH3), primarily interacts with Putative Thaumatin-Like Proteins and NPR1 and Pathogenesis-Related Leaf Protein 6. These interactions suggest a role for PR5 in the defense against pathogens and acquired pathogen resistance in S. tuberosum.

Common Interactions Between ATPR5 and SlPR5 and Comparison Between SlPR5 and StPR5

We observed a recurring connection between PR5 in AtPR5 and SlPR5, involving NPR1. This repeated interaction implies a preserved role of PR5 and NPR1 in A. thaliana and S. lycopersicum. However, this interaction is not present in StPR5. By comparing the interactions of PR5 in SlPR5 and StPR5, we found some commonalities, especially in interactions with Putative Thaumatin-Like Proteins, hinting at a potentially conserved function of PR5 with these proteins in Solanum species.

PR10 Interactions

The analysis of PR10 in Fig. 4 indicates that MLP423 in AtPR10 interacts with MLP-like protein 423, suggesting a potential functional link within the MLP protein family. Additionally, interactions with Putative membrane lipoprotein were observed, albeit with undefined functions. Importantly, MLP423 exhibits interaction with a Cellulase (Glycosyl hydrolase family 5) protein, implying its involvement with glycosyl hydrolase family 5. Furthermore, an interaction with Heat shock 70 kDa protein 1, known for its role in protein folding and stress responses, was noted. TSI-1 (PR10) in SlPR10 exhibits a distinct interaction profile. It engages with a Bet\_v\_1 domain-containing protein, a characteristic feature of the BetVI family. Additionally, TSI-1 interacts with Glyco\_hydro\_18 domain-containing proteins, aligning it with the glycosyl hydrolase 18 family. Further interactions involve 9-divinyl ether synthase, Ethylene-responsive transcription factor 1, RAP2.2-1 transcription factor, and multiple Pathogenesis-related genes transcriptional activators. These diverse interactions suggest a broad range of functions, particularly in stress responses and disease resistance. M0ZMA9\_SOLTU (PR10) in StPR10 shares an interaction with a Bet\_v\_1 domain-containing protein, a hallmark of the BetVI family. Additionally, it interacts with Phytocyanin domain-containing proteins, Adenosylhomocysteinase, and Ethylene responsive factor 1. This interaction profile is comparable to SLPR10, but the specific interactions differ, emphasizing the unique roles and associations of M0ZMA9\_SOLTU in S. tuberosum.

No common interactions have been observed between MLP423 in AtPR10, TSI-1 in SlPR10, and M0ZMA9\_SOLTU in StPR10. This suggests that these proteins may have unique roles in their respective plant species, with minimal overlap in their protein interaction networks. Interestingly, the only shared interaction is with a Bet\_v\_1 domain-containing protein in both SlPR10 and StPR10, which implies a potential conserved function associated with the BetVI family in Solanum species.

Discussion

PR proteins are indispensable components of the plant immune system, playing a vital role in defending plants against diverse biotic and abiotic stresses [28]. Their functions extend to pathogen recognition, stress response regulation, and overall plant health maintenance [29].

In this comprehensive study, we conducted an extensive analysis of PR proteins, focusing on PR1, PR5, and PR10. Our investigation encompassed diverse physico-chemical properties, structural analyses using state-of-the-art tools, and subcellular localization predictions through bioinformatics. These analyses unveiled critical insights into the structural and functional aspects of PR proteins across different plant species, augmenting our knowledge of their roles in plant defense mechanisms.

The main finding of our study reveals that the SlPR10–StPR10 protein pair exhibits the highest degree of similarity compared to all other protein pairs analyzed. This conclusion is based on a comprehensive assessment of sequence identity, structural elements, and physicochemical properties. The remarkable 80% sequence identity and extensive residue overlap of 160 residues between SlPR10 and StPR10 protein suggest a strong evolutionary conservation. Moreover, our structural analysis highlights the significance of extended strand (Ee) and random coil (Cc) elements contributing to this remarkable similarity along with the least RMSD value (0.307 Å). PR-10 proteins are involved in plant defense against biotic and abiotic stressors in [30]. CaPR10 protein from the Solanaceae family exhibited ribonucleolytic activity against Tobacco Mosaic Virus (TMV) [31]. Previous research found that phosphorylating CaPR10 greatly increased its antiviral efficacy against TMV [32]. These findings underscore the potential functional and evolutionary relevance of this protein pair in the context of plant defense mechanisms in Solanaceae.

