Approach towards sustainable leather: Characterization and effective industrial application of proteases from Bacillus sps. for ecofriendly dehairing of leather hide.

Tanneries are one of the most polluted industries known for production of massive amount of solid and liquid wastes without proper management and disposal. In this project we demonstrated the ecofriendly single step dehairing of leather hides with minimum pollution load. In this study, Bacillus species (Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P) capable of producing proteases was successfully isolated by employing the new optimized selective media named M9-PEA as confirmed by 16sRNA genes sequencing. Sequence of 1493 bp long 16S rRNA genes of Bacillus paralicheniformis strain BL.HK and Bacillus cereus strain BS. P was submitted to GenBank under the accession number OP612692.1, OP612721.1 respectively The Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P produced extracellur proteases of 28 and 37 KDa as resolved by SDS-PAGE respectively. The enzymes showed temperature optima at 50 °C and 55 °C and pH optima at 8.5, 9.5 respectively. The Proteases of Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P were employed for dehairing of animal hides. The process resulted in significant removal of interfibriller substances without damage to collagen layer after one hour treatment, which was confirmed by histology, scanning electron microscopy. The quantification of various skin constituents (collagen, uronic acid, hexosamines, and GAGs) and pollution load parameters revealed that enzymatic treatment are more reliable. The results of skin application trials at industrial level with complete elimination of chemicals remark the biotechnological potential of these proteases for ecofriendly dehairing of animal hides without affecting the quality of the leathers produced.

Mentha piperita silver nanoparticle-loaded hydrocolloid film for enhanced diabetic wound healing in rats.

OBJECTIVE: To investigate the role of Mentha piperita silver nanoparticle-loaded carbopol gel for enhanced wound healing in a diabetic rat model. This research further aims to explore bioactive compounds derived from Mentha piperita obtained from high altitude. METHOD: Methanolic extracts of Mentha piperita (MP), Mentha spicata (MS) and Mentha longifolia (ML) were used to synthesise silver nanoparticles (AgNP). AgNP synthesis was confirmed by ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antioxidant activity was assessed by 2, 2-diphenyl-1-picrylhydrazyl (DDPH) assay. Antiglycation potential was determined by measuring the fluorescent advanced glycation end products. The bioactive compound identified in the Mentha piperita methanolic (MPM) fraction through electrospray ionisation tandem mass spectrometric analysis (ESI-MS) was responsible for the highest antiglycation. The effects of MPM and MPM.AgNP-loaded Carbopol (Sanare Lab, India) on wound healing were compared in male, alloxan-induced, diabetic albino rats (200-250g), divided into control and treated groups. Effects on wound healing were assessed via histopathology. RESULTS: UV-Vis and FTIR confirmed NP synthesis with peaks for flavonoids and polyphenols. SEM and XRD explored the cubical, 30-63nm crystalline NP. The maximum antioxidant and antiglycation potential was observed in order of; MP.AgNP>MS.AgNP>ML.AgNP. The highest antioxidant activity was observed by methanolic and aqueous MP.AgNPs (88.55% and 83.63%, respectively) at 2mg.ml-1, and (75.16% and 69.73%, respectively) at 1mg.ml-1, compared to ascorbic acid (acting as a positive control, 90.01%). MPM.AgNPs demonstrated the best antiglycation potential of 75.2% and 83.3% at 1mg.ml-1 and 2mg.ml-1, respectively, comparable to positive control (rutin: 88.1%) at 14 days post-incubation. A similar trend was observed for antimicrobial activity against Bacillus subtilis, Micrococcus luteus and Escherichia coli with an inhibition zone of 21mm, 21.6mm and 24.6mm. Rosmarinic acid was the active compound present in Mentha piperita, as identified by ESI-MS. MPM.AgNP-loaded Carbopol resulted in 100% wound closure compared with control at 20 days post-wounding. In the treatment group, re-epithelialisation was achieved by day 18, compared with 25 days for the positive control group. CONCLUSION: MPM.AgNP-loaded Carbopol demonstrated safer and more effective biological properties, hence accelerating the diabetic excision wound healing process in alloxan-induced diabetic rats.

Real-time expression and in silico characterization of pea genes involved in salt and water-deficit stress.

