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. 2020 Sep 28;15(9):e0239836. doi: 10.1371/journal.pone.0239836

Physico-chemical characterization and topological analysis of pathogenesis-related proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches

Amritpreet Kaur 1, Pratap Kumar Pati 2,*, Aparna Maitra Pati 3,*, Avinash Kaur Nagpal 1,*
Editor: Diego F Gomez-Casati4
PMCID: PMC7521741  PMID: 32986761

Abstract

Plants are constantly under the threat of various biotic and abiotic stress conditions and to overcome these stresses, they have evolved multiple mechanisms including systematic accumulation of different phytohormones, phytoalexins and pathogenesis related (PR) proteins. PR proteins are cluster of proteins with low molecular weight which get incited in plants under different stresses. In this paper, in-silico approaches are used to compare the physico-chemical properties of 6 PR proteins (PR1, PR2, PR5, PR9, PR10, PR12) of Arabidopsis thaliana and Oryza sativa. Topological analysis revealed the presence of transmembrane localization of PR2 and absence of transmembrane domain in PR10 of both model plants studied. Amino acid composition shows the dominance of small aliphatic amino acids i.e. alanine, glycine and serine in both plants studied. These results highlights the similarities and differences between PRs of both model plants, which provides clue towards their diversified roles in plants.

Introduction

Ever increasing human population and drastic climate change being observed in recent decades continue to pose serious threat to growth and productivity of agricultural crops. The latter encounter different types of environmental stresses, mainly categorized as biotic or abiotic. The abiotic stresses include salinity, cold, heat, drought, floods, heavy metals etc. whereas, biotic stresses include attack by pathogens (bacterial, fungal, viral). Both biotic and abiotic stresses are detrimental to plant growth and development because they are known to cause several metabolic dysfunctions in plants and in extreme cases can also cause death of the plant [1, 2]. During the course of evolution, plants have evolved a broad range of defense mechanisms for survival under various stressful conditions. These mechanisms involve the responses like activation of Reactive Oxygen Species (ROS); accumulation of different phytohormones like abscisic acid (ABA), ethylene (ET), jasmonic acid (JA), methyl jasmonate (MeJA) and salicylic acid (SA); production of pathogenesis related (PR) proteins and accumulation of phytoalexins. Out of these, PR protein production and accumulation in plants during biotic and abiotic stresses is very crucial. They are not only induced by different stresses but also accumulate in plant tissues during different developmental stages like flowering, senescence etc.. First PR protein was isolated from tobacco leaves but now its presence is reported in many plants. Initially PR proteins were classified into 17 families based on their serological relationships, biological or enzymatic activity and sharing of amino acid sequences [3]. Among them, many PRs are antifungal (PR1, PR2, PR3, PR4, PR5, PR7, PR12, PR13 and PR14); a few possess endochitinase activity (PR8 and PR11); whereas two others are oxalate oxidases (PR15 and PR16). PR17 proteins are basic secretary proteins [4–7]. Custers et al., [8] characterized a class of carbohydrate oxidases with a defensive role in higher plants, later on these were categorized as PR18 [9]. Sooriyaarachchi et al., [10] purified new antimicrobial protein from Pinus Sylvestris and categorized them as PR19. The present study is focused on PR1, PR2, PR5, PR9, PR10 and PR12; with diverse functions as mentioned below.

PR1 is the first discovered class of pathogenesis related proteins with molecular weight ranging from 14–17 KDa. The first PR1 protein was isolated from tobacco after which PR1 proteins were identified in number of plant species including Arabidopsis, barley, maize, pepper, tomato, wheat, rice etc. [11]. Gamir et al., [12], for the first time described mode of action of PR1 proteins which were shown to inhibit the sterol-auxotrophic pathogenic microorganisms by binding and sequestering sterols. PR1 proteins are known to have antifungal activity and their increased expression is considered as hallmark for activation of SA mediated signaling pathway [11, 13]. PR2 proteins are low molecular weight proteins in the range of approximately 6–43 KDa. They have been identified in various plants species such as tomato, barley, rice etc. [14–16]. PR2 proteins are strong anti-fungal proteins, possessing β-1, 3-glucanase activity, catalyzing hydrolytic cleavage of β-1,3-glucosidic linkages present in the β-1,3-glucan, resulting in breakdown of fungal cell wall [17]. They have role not only in providing defense against the pathogen attack but also in different developmental processes like pollen germination, embryogenesis, fruit ripening, seed germination and development etc. [18]. PR5 proteins are thaumatin like proteins (TLPs) because of their sequence similarity with thaumatin, which is sweet protein from Thaumatococcus danielli, a west African shrub. They have diverse roles in managing biotic and abiotic stresses and have antifungal, antipest, antifreeze activities and also exhibit tolerance to osmotic stress [19]. PR9 proteins are peroxidases with molecular weight ranges from 32–42 KDa involved in lignification of plant cell wall [20]. PR10 proteins are ribonuclease like proteins having antibacterial activity against number of bacterial species including those of Pseudomonas, Agrobacterium, Serratia etc. [3, 21, 22]. They are also known to possess antiviral activity. Their role in abiotic stress management has been demonstrated in rice and maize [23, 24]. PR12 (defensins) are small antimicrobial peptides with very low molecular weight ranging from 3–5 KDa. They provide protection against wide range of microbial pathogens including bacteria and fungi. They have role in management of abiotic stress like cold, drought, heavy metals etc. [25]. Many PR proteins are induced through the action of many phytohormones like JA, SA and ET and the level of these hormones is also known to increase at the site of infection in a plant [6]. It is clear that PR proteins are associated with resistance against various kinds of stresses (abiotic and biotic); and plant development and morphogenesis related signaling pathways but the mechanism of their action is yet to be elucidated. Hence, the detailed analysis of their varied physico-chemical properties could throw light on their useful multiplicity.