Physico-chemical Properties

Our analysis revealed significant variations in protein length, molecular weight, isoelectric point (pI), and hydrophobicity among these PR proteins, as reported by [7]. For instance, PR5 proteins generally exhibited longer lengths and higher molecular weights compared to PR1 and PR10. These differences can be attributed to the diverse functions of PR proteins in plant defense mechanisms [33], with PR5 proteins often associated with broader pathogen recognition [34].

Moreover, the variation in pI values suggests differences in the charge distribution among these proteins, potentially influencing their interactions with other cellular components [35]. The hydrophobicity profiles, as indicated by the GRAVY score, also contribute to the functional diversity of PR proteins [36]. Notably, PR1 proteins displayed more negative GRAVY scores, indicating higher hydrophilicity, while PR10 proteins tended to be more hydrophobic. This distinction may correlate with the subcellular localization of PR proteins and their roles in specific cellular compartments. These findings align with previous studies that have emphasized the importance of physicochemical properties in determining the functions and localization of proteins within the cell [37]. Furthermore, understanding these variations can aid in predicting the potential roles of PR proteins in plant defense and immunity, highlighting the need for further investigation into the functional significance of these properties and their implications for plant-pathogen interactions.

Topological Analysis and Secondary Structure Prediction

Zhou et al. [10] reported that transmembrane proteins participate in various physiological activities in plants, including signal transduction, substance transport, and energy conversion. Our investigation into the presence of transmembrane domains revealed occurrence of transmembrane domains solely in AtPR5-1, highlighting its distinctive structural characteristic compared to the other PR proteins. In contrast, all other PR proteins across the three plant species consistently lacked transmembrane domains, suggesting a commonality in their cellular localization and functional roles.

Rusling and Kumosinski [38] study suggests protein's secondary structure, which includes helices and sheets, is critical to how it folds and functions. Rose [39] emphasizes the importance of secondary structures, including as alpha-helices and beta-strands, in the formation of protein folds. We observed a prevalence of random coils across all PR proteins of three species, with percentages ranging from 27.10% in AtPR10 to 60.59% in SlPR5. This prevalence of random coils indicates the flexible and dynamic nature of PR proteins. It also acts as "connecting bridges" for the alpha-helix and beta-strands, stabilising the unfolded protein state and opposing protein folding [40], which is crucial for their functionality in plant defense mechanisms. α-Helices are the most prevalent structures identified within proteins and serve a key role in determining protein global structure and function [41], we observed variations in the occurrence of alpha helices and extended strands among the proteins. SlPR5 displayed the lowest occurrence of alpha helices (4.71%), while StPR10 exhibited the highest (39.38%). In contrast, AtPR1 showed the minimum proportion of extended strands (16.77%), while SlPR1 displayed the maximum (30.48%). Extended strands are frequently distinguished by the presence of main-chain amide and carbonyl groups that form hydrogen bonds with polar side chains or water [42]. Overall, our topological and secondary structure analyses provide valuable insights into the structural and functional diversity of PR proteins in different plant species.