BACKGROUND: To tolerate salt and water-deficit stress, the plant adapts to the adverse environment by regulating its metabolism and expressing certain stress-induced metabolic pathways. This research analyzed the relative expression of four pea genes (P5CR, PAL1, SOD, and POX) in three pea varieties (Climax, Green grass, and Meteor) under different levels of salt and water-deficit stress. METHODS AND RESULTS: The experiments on salt stress and water-deficit stress were carried out within greenhouse settings under controlled environment. The saturation percentage was employed to create artificial salinity conditions: Control without NaCl treatment, Treatment 1: 50 mM NaCl treatment, Treatment 2: 75 mM NaCl treatment, and Treatment 3: 100 mM NaCl treatment. Field capacity (FC) was used for the development of artificial water-deficit treatments in the pots, i.e., Treatment 1 (Control; water application 100% of FC), Treatment 2 (water application 75% of FC), and Treatment 3 (water application 50% of FC). Pea genes involved in biosynthetic pathways of proline, flavonoids, and enzymatic antioxidant enzymes including P5CR, PAL1, SOD, and POX were selected based on literature. Quantitative real-time PCR using cDNA as a template was used to analyze the gene expression. Pea genes were analyzed for phylogenetic analysis in closely related crops having similarity percent identity 80% and above. In silico characterization of selected proteins including the family classification was done by the NCBI CDD and INTERPRO online servers. Results from RT-qPCR analysis showed increased expression of P5CR, PAL1, and POX genes, while SOD gene expression decreased under both stresses. Climax exhibited superior stress tolerance with elevated expression of P5CR and PAL1, while Meteor showed better tolerance through increased POX expression. Phylogenetic analysis revealed common ancestry with other species like chickpea, red clover, mung bean, and barrel clover, suggesting the cross relationship among these plant species. Conserved domain analysis of respective proteins revealed that these proteins contain PLNO 2688, PLN02457, Cu-Zn Superoxide dismutase, and secretory peroxidase conserved domains. Furthermore, protein family classification indicated that the oxidation-reduction process is the most common chemical process involved in these stresses given to pea plant which validates the relationship of these proteins. CONCLUSIONS: Salt and water-deficit stresses trigger distinct metabolic pathways, leading to the up-regulation of specific genes and the synthesis of corresponding proteins. These findings further emphasize the conservation of stress-tolerance-related genes and proteins across various plant species. This knowledge enhances our understanding of plant adaptation to stress and offers opportunities for developing strategies to improve stress resilience in crops, thereby addressing global food security challenges.

GC-MS metabolomics profile of methanol extract of Acacia modesta gum and gum-assisted fabrication and characterization of gold nanoparticles through green synthesis approach.

Hereunder, for the first time, we reported phytocompounds in the methanolic extract of Acacia modesta (AM) gum through Gas chromatography-mass spectrometry (GS-MS). Further, the AM gum aqueous solution was used for gold nanoparticles (AuNPs) synthesis through a simple, swift, eco-friendly, and less costly green synthesis approach. A total of 108 phytocompounds (63 with nonpolar, 45 with polar column) were identified in the gum extract, which includes fatty acids, alcohols, sterols, aldehyde/ketones, furans, aromatic compounds, esters, phenols, terpenes, sugar derivatives, alkaloids, and flavones. From three used concentrations (5, 10, and 15 mg/mL) of the AM gum aqueous solution, the 15 mg/mL gum solution resulted in more successful AuNP synthesis with a smaller size, which was visualized by a rusty red color appearance. UV-Visible absorption spectroscopy revealed the characteristic surface plasmon resonance (SPR) of AuNPs in aqueous solution at 540 nm. Dynamic light scattering (DLS) measurement of NPs solution revealed a hydrodynamic diameter of 162 ± 02 nm with the highest gum concentration where core AuNPs diameter was 22 ± 03 nm, recorded by Transmission electron microscopy. Zeta potential revealed fair stability of AuNPs that was not decreased with time. Catalytic activity experiments revealed that AM gum-based AuNPs can increase the rate of the reduction of methylene blue 10 times in comparison with AM gum extract alone. Results from this study showed that a diverse array of phytocompounds in AM gum can successfully reduce gold ions into gold nanoparticles, which can be used further in different pharmaceutical and industrial applications.

Production of Sitobion avenae-resistant Triticum aestivum cvs using laccase as RNAi target and its systemic movement in wheat post dsRNA spray.