Apart from different experimental methods, many in-silico methods and online tools are available for the analysis and characterization of protein sequences. Bioinformatics tools offer researchers a quick and cost effective information on the physico-chemical properties of proteins that is useful in planning laboratory experiments.. Different physico-chemical properties like protein length, amino acid composition, molecular weight, aliphatic index, extinction coefficient, isoelectric point, half-life, instability index and grand average of hydropathicity can be analyzed. Several studies have shown the presence and expression of PR genes in various plants including Arabidopsis thaliana and Oryza sativa. In our earlier study, we compared cis-elements in the promoter regions of PR proteins using in-silico tools [26]. To the best of our knowledge, there is no report on physicochemical properties and topology of various PRs of A. thaliana and O. sativa. Hence, the present study was planned to analyze and compare physicochemical properties and topology of 6 PR (PR1, PR2, PR5, PR9, PR10 and PR12) proteins of each of the two model plants. This study will aid in understanding the occurrence of diversification in different PR proteins of A. thaliana and O. sativa. Further, it will also throw light on the similarities and differences between A. thaliana and O. sativa PR protein sequences.

Materials and methods

Retrieval of protein sequences

Six PR protein sequences viz. PR1, PR2, PR5, PR9, PR10 and PR12 of A. thaliana and O. sativa were downloaded from UniProt (http://www.uniprot.org) [27] in FASTA format for further analysis.

Analysis of physico-chemical properties of PRs

Physico-chemical properties of PR1, PR2, PR5, PR9, PR10 and PR12 of A. thaliana and O. sativa were computed using ExPASy ProtParam tool (https://web.expasy.org/protparam/) [28]. Cellular localization of PR proteins was predicted by using web servers like: CELLO v.2.5 (http://cello.life.nctu.edu.tw/) [29]; WoLF PSORT (http://wolfpsort.seq.cbrc.jp/) [30]; and EuLoc (http://euloc.mbc.nctu.edu.tw/) [31]. For detection of signal peptides, PrediSi (http://www.predisi.de/) [32] and SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) [33] were used.

Topological analysis

Topological analysis of each PR protein of A. thaliana and O. sativa was carried out using online tools viz. TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) [34]; TMAP (http://www.bioinformatics.nl/cgi-bin/emboss/tmap) [35]; PHDhtm (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_htm.html) [36]; DAS (http://www.sbc.su.se/~miklos/DAS/) [37], HMMTOP (http://www.enzim.hu/hmmtop/) [38]; TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) [39]; Phobius (http://phobius.sbc.su.se/) [40]; WHAT (http://saier-144-21.ucsd.edu/barwhat.html) [41] and MEMBRAIN (http://www.csbio.sjtu.edu.cn/bioinf/MemBrain) [42].

Secondary structure prediction

Secondary structures of PR proteins were predicted with online Expasy SOPMA tool (Self-Optimized Prediction Method and Alignment) (https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) [43]. This tool provides details about different conformations of proteins from the given sequences such as percentages of α-helices, β-sheets, turns, extended strands and random coils.

Results

The results on different PR proteins of Arabidopsis thaliana and Oryza sativa viz. PR1, PR2, PR5, PR9, PR10 and PR12 with respect to their accession numbers, physico-chemical properties, cellular localization, topology and signal peptides are presented in Tables 1–3.

Table 1. Physico-chemical properties of Pathogenesis Related proteins of A. thaliana and O. sativa computed using ProtParam tool.

PR Accession no. Length M.Wt. pI (-) R (+) R Eca II AI GRAVY
At Os At Os At Os At Os At Os At Os At Os At Os At Os At Os
PR1 P33154 Q6YSF8 161 168 17676.94 17534.13 9 4.55 10 17 16 8 38765 43930 25.57 46.09 73.85 59.94 -0.288 -0.176
PR2 P33157 Q94CR1 339 334 37338.92 35682.43 4.85 9.40 39 23 27 31 41830 40800 48.07 31.95 81.68 85.96 -0.266 -0.061
PR5 P50700 Q7XST4 244 278 26632.76 29863.03 6.08 7.98 18 21 17 24 34920 42285 44.2 46.81 59.63 63.53 -0.295 0.040
PR9 P0DI10 Q7F1U0 325 317 35624.39 42889.77 9.35 5.77 31 23 44 20 23420 11960 35.32 30.35 97.17 83.56 -0.049 0.028
PR10 Q93VR4 Q9LKJ9 155 158 17045.5 16656.91 5.09 4.95 23 24 17 16 19940 16180 21.89 30.69 92.45 83.42 -0.163 -0.118
PR12 P30224 Q6K209 80 80 8709.22 8784.33 8.47 9.08 4 6 7 12 8980 500 27.49 47.84 75.5 65.88 0.339 -0.041
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At = A. thaliana Os = O. sativa.