Functional Annotation and Comparative Analysis

Understanding the molecular basis of life requires very accurate functional annotation of protein sequences [43, 44], and has significant biological [45–47], and pathological [48–50] implications. In the functional annotation, AtPR1 and StPR1 shared several common features, including the presence of CRISP family signatures and membership in the Cysteine-rich secretory protein-related family. In plant-pathogen interactions, cysteine-rich secretory proteins (CRiSPs) are essential to modulate host immunity [51]. Thaumatin-like proteins (TLPs) are large protein family that is involved in host defence and development in plants and fungi, and are highly diverse in angiosperms [52]. The analysis of PR5 proteins revealed their association in the Thaumatin family and the Osmotin/thaumatin-like superfamily, with a shared Thaumatin-related site. AtPR5 stood out with additional domains like TMhelix and G3DSA:2.60.110.10, suggesting unique functional elements not found in SlPR5 and StPR5. Transmembrane proteins have intricate structures, mainly in their transmembrane domains, which greatly differ among proteins, making it difficult to examine their interactions and functions [53]. Most PR1 family members have only a CAP domain and short C- and N-terminal extensions, indicating that their activity in plant pathogen defence is determined by the CAP domain. [54]. Specific domains such as the CAP domain and Venom allergen 5 signature in AtPR5 differentiated it from SlPR5 and StPR5, while StPR5 exhibited multiple instances of the Pathogenesis-related protein signature, not observed in the other two proteins. Moving on to PR10 proteins, commonalities and distinctions emerged. The Bet v 1 allergen is a member of the plant pathogenesis-related proteins family PR-10 [55], and may be important for plant defense [56]. All three proteins shared a Major Pollen Allergen Bet V1 domain, and a START-like domain superfamily, indicating potential involvement in metabolic pathways. Proteins with START domains can bind various ligands such as sterols (StAR protein) and phosphatidylcholine (PC-TP) [57].

Sequence Alignment and Comparative Analysis

Multiple alignments of protein sequences are important in many applications, including phylogenetic tree estimation, secondary structure prediction and critical residue identification [58]. The sequence alignment and comparative analysis of PR proteins across three species using MUSCLE and Jalview yielded insights into their evolutionary relationships and percentage similarity. Notably, SlPR10 and StPR10 exhibited the highest percentage identity (80%), along with substantial residue overlap (160 residues), suggesting a close evolutionary relationship and functional similarity. In contrast, Slpr1–Stpr1 showed the lowest identity (30.00%) and occupied a separate clade, Protein similarity can be low due to an array of factors such as conformational plasticity, mutations, solvent effects, ligand binding, and low-complexity areas [59]. For PR5 proteins, SlPR5 and StPR5 displayed a significant percentage identity (62.70%) and a considerable residue overlap (169 residues), indicating evolutionary proximity and potential functional convergence as Proteins with a high degree of sequence identity and structural similarity have similar functions and evolutionary links [59]. AtPR1 and StPR1 also exhibited very close identity value (60.90%) and formed the same clade, these findings, combined with the sequence identity data, provide valuable insights into the evolutionary relationships and potential functional roles of PR proteins across different plant species.

Structure Prediction and Homology

Estimating the accuracy of predicted protein structures is critical for understanding how the models will be function in biological system [60], the structural predictions and homology analysis conducted in this study using AlphaFold2 and PyMOL provided valuable insights into the structural characteristics and interactions of PR proteins. AlphaFold is a combination of the bioinformatics and physical approaches: using physical and geometric inductive bias to build components that learn from PDB data [12]. Our examination predicted the structures of PR protein along with predicted local distance difference test (pLDDT) scores, aligned error, and sequence coverage. Higher pLDDT scores corresponds to higher confidence, and corresponds to the model's predicted per-residue scores on the lDDT-Cα metric [61]. The root-mean-square deviation (RMSD) is a quantitative measure of protein structural similarity between two or more proteins [62]. An essential aspect of this analysis was the evaluation structural similarity through (RMSD) values and assessing the Ramachandran plot. Notably, the SlPR10-StPR10 homology demonstrated the lowest RMSD value (0.307 Å), the smaller the RMSD, the more similar the two structures [63] signifying a high degree of structural alignment and suggesting a potential functional relevance in their interaction.

