The Wheat Pathogenesis Related Protein (TdPR1.2) Ensures Contrasting Behaviors to E. coli Transformant Cells under Stress Conditions

The pathogenesis-related proteins 1 (PR-1) gene family play important roles in the plant metabolism in response to biotic and abiotic stresses. The wheat TdPR1.2 has been previously isolated and characterized. Here we showed by bio-informatic analysis that TdPR1.2 contains six cysteine residues that are conserved between all PR-1 proteins tested. Using ScanProsite tool, we found that TdPR1.2 structure has a CRISP family signature 1 and 2 located at the C-terminal part of the protein. Those two domains are conserved in many identified PR1.2 proteins in plants. Moreover, SignalIP-5.0 analysis revealed that TdPR1.2 contains a putative signal peptide formed by 25 amino acids at the N-terminal extremity. The presence of this signal peptide suggested that the mature proteins will be secreted after the cleavage of the signal sequence. Further, we investigate the role of the TdPR1.2 proteins in the growth of Esche-richia coli transformants cells under different abiotic stresses. Our results showed that the full-length form of TdPR1.2 enhanced tolerance of E. coli against salt and osmotic stress but not to KCl. Moreover, TdPR1.2 protein confers bacterial tolerance to heavy metals in solid and liquid mediums. Based on these results, we suggest that the TdPR1.2 protein could play an important role in response to abiotic stress conditions.