M.wt. = Molecular weight; pI = Isoelectric point; (-) R = total number of negatively charged residues; (+) R = total number of positively charged residues; Eca = Extinction coefficient (aunits of M-1 cm -1at 280 nm measuring in water); II = Instability index; AI = Aliphatic index; GRAVY = Grand average of hydropathicity.

Table 3. Comparative topological analysis of PR proteins of A. thaliana and O. sativa indicating the presence or absence of transmembrane domains using different online tools.

PR protein
TOOL		 PR1 PR2 PR5 PR9 PR10 PR12
At Os At Os At Os At Os At Os At Os
TMHMM 0 0 1 1 1 0 1 1 0 0 1 0
Phobius 0 0 1 1 0 1 1 1 0 0 0 0
HMMTOP 1 0 1 1 1 0 1 1 0 0 1 1
TMpred 1 1 1 1 1 1 1 1 0 0 1 1
PHDhtm 1 1 1 1 1 1 1 1 0 0 1 1
TMAP 1 1 1 1 1 1 1 1 0 0 1 1
DAS 1 1 1 1 1 1 1 0 0 0 1 1
MEMBRAIN 1 0 1 1 0 1 0 1 0 0 1 1
Various tools predicting the percentage of transmembrane domains 75 50 100 100 75 75 87.5 87.5 0 0 87.5 75
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At: Arabidopsis thaliana; Os: Oryza sativa (0-absent; 1-Present).

Physico-chemical properties

Physico-chemical properties like protein length, molecular weight, isoelectric point (pI), total number of negatively and positively charged residues, extinction coefficient, instability index (II), aliphatic index (AI) and grand average of hydropathicity (GRAVY) for PRs of A. thaliana and O. sativa were computed using ExPASy ProtParam tool (Table 1). Among all PRs of both species; A. thaliana and O. sativa, minimum length was observed in AtPR12 and OsPR12 (80) while maximum length was observed for PR2 (339 in AtPR2 and 334 in OsPR2). Accordingly minimum molecular weight for PRs of both plants was observed for PR12 (8709.22 for AtPR12 and 8784.33 for OsPR12) whereas, maximum molecular weight was observed for PR2 (37338.92) in case of A. thaliana and PR9 (42889.77) for O. sativa. On the basis of computed pI values, three PR proteins of A. thaliana (AtPR2, AtPR5 and AtPR10) were found to be acidic while other three (AtPR1, AtPR9 and AtPR12) were basic in nature whereas in case of O. sativa, OsPR1, OsPR9 and OsPR10 were acidic and OsPR2, OsPR5 and OsPR12 were basic in nature. In case of AtPRs, number of positively charged residues were more as compared to negatively charged residues for PR1, PR9 and PR12, whereas, in case of OsPRs, more positively charged residues were observed for PR2, PR5 and PR12. Extinction coefficient (EC) values ranged from 8980 (AtPR12) to 41830 M-1 cm -1 (AtPR2) for AtPRs and 500 (OsPR12) to 43930 M-1 cm -1 (OsPR1) for OsPRs. Instability index of four PRs of A. thaliana (AtPR1, AtPR9, AtPR10 and AtPR12) and three PRs of O. sativa viz. OsPR2, OsPR5 and OsPR12 was less than 40, which means that they are stable. AI values ranged from 59.63 (AtPR5) to 97.17 (AtPR9) for A. thaliana PR proteins and 59.94 (OsPR1) to 85.96 (OsPR2) for O. sativa PR proteins. GRAVY score of five (PR1, PR2, PR5, PR9, PR10) of the six PR proteins analyzed in A. thaliana was found to be negative with range from -0.049 to –0.295 with the exception of AtPR12 whose GRAVY score was 0.339. GRAVY score of OsPR proteins was found to be negative (-0.061 to -0.176) for OsPR2, OsPR12, OsPR10, OsPR1 and score was positive for OsPR5 (0.040) and OsPR9 (0.028). Amino acid composition of PR proteins of A. thaliana and O. sativa, with respect to aromatic, polar, non-polar, positively and negatively charged amino acids, is given in Fig 1. S1 Fig shows the comparison of percentage of each amino acid in different PR proteins of A. thaliana and O. sativa. Heat map representation of the comparison of amino acid composition of PR proteins of A. thaliana and O. sativa is shown in Fig 2.

Fig 1. Comparison of amino acid composition of PR proteins of A. thaliana (A) and O. sativa (B) with respect to their number of each amino acid in their respective class.

Fig 1

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Fig 2. Heat map representation showing the comparison of amino acid composition of PR proteins of A. thaliana and O. sativa.