Among the wheat biotic stresses, Sitobion avenae is one of the main factors devastating the wheat yield per hectare. The study's objective was to find out the laccase (lac) efficacy; as a potential RNAi target against grain aphids. The Sitobion avenae lac (Salac) was confirmed by Reverse Transcriptase-PCR. Gene was sequenced and accession number "ON703252" was allotted by GenBank. ERNAi tool was used to design 143 siRNA and one dsRNA target. 69% mortality and 61% reduction in lac expression were observed 8D-post lac DsRNA feeding. Phylogenetic analysis displayed the homology of grain aphid lac gene with peach potato, pea, and Russian wheat aphids. While Salac protein was found similar to the Russian grain, soybean, pea, and cedar bark aphid lac protein multi-copper oxidase. The dsRNAlac spray-induced silencing shows systematic translocation from leaf to root; with maximum lac expression found in the root, followed by stem and leaf 9-13D post-spray; comparison to control. RNAi-GG provides the Golden Gate cloning strategy with a single restriction ligation reaction used to achieve lac silencing. Agrobacterium tumefaciens mediated in planta and in-vitro transformation was used in the study. In vitro transformation, Galaxy 2012 yielded a maximum transformation efficiency (1.5%), followed by Anaj 2017 (0.8%), and Punjab (0.2%). In planta transformation provides better transformation efficiencies with a maximum in Galaxy 2012 (16%), and a minimum for Punjab (5%). Maximum transformation efficiency was achieved for all cultivars with 250 µM acetosyringone and 3h co-cultivation. Galaxy 2012 exhibited maximum transformation efficiency, and aphid mortality post-feeding transgenic wheat.

Environmental impact and diversity of protease-producing bacteria in areas of leather tannery effluents of Sialkot, Pakistan.

Massive discharge of wastes produced by the processing of leather so far confers the most important environmental challenge facing the tanneries worldwide. Waste material from tanneries mostly consists of skin remnants and proteinaceous substances as by-products of leather processing. In these conditions, protease-producing bacteria play a vital role in degrading wastes in this sludge. Therefore, an investigation was made to study the effect of long-term tannery sludge contamination on the diversity of both protease-producing microbes and of bacterial extracellular proteases near tanneries of Sambrial and Sialkot. The high amount of carbon and nitrogen in the soil samples reflected their effect on the diversity of the microbial communities in these areas. Phylogenetic analysis based on 16S rRNA gene sequences suggest that the isolated proteolytic bacteria belonged to 9 different genera including Pseudomonas (26.19%), Proteus (19.04%), Serratia (16.66%), Klebsiella (14.28%), Providencia (9.52%), Achromobacter (7.14%), Enterobacter (2.38%), Myroides (2.38%), and Acinetobacter (2.38%). Enzyme activity showed that among all Pseudomonas and Proteus showed relatively high protease production, and inhibition studies revealed that proteases produced by all isolates were strongly inhibited by serine and/or metalloprotease inhibitors, and a smaller proportion was inhibited by inhibitors of cysteine and/or aspartic proteases. Furthermore, isolated bacteria revealed promising degradation activities against casein and/or gelatin with only a few that could hydrolyze elastin, suggesting proteases produced by these isolated bacteria belong to different classes of proteases, i.e., serine and metalloproteases. This study provided new insights on the community structure of cultivable protease-producing bacteria near tannery sludge of Sambrial and Sialkot. This study would be beneficial not only for establishing the way for effective degradation of tannery slugs but also for questing the novel properties of proteases for a future technological application.

A novel avian isolate of hepatitis E virus from Pakistan.

BACKGROUND: Avian hepatitis E virus (aHEV) has been associated with hepatitis-splenomegaly syndrome (HSS) in chickens along with asymptomatic subclinical infection in many cases. So far, four genotypes have been described, which cause infection in chickens, specifically in broiler breeders and layer chickens. In the present study, we isolated and identified two novel aHEV strains from the bile of layer chickens in Pakistan evincing clinical symptoms related to HSS. METHODOLOGY: Histology of liver and spleen tissues was carried out to observe histopathological changes in these tissues. Bile fluid and fecal suspensions were used for viral RNA isolation through MegNA pure and Trizol method which was further used for viral genome detection and characterization by cDNA synthesis and amplification of partial open reading frame (ORF) 1, ORF2 and complete ORF3. The bioinformatics tools; Molecular Evolutionary Genetics Analysis version 6.0 (MEGA 6), Mfold and ProtScale were used for phylogenic analysis, RNA secondary structure prediction and protein hydropathy analysis, respectively. RESULTS: Sequencing and phylogenetic analysis on the basis of partial methyltranferase (MeT), helicase (Hel) domain, ORF2 and complete ORF3 sequence suggests these Pakistani aHEV (Pak aHEV) isolates may belong to a Pakistani specific clade. The overall sequence similarity between the Pak aHEV sequences was 98-100%. The ORF1/ORF3 intergenic region contains a conserved cis-reactive element (CRE) and stem-loop structure (SLS). Analysis of the amino acid sequence of ORF3 indicated two hydrophobic domains (HD) and single conserved proline-rich domain (PRD) PREPSAPP (PXXPXXPP) with a single PSAP motif found in C-terminal. Amino acid changes S15 T, A31T, Q35H and G46D unique to the Pak aHEV sequences were found in the N-terminal region of ORF3. CONCLUSIONS: Our data suggests that Pak aHEV isolates may represent a novel Pakistani clade and high sequence homology to each other support the supposition they may belong to a monophyletic clade circulating in the region around Pakistan. The data presented in this study provide further information for aHEV genetic diversity, genotype mapping, global distribution and epidemiology.