Introduction
Plants have developed complex mechanisms to protect themselves against pathogens. Pathogenesis-related (PR) genes are the key elements of these mechanisms, and are activated in response to pathogen attacks. They regulate production of several proteins, peptides or compounds which are toxic to pathogens or prevent pathogen infections where they start [1]. PR proteins have been classified under 17 different families based on their main properties [2]. Having antifungal activities, PR-1 constitutes the main family of the PR proteins induced by pathogens or salicylic acid [3]. The first member of PR-1 family, PR1-a was identified in Nicotiana tabacum plants infected with Tobacco Mosaic Virus (TMV) [4]. Subsequently, several other PR-1 proteins have been identified and characterized in monocot and dicot plant species, such as tomato (Solanum lycopersicum) [5], Triticum aestivum [6], Piper nigrum [7], Grapevine [8], Arabidopsis thaliana, Oryza sativa [9] and cassava (Manihot esculenta) [10]. Their important roles were reported in response to stress conditions [5]- [10]. The PR-1 family belongs to PR proteins (known as antimicrobial peptides, AMPs) which are classified into 17 families based on their protein sequence similarities, enzymatic activities and other biological features [2]. PR-1 proteins are considered mainly secreted and accumulated in the extracellular/apoplastic space facilitated by means of their N-terminal secretion peptide. In contrast, it was shown that some PR-1 were proteins accumulated in the vacuoles of protoplasts in tobacco [1]. Alexander et al. [11] reported that approximately 2% of the total leaf proteins in pathogen-infected tobacco plants were PR-1 proteins produced through defense response. In tomato, the expression of BG124298 (a pathogenesis-related protein-like protein gene) was 5.57-fold upregulated in a resistant genotype while 1.63-fold upregulated in susceptible genotype under Alternaria solani infection [12]. In another study, three distinct alkaline 14-kD proteins, P14a, P14b and P14c related to the PR-1 family, showed antifungal activity against Phytophthora infestans both in vitro and in vivo conditions [13]. PR-1 genes also play important roles in response to abiotic stresses. In rice, stress-associated proteins 1 (OsSAP1) induce endogenous stress-related genes such as aminotransferase (OsAMTR1), SCP/TAPS or pathogenesis-related 1 class of protein (OsSCP) [14]. Besides, tomatoes present 13 SlPR-1 genes which were all up-regulated upon drought stress treatment [5]. Liu et al. [15] showed that a transcription regulator, Di19 (Droughtinduced) gene induced up-regulation of pathogenesis-related PR-1, PR-2 and PR-5 genes expressions in Arabidopsis. Seo et al. [16] reported that plasma membrane-tethered NAC (NAM/ATAF1/2/CUC2) transcription factor NTL6 induced some PR genes by directly binding to promoter sequences of cold-responsive PR genes, PR-1, PR-2 and PR-5. In Arabidopsis thaliana, NPR1 [16]. In wheat, TaPR-1-1 expression was induced by freezing, salinity and osmotic stresses, and the overexpression of TaPR-1-1 granted tolerance to them in yeast and Arabidopsis [19]. In tomato, pathogenesis-related protein 1b1 (PR-1b1) abundance increased and remained stable till day 15 post warming in both the 556HO and 579HO transgenic tomato lines exposed to chilling temperature [20]. Over-expression of PR-1a gene in tobacco ameliorates its response to the pathogens Phytophthora parasitica var. nicotianae and Peronospora tabacina [11]. In tomato, it has been demonstrated that PR-1 strongly inhibited Phytophthora infestans's zoospores germination and the development of symptoms [13]. In pepper, CABPR1 proteins were strongly expressed after ethephon treatment, and with a lesser extent after wounding and infection by the bacterial pathogen Xanthomonas campestris pv.
Vesicatoria [21]. CABPR1 proteins also ameliorate plant tolerance to heavy metal (CdCl 2 or HgCl 2 ) and pathogen (Pseudomonas syringae pv. Tabaci; Phytophthora nicotianae and R. solanacearum) stresses [22]. Durum wheat, Triticum turgidum L. ssp durum is a tetraploid cereal (2n = 4x = 28 chromosomes; AABB), used essentially for the production of pasta and other semolina [23] that was considered as a prominent crop for 2000 years ago [24] and considered as one of the 10 most important crops all over the world [25]. Like other crops, wheat could be subjected to different abiotic and biotic stresses such as osmotic stresses which are differentially perceived by different plant organs (spike, root and shoot) [26]. For example, application of heavy metal stress CuSO 4 at two different levels (5 mM and 25 mM) causes a reduction of fresh weight, dry matter and length. Durum wheat response to such stresses includes accumulation of soluble sugar and soluble proteins and nitrate reductase [26]. We have recently isolated and characterized a novel PR1.2 gene (TdPR1.2) from durum wheat [27]. TdPR1.2 gene was up-regulated in roots, stems and leaves after plant treatment with Salicylic Acid (SA). Besides, TdPR1.2 showed an antibacterial activity against some Gram + and Gram − bacteria and against the fungi Septoria tritici. Moreover, we provide experimental evidence that TdPR1.2 binds to calmodulins (CaMs) in Calcium dependent manner and that this binding stimulates the antimicrobial effect of TdPR1.2 in presence of both Ca 2+ and Mn 2+ cations [27]. In the present study, we show that TdPR1.2 protein represents a putative SCP_PR-1_like domain that belongs to SCP superfamily, conserved in other PR1 homologues. Besides, TdPR1.2 protein is much closed to monocotyledonous and dicotyledonous PR1 proteins isolated from many species indicating evolutionary conservation of PR1 genes among species. Further, we demonstrate that TdPR1.2 confers bacterial tolerance to salt and osmotic stress and not for KCl in E. coli transformant cells. Moreover, TdPR1.2 protein confers bacterial tolerance to heavy metals in solid and liquid mediums. As far as we know, this

TdPR1.2 Protein in silico Analysis
The 3D structure of TdPR1.2 protein was performed via the Swiss-Model server [28]. The presence of transmembrane structures was revealed by PSIPRED da-

TdPR1.2 Protein Expression
In order to construct the expression vector pET28a-TdPR1.2, specific primers with restriction enzyme sites (EcoRI) were designed as follows in order to ensure an in-frame cloning sense, PR1_Ec1_Fw (5'-GAATTCATGGCATCTTCCAAGAGT-3') and PR1_Ec1_Rev (5'-GAATTCGGCTTCGGCGTCAAG-3'). The amplified products were cloned into the pET28a vector at the EcoRI site to express the pET28a-TdPR1.2 fusion protein, which has a poly-Histidine tag at the N-terminus [27]. The recombinant protein His_TdPR1.2 was produced in the Rosetta E. coli strain (DE3) (Novagen) after cloning of the corresponding ORF in-frame with a poly histidine tag into the pET28a expression vectors (Novagen, Madison).