Fig 2

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Three different tools (Cello, Euloc and Wolfpsort), used to determine subcellular localization of different PR proteins in A. thaliana and O. sativa, revealed the extracellular localization of PR12 in both A. thaliana and O. sativa, PR5 in O. sativa and PR9 in A. thaliana. PR10 protein was found to be localized in cytoplasm of both the plants as predicted by all the three tools. Variable results were observed for PR1 and PR2 in both A. thaliana and O. sativa; PR5 in A. thaliana and PR9 in O. sativa, with regard to their localization (Table 2). The tools, PrediSi and SignalP 4.1 predicted the presence of signal peptides in all PR proteins of A. thaliana and O. sativa except PR10 protein i.e. AtPR10 and OsPR10.

Table 2. Subcellular localization of PR proteins predicted by different tools.

Tool PR Cello Euloc Wolfpsort
PR1 At Extracellular Extracellular Extracellular/Vacuole
Os Extracellular Vacuole Extracellular
PR2 At Vacuole Vacuole Vacuole/Extracellular
Os Vacuole Vacuole Chloroplast
PR5 At Extracellular Vacuole Chloroplast
Os Extracellular Extracellular Extracellular
PR9 At Extracellular Extracellular Extracellular
Os Extracellular Extracellular Chloroplast
PR10 At Cytoplasmic Cytoplasmic Cytoplasmic
Os Cytoplasmic Cytoplasmic Cytoplasmic
PR12 At Extracellular Extracellular Extracellular
Os Extracellular Extracellular Extracellular
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At: Arabidopsis thaliana; Os: Oryza sativa.

Topological analysis

Comparative topological analysis of different PR proteins of A. thaliana and O. sativa using various online tools indicated the presence of transmembrane domains in all PR proteins studied except PR10 (Table 3). 100% of the tools used in the study predicted the presence of transmembrane domains in PR2, whereas, 87.5% tools for PR9 and 75% for PR5 indicated the presence of transmembrane domains for both species, A. thaliana and O. sativa.

Secondary structure prediction

SOPMA tool was used to predict percentage occurrence of secondary structure features (alpha helices, extended strands, beta turns and random coils) of PR proteins of A. thaliana and O. sativa (Figs 3 and 4).The analysis revealed occurrence of maximum frequency of random coils in PR1 and PR5 of both A. thaliana and O. sativa, whereas, alpha helices were found to be maximum in PR2, PR9 and PR12 proteins of both the plants. In case of PR10 protein, number of extended strands was maximum in A. thaliana, whereas alpha helices were found to be maximum in O. sativa.

Fig 3. Percentage occurrence of secondary structural features.

Fig 3

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Fig 4. Comparison of secondary structure features of PR proteins of A. thaliana and O. sativa.

Fig 4

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Discussion

The present study focused on in-silico analysis of physico-chemical properties of 6 PR proteins (PR1, PR2, PR5, PR9, PR10, PR12) w.r.t various parameters including their subcellular localization, topology and detection of signal peptides. Comparison of protein length and molecular weight of each type of PR in both species (A. thaliana and O. sativa) showed little variation. However, protein lengths and molecular weights of different PRs (PR1, PR2, PR5, PR9, PR10 and PR12) within each plant were significantly different. Subcellular localization, interactions and solubility depend upon isoelectric point and number of positively and negatively charged residues. pI is the pH value at which proteins carry no charge or the sum of negatively and positively charges is equal. pI value more than 7 was observed for PR12 of both the species; PR1 and PR9 of A. thaliana; and PR2 and PR5 of O. sativa. For PR10 of both the plants, the pI value was less than 7. This study is in line with some of the earlier studies which show that PR1 and PR2 can either be acidic or basic in nature [6, 44]. The acidic nature of PR10 observed for both the species studied is in confirmation with an earlier study which also showed PR10 to be acidic [45]. The EC value of a protein solution is an important parameter based on amount of light absorbed per mole of protein at a certain wavelength, most commonly 280 nm wavelength is used. EC value of protein is calculated from the number of tryptophan, tyrosine and cysteine residues per molecule because these residues contribute significantly to measured optical density of denatured protein at 276–282 nm range [28, 46, 47]. In the present study, minimum EC value (500 M-1 cm -1) was observed for OsPR12 which is mainly due to four cystine amino acids (two cysteines joined by disulphide bond form cystine). For, AtPR12 relatively higher value of EC (8980 M-1 cm -1) was observed which is due to the presence of 1 tryptophan and 2 tyrosine residues in addition to four cystines (8 cysteines). Among different PR proteins analysed, PR2 of both the species was found to be tyrosine rich and PR5 was cystine rich.