Characterization of recombinant endo-1,4-ß-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach.

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-ß-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant Km of 1.32 mg ml-1 birchwoodxylan (soluble form) and maximum reaction velocity (Vmax) 73.53 mmol min-1 mg-1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

Biosynthesis of silver nanoparticles from leaf extract of Litchi chinensis and its dynamic biological impact on microbial cells and human cancer cell lines.

Green synthesis of metallic nanoparticles has attracted a great deal of attention from scientific community due to its biocompatibility and environment friendly nature. In the present study, silver nanoparticles were biologically synthesized using leave extracts of Litchi chinensis. Biosynthesized silver nanoparticles were characterized and their applications were observed by different methodologies. Bio-reduction reaction was confirmed by the surface plasmon resonance of silver nanoparticles at 417 nm through UV-VIS spectrophotometer. FTIR analysis revealed that the amine groups present in the leaf extracts were responsible for the reduction of silver ions to silver nanoparticles. X-ray diffraction analysis was used to determine the crystalline nature of silver nanoparticles and their diameter was noted in the range of 41-55 nm by scanning electron microscopy. Antibacterial activity was observed against gram positive and gram negative strains of bacteria. Furthermore, human epithelial type 2 cancer cells (HEp-2) and Human breast adenocarcinoma cells lines (MCF-7) were treated with the biosynthesized silver nanoparticles using MTT assay. The resulting cell death rate was noted up to 40.91+1.99%. This study concludes that plant mediated biosynthesis of nanoparticles is the superior alternative compared to chemical and physical approaches, to utilize them as drug delivery tool and need to conjugate apoptosis inducing biological agents with silver nanoparticles to suppress the uncontrolled division of cancer cells.

Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Differential proteome analysis of diabetes mellitus type 2 and its pathophysiological complications.

The prevalence of Diabetes Mellitus Type 2 (DM 2) is increasing every passing year due to some global changes in lifestyles of people. The exact underlying mechanisms of the progression of this disease are not yet known. However recent advances in the combined omics more particularly in proteomics and genomics have opened a gateway towards the understanding of predetermined genetic factors, progression, complications and treatment of this disease. Here we shall review the recent advances in proteomics that have led to an early and better diagnostic approaches in controlling DM 2 more importantly the comparison of structural and functional protein biomarkers that are modified in the diseased state. By applying these advanced and promising proteomic strategies with bioinformatics applications and bio-statistical tools the prevalence of DM 2 and its associated disorders i-e nephropathy and retinopathy are expected to be controlled.

Dermatomycoses: challenges and human immune responses.

The most prevalent skin infections are mainly caused by species of dermatophytes of the genera Trichophyton, Microsporum, and Epidermophyton that infect keratinized tissues and stratum corneum of skin and hair. Besides proteases with putative role of kinases and other enzymes, immune modulators are abundantly secreted during infection as well. The molecular mechanism used by the dermatophytes to infect and counteract the host immune response is not well understood. The defense against infections basically depends on the host's immune responses to metabolites of the fungi, virulence of the infecting strain or species and anatomical site of the infection. The two aspects of the immune system, the immediate hypersensitivity and delayed-type hypersensitivity against dermatophytes may be crucial to the progression and severity of skin infection. Management of the infection through species identification and molecular diagnostic techniques as well as use of novel targeted drugs in addition to conventional anti-fungal compounds is of great importance in dealing with disease onsets and outbreaks. Here we reviewed the fungal skin infections elucidating their biologic and immunologic characteristics. Reaction to fungal invasion by the infected epithelial tissue on the host side is also discussed. Moreover, determinants of protective immunity and treatment options are focused that could confer long-lasting resistance to infection.