Assays for Abiotic Stress Tolerance of E. coli Transformant Cells
The pET28a vector and pET28a-TdPR1.2 plasmid were transformed into E. coli BL21 (

Statistical Analysis
Each experiment was carried out in at least three replicates. Student's t was performed to determine significant differences between the means of bacterial growth in unstressed and stressed mediums. The percentage presented in the following figures was calculated by the data of survival of bacteria transformed by empty pET-28a or pET-28a_TdPR1.2 grown in LB medium supplemented or not with the mentioned stress. The results were compared statistically by bacteria grown in unstressed medium and differences were considered significant at p < 0.01. Mean values that were significantly different at p < 0.01 from each other are marked with asterisks (*).

In silico Sequence Analysis of TdPR1.2 Protein
The full-length cDNA sequence of TdPR1.  (Figure 1(a)). Besides, TdPR1.  To gain insights into the occurrence of signatures, the TdPR1.2 protein was analyzed by ScanProsite tool. This analysis showed that TdPR1.2, present two well conserved CRISP domains (CRISP family signature 1 and 2 (Figure 2(a)).
Those domains are also conserved in some other studied proteins (Figure 2(b)).
In fact, the first domain is well conserved in monocotyledonous and dicotyledonous plants investigated whereas the second domain is less conserved between monocyledonous and dicotyledonous (Figure 2

Recombinants TdPR1.2 Proteins Confers Contrasting Bacterial Tolerance to Ionic and Osmotic Stress
The biological role of TdPR1.2 in abiotic stress tolerance has been studied in vivo. For this, we used heterologous expression in E. coli cells. The full-length cDNA was cloned in the pET28a expression vector transformed into E. coli (BL21 strain) as previously described [27].

TdPR1.2 Have Dual Role in Response to Heavy Metal Stress of E. coli Cells
The growth of E. coli cells containing pET28a-TdPR1.2 vectors or the empty

Discussion
Abiotic stress is one of the major threats to the modern agriculture that causes not only enormous yield losses [31] [32] [33] [34], but also provides the entry points to various microbial pathogens. Subsequently, the global climate change is another threat to crop system because of increased emergence of more virulent and broad host range pathogenic strains. Therefore, studying the molecular mechanisms of plant resistance or tolerance to either biotic and abiotic stresses or multiple stresses will provide novel opportunities to develop multiple stress tolerance crops [31] [32] [33] [34] [35]. Pathogenesis-related proteins are induced by multiple stresses and seem to be important candidates for generating multiple stress tolerant crop varieties [31]- [37]. Abiotic stress mediated expression of PR  of PR-1 type proteins from vertebrates has been described as cysteine-rich secretory proteins (CRISPs) [32].  [21] demonstrated that TaPR-1-1 expression was also induced by freezing, salinity, and osmotic stresses. Overexpression in yeast and Arabidopsis showed that TaPR-1-1 conferred tolerance to these stresses. Hence at least some PR-1 proteins have function in biotic and/or abiotic stress tolerance. Although expression profiles of some PR-1 members were tested under pathogen attack, expression profiles of PR-1 members under abiotic stress are still not adequately studied.
Some evidence indicates that PR proteins, such as PR-2 (glucanase), PR-3 (chitinase) and PR-5 (thaumatin-like protein), inhibit microbial growth through enzymatic activity [36] [37]. PR-1 proteins that contain a CAP-derived (cysteine-rich secretory protein, antigen 5, and pathogenesis-related-1) peptide 1 have been shown to confer stress tolerance [38]. However, the actual mechanism of PR-1 in abiotic stress tolerance and whether PR-1 takes part in tolerance to both abiotic and biotic stress through the same pathway remains to be addressed.

Conclusion and Future Prospects
Although it was suggested that all proteins of the PR protein superfamily could, on the basis of present knowledge of the molecular structure, have similar functions, the most convincing homology is observed with the proteins from yeast, To the best of our knowledge, this is the first report of a PR1 gene from wheat to describe its functional accreditation in imparting defense against abiotic stress in vitro. Further characterization of TdPR1.2 and its regulation under ambient and stress environments in planta will enhance our understanding of the molecular cross-talk among various signaling pathways mediating plant defense responses.