Instability index (II) indicates about the protein stability under both in-vivo and in-vitro conditions. Proteins with instability index (II) <40 are considered to be stable and those with II value >40 are referred to as unstable [48]. Instability index of PR9 and PR10 of both species studied; and PR1 and PR12 of A. thaliana only were found to be less than 40 indicating their stable nature. PR5 of both plants; PR2 of A. thaliana only; PR1 and PR12 of O. sativa only had stability index of more than 40, indicating them to be unstable proteins. Apart from instability index (II), aliphatic index (AI) is another parameter to check the stability of the proteins. For a protein, AI can be defined as the relative volume captured by aliphatic side chains of amino acids like A (alanine), V (valine), L (leucine) and I (isoleucine). Earlier a good correlation was established between AI and thermostability of proteins by Ikai [49]. Among the PRs of A. thaliana and O. sativa, AtPR5 (59.63) and OsPR1 (59.94) have lower values of AI as compared to other PRs of A. thaliana and O. sativa; indicating that they are less thermostable and have more flexible protein structure. The high AI value indicates that under wide range of temperature conditions, the protein is stable. Apart from studying protein concentration and stability, its hydrophobic or hydrophilic character is also analyzed with the GRAVY score. GRAVY score for particular protein is calculated as the sum of hydropathy values of all amino acids present in the protein, divided by the number of residues in that protein. Its value lies between -2 to +2 where; negative score means hydrophilicity and positive score indicates hydrophobicity [50]. Proteins with more negative GRAVY score are considered to be hydrophilic in nature with good solubility and vice-versa. If a protein has GRAVY score more than 0.4, suggest its hydrophobic nature and difficult to detect on 2-D gels [51].

In-silico approaches have been used to determine subcellular localization of proteins which plays an important role to depict their function. Three different tools viz., Cello, Euloc and Wolfpsort were used to determine subcellular localization of different PR proteins analysed in this study. Cello tool is a two level support vector machine (SVM) classifier system and its prediction regarding subcellular localization for a particular protein is considered accurate/acceptable if its reliability/confidence value is at least 1 [52]. In our study, we observed minimum confidence score of 1.5 in case of AtPR9 and 2.9 for OsPR10. Whereas, maximum score of 4 was obtained for AtPR1, OsPR1, OsPR5, OsPR9 and OsPR12. Euloc is a hybrid tool that integrates three different approaches like homology search, Hidden Markov Model and SVM for detection of subcellular localization of proteins [31]. Wolfpsort predicts multi-site (nine sites) localization of a protein. It is a Sequence-based prediction method which along with homology/ functional motifs and sorting signals has greatly improved the accuracy of the prediction of subcellular localization of proteins [53].

Subcellular localization of PR proteins as predicted by Cello and Euloc was similar for all the PR proteins of A. thaliana and O. sativa except for PR1 of O. sativa and PR5 of A. thaliana. The results obtained by Wolfpsort for PR1, PR2, PR5 and PR9 were different from either predicted by Euloc and Cello or both. Subcellular localization of PR10 or PR12 was found to be cytoplasmic and extracellular, respectively and was uniformly predicted by all the three tools used. PR1 is an important antifungal protein and is known to be localized in extracellular space. Previously the localization of PR1 is detected in vacuoles, vesicles of cortical cytoplasm, Endoplasmic reticulum bodies etc., using prolonged dark incubation in combination with salicylic acid treatment of seedlings of A. thaliana by Pecenkov et al., [54]. In our study also, this protein (PR1) was shown to be localized in vacuoles (Euloc in O. sativa and Wolfpsort in A. thaliana). PR2 are group of proteins involved in number of developmental processes as well as in defense against biotic stress. Many studies reported the difference in localization of PR2 proteins in potato cultivars susceptible or resistant to PVY infections. The level of PR2 was found to be higher in cell walls, chloroplasts and vacuoles of susceptible cultivar [55]. In a number of in vivo studies, PR5 proteins have shown anti-microbial activities [56, 57]. PR5 has been shown to exhibit sequence similarity with a sweet tasting protein, thaumatin, which is derived from Thaumatococcus daniellii, a shrub from South Africa. Hence, they are also called as Thaumatin-like proteins (TLPs) [58]. Though PR5 proteins are called as TLPs, but none of them have a sweet taste like thaumatin. Based on their molecular weight, TLPs have been shown to fall into two categories viz. high molecular weight group with molecular weight range between 22–26 kDa and low molecular weight group with range less than 18 kDa. High molecular weight TLPs have been shown to get accumulated in cell vacuoles whereas, low molecular weight TLPs are extracellular [59]. PR5 of Gossypium hirsutum (GhPR5) has been shown to contain 242 amino acids with a signal peptide at the N-terminal end that facilitates its secretion into the extracellular space and a signal peptide at C-terminal signal to transport it to vacuoles [60]. In our study, PR5 proteins were found to be of high molecular weight with 26 kDa for A. thaliana and 29 kDa for O. sativa. Though being of high molecular weight PR5 of O. sativa was predicted to be extracellular by three different tools used in the study. However, one of the tools, Euloc indicated the localization of PR5 protein in the vacuoles of A. thaliana. The extracellular location of PR5 is indicative of presence of signal peptide in the N-terminal end of O. sativa as well as in A. thaliana as predicted by Cello tool. The vacuolar location of PR5 in A. thaliana as predicted by Euloc tool is the indication of presence of signal peptide at C-terminal end of protein. PR9 are peroxidases and provide resistance against pathogens. They are extracellular or transmembrane proteins playing an important role in plant cell wall construction by catalyzing lignification [61]. Earlier the subcellular localization of peroxidases from sweet potato (swpa4) using fluorescence microscopy was evaluated, and observed the expression of swpa4 in the extracellular space of cell [62]. PR10 are localized in the cytoplasm and are non-transmembrane proteins getting induced during several biotic and abiotic stresses [63]. In our study, we also found localization of PR10 in the cytoplasm of A. thaliana and O. sativa. Defensins are small and globular proteins belong to PR12 type of PR proteins present in extracellular spaces of plant cells. They are known to provide first line of immunity against pathogen attack [64, 65]. All the three tools used in the present study revealed the extracellular localization of PR12 of both A. thaliana and O. sativa.