Plant-bacterium interactions analyzed by proteomics.

The evolution of the plant immune response has resulted in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as pathogen associated molecular pattern (PAMP) triggered immunity and has evolved to recognize common features of microbial pathogens. In response to the delivery of pathogen effector proteins, plants acquired R proteins to fight against pathogen attack. R-dependent defense response is important in understanding the biochemical and cellular mechanisms and underlying these interactions will enable molecular and transgenic approaches for crops with increased biotic resistance. Proteomic analyses are particularly useful for understanding the mechanisms of host plant against the pathogen attack. Recent advances in the field of proteome analyses have initiated a new research area, i.e., the analysis of more complex microbial communities and their interaction with plant. Such areas hold great potential to elucidate, not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa, symbiotic, pathogenic bacteria, and commensal bacteria. During biotic stress, plant hormonal signaling pathways prioritizes defense over other cellular functions. Some plant pathogens take advantage of hormone dependent regulatory system by mimicking hormones that interfere with host immune responses to promote virulence (vir). In this review, it is discussed the cross talk that plays important role in response to pathogens attack with different infection strategies using proteomic approaches.

Application of proteomics to investigate stress-induced proteins for improvement in crop protection.

Proteomics has contributed to defining the specific functions of genes and proteins involved in plant-pathogen interactions. Proteomic studies have led to the identification of many pathogenicity and defense-related genes and proteins expressed during phytopathogen infections, resulting in the collection of an enormous amount of data. However, the molecular basis of plant-pathogen interactions remains an intensely active area of investigation. In this review, the role of differential analysis of proteins expressed during fungal, bacterial, and viral infection is discussed, as well as the role of JA and SA in the production of stress related proteins. Resistance acquired upon induction of stress related proteins in intact plant leaves is mediated by potentiation of pathogens via signal elicitors. Stress related genes extensively used in biotechnology had been cited. Stress related proteins identified must be followed through for studying the molecular mechanism for plant defense against pathogens.

Determination of proteins induced in response to jasmonic acid and salicylic acid in resistant and susceptible cultivars of tomato.

Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and down-regulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

Analyses of the proteomes of the leaf, hypocotyl, and root of young soybean seedlings.

The functions of organs in young soybean seedling were determined by means of proteomic analysis. Extracts from leaves, hypocotyls, and roots were separated by two-dimensional polyacrylamide gel electrophoresis, and the proteins were identified by mass spectrometry and protein sequencing. The identified proteins were categorized into various groups according to their function. The leaf was abundant in proteins associated with energy production (50.0%), the hypocotyl was rich in defense proteins (31.8%), and the root contained defense-related proteins (16.7%) and destination and storage proteins (26.7%). Stem 31-kDa glycoprotein, 20 kDa chaperonin, 50S ribosomal protein, and trypsin inhibitor were common to all three tissues. The sequence information obtained from the soybean proteome should be helpful in predicting the functions of unknown proteins.

Comparative proteomic analysis of bacterial wilt susceptible and resistant tomato cultivars.

To investigate the molecular mechanisms of bacterial resistance in susceptible and resistant cultivars of tomato, a proteomic approach was adopted. Four cultivars of tomato were selected on the basis of their response to bacterial (Pseudomonas solanacearum) inoculation wherein cultivar Roma and Riogarande, and cultivar Pusa Ruby and Pant Bahr were considered as resistant and susceptible cultivars, respectively. Proteins were extracted from leaves of 3-week-old seedlings of the four cultivars and separated by 2-DE. A total of nine proteins were found to be differentially expressed between the susceptible and resistant cultivars. Amino acid sequences of these proteins were determined with a protein sequencer. The identified proteins belongs to the categories of energy, protein destination and storage, and defense. Of these proteins, a 60kDa chaperonin and an apical membrane antigen were significantly upregulated in resistant cultivars compared with susceptible cultivars. Application of jasmonic acid and salicylic acid resulted in significant changes in levels of apical membrane antigen and protein disulfide-isomerase. Taken together, these results suggest that apical membrane antigen might be involved in bacterial resistance process through salicylic acid induced defense mechanism signaling in tomato plants.