Protein–Protein Interactions

Proteins and their functional interactions are fundamental to cellular machinery, essential for understanding biological events comprehensively [64]. Our analysis of protein–protein interactions revealed intriguing patterns among PR proteins across different plant species. For instance, PR1 proteins interacted with various defense-related proteins, including Thaumatin-like proteins (TLPs), with AT-PR1-2 and ST-PR1 showing distinct sets of interactors, indicating unique functional roles [65]. Similarly, PR5 proteins exhibited diverse interaction profiles, with AT-PR5 interacting with multiple defense-related proteins in A. thaliana, while SL-PR5 showed interactions linked to phenolic compound synthesis and pathogenesis-related leaf proteins in S. lycopersicum [66], Notably, ST-PR5 primarily interacted with Putative Thaumatin-Like Proteins and NPR1, suggesting significant roles in systemic acquired resistance [66]. Additionally, SL-PR10 (TSI-1) exhibited a range of interactions, including interactions with Bet_v_1 domain-containing proteins crucial for plant responses to biotic or abiotic stress, growth, and development, including disease and stress resistance [67]. Moreover, interactions with Pathogenesis-related genes transcriptional activators indicate diverse functions, particularly in stress responses and disease resistance. Similarly, ST-PR10 interacted with Phytocyanin domain-containing proteins, which play a pivotal role in plant development and defense responses to abiotic stressors [68, 69]. These findings shed light on the intricate protein interaction landscapes and potential functional distinctions among PR proteins in different plant species.

Limitations

Our study, while providing valuable insights into PR protein properties, sequence identity, structures, functional annotations, and interactions across plant species, has limitations. It predominantly examines PR proteins from three species, limiting its scope. Computational tools and databases were used extensively, necessitating experimental validation for robustness. Structural predictions offer static snapshots, and molecular dynamics simulations could provide a more dynamic view. Functional roles were predicted, but experimental validation is vital. Stringent criteria were applied for protein pair selection, and additional measures of similarity could be explored. Subcellular localization predictions might benefit from experimental techniques. Finally, mechanistic insights into protein interactions require further investigation.

In conclusion, our study provides a comprehensive overview of PR proteins, but it is crucial to view the results as a starting point for further research. Overcoming these limitations and integrating experimental data will be essential to deepen our understanding of PR proteins' roles in plant defense and their potential applications in agriculture and biotechnology.

Implications

The implications of this study are twofold: first, it enhances our understanding of PR proteins and their roles in plant defense mechanisms, offering valuable insights into the structural, functional, and topological aspects of these proteins across different plant species. Second, these findings hold great promise for applied biotechnology, as they provide a foundation for the targeted engineering of PR proteins with improved characteristics for crop protection, stress tolerance, and sustainable agriculture. By unraveling the intricate details of PR proteins and their interactions, this research not only contributes to fundamental plant biology but also opens avenues for the development of innovative solutions to address pressing challenges in agriculture and crop improvement.

Conclusion

Our investigation into Pathogenesis-Related (PR) proteins across A. thaliana, S. lycopersicum, and S. tuberosum has revealed their structural, functional, and evolutionary intricacies. Through our analysis, we have gained valuable insights into plant–pathogen interactions, emphasizing the nuanced variations in physico-chemical properties among different species. The identification of species-specific adaptations underscores the critical role of PR protein functionality in plant defense mechanisms. Particularly noteworthy is the convergence in subcellular localization observed between 'AtPR5' and 'SlPR5,' prompting intriguing inquiries into their potential cooperative roles in plant defense. Furthermore, our examination of secondary structure compositions and protein interaction networks has revealed the diverse functional repertoires harbored by PR proteins.

The varying degrees of similarity identified between protein pairs, with 'SlPR10' and 'StPR10' demonstrating compelling resemblance, underscore the multi-faceted nature of these proteins. This study not only advances our understanding of PR proteins but also lays the groundwork for future research and applications in crop protection. By emphasizing the pivotal roles of PR proteins in safeguarding plants against pathogens, our findings contribute to the ongoing efforts to enhance crop resilience and global food security. Moving forward, this research serves as a springboard for further investigations and innovations in agriculture, with the ultimate aim of improving crop yields and ensuring sustainable food production for generations to come.

Supplementary Information

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Author Contributions

The author(s) certify that they have read and approved the final version of the manuscript, and have made substantial contributions to the submitted work.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.