For the topological analysis, 8 different tools were used (Table 3). Out of these 8 tools, 3 tools viz TMpred, PHDhtm and TMAP gave similar results with respect to presence or absence of transmembrane domains for all the 6 PR proteins in both the species. Whereas, other tools showed variable results for one or the other PR protein. The tool TMHMM indicated the presence of transmembrane domain in AtPR5 as well as AtPR12 but not in OsPR5 and OsPR12. This might be due to some prediction error of the tool itself [66] or may be the transmembrane domain of OsPR5 and OsPR12 did not meet the cutoff of the tool [67].

Analysis of amino acid composition of all PR proteins of A. thaliana and O. sativa revealed the dominance of amino acids with small aliphatic side chains (alanine, serine, glycine and threonine). Glycine is the smallest amino acid without any side chains and is often found in loop regions. The frequent presence of glycine has been reported in membrane proteins mainly in the transmembrane helices, suggesting its structural role [68, 69]. In the present study, among different PR proteins maximum percentage of glycine was observed for PR1 which has been shown to be transmembrane protein by 6/8 tools in A. thaliana and 4/8 tools in O. sativa. PR10 protein has been localized in cytoplasm by three software’s used in the study. However, it also shows high percentage of glycine amino acid in both plants. Alignment of PR10 of A. thaliana and O. sativa revealed the presence of glycine motif (GXGGXG), which is also known as RNA binding site. The glycine motif has been shown to be involved in different enzymatic processes, membrane binding and transport, biosynthesis of secondary metabolites, binding to phytohormones etc [70]. Side chain of alanine being non-reactive is not directly involved in the function of the protein but has significant role in substrate recognition. Serine and threonine have a fairly reactive hydroxyl group which forms hydrogen bonds with number of polar substrates. Serine forms a catalytic triad along with histidine and aspartic acid (Asp-His-Ser) in many hydrolases. In rare cases, serine is replaced by cysteine in catalytic triad to fulfil same role. Proline has been shown to act like a molecular chaperon which provide protection against abiotic and biotic stresses by enhancing activities of some enzymes as well as maintaining integrity of proteins [71].

Conclusion

PR proteins are defense related inducible proteins associated with resistance to various kinds of biotic and abiotic stresses in plants. In the present study, several bioinformatics tools were used to study the variations in the physicochemical properties and topology of 6 PRs each of A. thaliana and O. sativa. The results of this study demonstrated that PR2 protein of both A. thaliana and O. sativa are the larger proteins with molecular weights of 37KDa and 35KDa, respectively followed by PR9, in both species viz. A. thaliana (35KDa) and O. sativa (42 KDa). Among A. thaliana and O. sativa PRs, maximum AI was observed for AtPR9 (97.17) and OsPR2 (85.96), respectively. For the subcellular localization prediction, we used 3 tools viz. Cello, Euloc and Wolfpsort. All these tools gave similar results for almost all of the PRs except for AtPR5, OsPR1 and OsPR2. This study throws light on the similarities and differences among the physio-chemical properties, topology, amino acid composition and secondary structure features of 6 PRs (PR1, PR2, PR5, PR9, PR10, PR12) of A. thaliana and O. sativa. This study will help in understanding the occurrence of diversification and functional multiplicity of various PR proteins of A. thaliana and O. sativa.

Supporting information

S1 Fig. Comparison of percentage of each amino acid in different PR proteins of A. thaliana and O. sativa.

(TIF)

Click here for additional data file. (512.5KB, tif)

Data Availability

All relevant data are within the manuscript. The study involves in-silico analysis of 6 PRs of Arabidopsis thaliana and Oryza sativa. In the analysis, the data was tabulated from annotations of Uniprot files of each PR and their accession numbers are given in Table 1.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Diego F Gomez-Casati

24 Jun 2020

PONE-D-20-00611

Physico-chemical Characterization and Topological Analysis of Pathogenesis-related Proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches

PLOS ONE

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Reviewer #2: Partly

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Reviewer #1: Literature review was too shallow and it rarely referenced from pools of already published relevant research. This should be rewritten. The analysis was done without adequate statistical packages to bring home the point. The conclusion contains flying general statements without concluding in specific terms on the results. This conclusion should be rewritten. The entire manuscript should be subjected to editing by native speaker of English language

Reviewer #2: I the view of reviewing manuscript “Physico-chemical Characterization and Topological Analysis of Pathogenesis-related Proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches ” has been looked thoroughly and observed following points.

1. Author justify that Arabidopsis thaliana and Oryza sativa are different family member of plant so that why compere these of PR proteins.

2. Compare the physico-chemical properties of 12 PR proteins 6 PRs of Arabidopsis

thaliana and 6 PRs of Oryza sativa .while as table no 1, 2 and 3 mentioned only 6 PRs in both Arabidopsis and Oryza PR1, PR2, PR5, PR9, PR10 , PR12 remaining are Figures 1, 2 and 3 why not all 12 PRs mentioned both table and Figures?

3. In introduction section line no.31 Reactive Oxygen Plants (ROS) : Reactive Oxygen Species (ROS).

4. Line no 40-41. many phytohormones like JA, SA and ET: write the full name of JA, SA and ET first time in manuscript.

5. Line No 55- 60 : include appropriate references in introduction section.

6. In table no 1 all entries of At are bold and Os are bold this demarcation pattern create confusion of readers like bold letter have other scientific meaning but this not view of author only differentiate to At and Os. So that table column may shadow.

7. In table no 1. II value of PR 9 and PR10 not stable disused.

8. In table no 2 . Cello, Euloc and Wolfpsort data are not presenting Subcellular localization of PR proteins predicting uniform result .

9. In table no 3. Comparative topological analysis of PR proteins presence or absence of transmembrane domains homogenious resu;t are showing such as TMHMM of PR12 At (1) and Os (0) why.

10. Conclusion should elaborate . Present conclusion no such pin pointing.

11. 5) Kaur A, Pati PK, Pati AM, Nagpal AK . In-silico analysis of cis-acting regulatory elements of

pathogenesis-related proteins of Arabidopsis thaliana and Oryza sativa. PLoS ONE. 2017; 12(9):

e0184523. https://doi.org/10.1371/journal.pone.0184523. Page no Missing.

12. Hofmann K, Stoffel W. TMbase - A database of membrane spanning proteins segments. Biological Chemistry Hoppe-Seyler. 1993; 374:166. doi: citeulike-article-id:9087200. Vol. missing

13. Shen H, Chou JJ. MemBrain: Improving the Accuracy of Predicting Trans membrane Helices. PLoS

ONE. 2008; 3(6): e2399. doi:10.1371/journal.pone.0002399. Page no Missing.

14. No any such current year 2019 and 2020 are present in this manuscript. May some refrences should be add.

15. All figure are not self explainer specially fig. no 3.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Sep 28;15(9):e0239836. doi: 10.1371/journal.pone.0239836.r002

Author response to Decision Letter 0

14 Aug 2020

Response to Reviewers

Dear Editor,

Thank you for your mail and valuable suggestions for improvement of our manuscript entitled “Physico-chemical Characterization and Topological Analysis of Pathogenesis-related Proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches”. We have studied your comments carefully and made major corrections as suggested. The answers to the questions of reviewers are given below.

Journal Requirements:

1) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: Manuscript has been edited according to PLOS ONE’s style.

2) Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Response: Caption has been added for Supporting Information file at the end of revised manuscript with track changes (lines 560-562).

3) In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available.

Response: The study involves in-silico analysis of 6 PRs of Arabidopsis thaliana and Oryza sativa. In the analysis, the data was tabulated from annotations of Uniprot files of each PR and their accession numbers are given in Table 1.

Reviewer Comments

Reviewer # 1.

1) Query: Literature review was too shallow and it rarely referenced from pools of already published relevant research. This should be rewritten. The analysis was done without adequate statistical packages to bring home the point. The conclusion contains flying general statements without concluding in specific terms on the results. This conclusion should be rewritten. The entire manuscript should be subjected to editing by native speaker of English language.

Response: The manuscript has been strengthened taking into account the suggestions of the reviewer. Whole introductory part and conclusion have been rewritten completely (pages 2-4, lines 31-99; pages 16-17, lines 331-343, respectively) in the revised manuscript with track changes. Manuscript has been checked for language usage, spelling and grammar by Vasavi Garisetti, Former Language editor, Taylor n Francis, Scientific publishing services, Chennai (India) and changes have been marked in manuscript.

Reviewer # 2

1) Query: Authors justify that Arabidopsis thaliana and Oryza sativa are different family member of plant so that why compere these of PR proteins.

Response: Arabidopsis thaliana and Oryza sativa are model plants belonging to two different groups i.e. dicots and monocots, respectively. The aim was to determine similarities and differences in PR proteins of these two groups.

2) Query: Compare the physico-chemical properties of 12 PR proteins 6 PRs of Arabidopsis thaliana and 6 PRs of Oryza sativa .while as table no 1, 2 and 3 mentioned only 6 PRs in both Arabidopsis and Oryza PR1, PR2, PR5, PR9, PR10 , PR12 remaining are Figures 1, 2 and 3 why not all 12 PRs mentioned both table and Figures?

Response: The study relates to 6 PR proteins (PR1, PR2, PR5, PR9, PR10, PR12) each for Arabidopsis thaliana and Oryza sativa. The necessary corrections have been made in the discussion section, in the revised manuscript with track changes (page, 11 and lines 204-205).

3) Query: In introduction section line no.31 Reactive Oxygen Plants (ROS) : Reactive Oxygen Species (ROS).

Response: Correction have been done in the revised manuscript with track changes (line 40).

4) Query: Line no 40-41. many phytohormones like JA, SA and ET: write the full name of JA, SA and ET first time in manuscript.

Response: Full names are already given in the beginning of the introduction section of the revised manuscript with track changes (lines 41-42).

5) Query: Line No 55- 60 : include appropriate references in introduction section.

Response: These lines highlight the objectives of the present work. (Lines 94-99 in the revised manuscript with track changes).

6) Query: In table no 1 all entries of At are bold and Os are bold this demarcation pattern create confusion of readers like bold letter have other scientific meaning but this not view of author only differentiate to At and Os. So that table column may shadow.

Response: As per the suggestion of the reviewer, alternate columns in table 1 have been shaded, in the revised manuscript with track changes (page 8).

7) Query: In table no 1. II value of PR 9 and PR10 not stable disused.

Response: Lines 230-231, indicate that PR9 and PR10 of both species; and PR1 and PR12 of A. thaliana only were found to be less than 40 indicating their stable nature.

8) Query: In table no 2 . Cello, Euloc and Wolfpsort data are not presenting Subcellular localization of PR proteins predicting uniform result.

Response: One of the aims of this study was to compare different online tools for subcellular localization of PR proteins and the results are discussed in the discussion section, in the revised manuscript with track changes (page 13, lines 249-261).

9) Query: In table no 3. Comparative topological analysis of PR proteins presence or absence of transmembrane domains homogeneous result are showing such as TMHMM of PR12 At (1) and Os (0) why.

Response: The tool TMHMM indicated the presence of transmembrane domain in AtPR5 as well as AtPR12 but not in OsPR5 and OsPR12. This might be due to some prediction error of the tool itself [1] or may be the transmembrane domain of OsPR5 and OsPR12 did not meet the cutoff of the tool [2]. The same has been added in discussion section in the revised manuscript with track changes (page 15, lines 304-310).

10) Query: Conclusion should elaborate. Present conclusion no such pin pointing.

Response: Conclusion has been elaborated taking into account the suggestions of the reviewer in the revised manuscript with track changes (page 16-17, lines 331-343).

11) Query: 5) Kaur A, Pati PK, Pati AM, Nagpal AK . In-silico analysis of cis-acting regulatory elements of pathogenesis-related proteins of Arabidopsis thaliana and Oryza sativa. PLoSONE. 2017; 12(9): e0184523. https://doi.org/10.1371/journal.pone.0184523. Page no Missing.

Response: PLOS One uses electronic location identifier instead of page numbers and its already mentioned (e0184523).

12) Query: Hofmann K, Stoffel W. TMbase - A database of membrane spanning proteins segments. Biological Chemistry Hoppe-Seyler. 1993; 374:166. doi: citeulike-article-id:9087200. Vol. missing.

Response: Volume number is already mentioned in the reference, its 374.

13) Query: Shen H, Chou JJ. MemBrain: Improving the Accuracy of Predicting Trans membrane Helices. PLoSONE. 2008; 3(6): e2399. doi:10.1371/journal.pone.0002399. Page no Missing.

Response: PLOS One uses electronic location identifier instead of page numbers and its already mentioned (e2399).

14) Query: No any such current year 2019 and 2020 are present in this manuscript. May some references should be add.

Response: As per suggestion, references for the year 2019 and 2020 have been added in the introduction section, line numbers 37, 62, 66 and 79 and References 1, 2, 13, 17 and 25 have been incorporated in the reference section of the revised manuscript with track changes.

15) Query: All figure are not self explainer specially fig. no 3.

Response: Fig 3 depicts the percentage of secondary structure features (Y axis) of PR proteins (X axis), and different features are shown in different colors as indicated in the figure.

References:

1) Abdullatypov AV. Hup-Type Hydrogenases of Purple Bacteria: Homology Modeling and Computational Assessment of Biotechnological Potential. International journal of molecular sciences. 2020; 21:366. doi: 10.3390/ijms21010366.

2) GENI-ACT; https://doi.org/10.25504/FAIRsharing.mhqkc7; Last accessed: Aug 07, 2020.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (25.1KB, docx)

Decision Letter 1

Diego F Gomez-Casati

15 Sep 2020

Physico-chemical Characterization and Topological Analysis of Pathogenesis-related Proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches

PONE-D-20-00611R1

Dear Dr. Nagpal,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Diego F. Gomez-Casati, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript was properly corrected and the authors took into account the suggestions made by the reviewers.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Author compliances of all my 15 comments and reviewer satisfied with all, specially origin of PR proteins , table formation and rewrite of discussion.

**********

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Reviewer #2: No

Acceptance letter

Diego F Gomez-Casati

17 Sep 2020

PONE-D-20-00611R1

Physico-chemical Characterization and Topological Analysis of Pathogenesis-related Proteins from Arabidopsis thaliana and Oryza sativa using in-silico approaches

Dear Dr. Nagpal:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Diego F. Gomez-Casati

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Comparison of percentage of each amino acid in different PR proteins of A. thaliana and O. sativa.

    (TIF)

    Click here for additional data file. (512.5KB, tif)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (25.1KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript. The study involves in-silico analysis of 6 PRs of Arabidopsis thaliana and Oryza sativa. In the analysis, the data was tabulated from annotations of Uniprot files of each PR and their accession numbers are given in Table 1.

    Articles from PLoS ONE are provided here courtesy of PLOS

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