Immunoinformatics analysis and evaluation of recombinant chimeric triple antigen toxoid (r-HAB) against Staphylococcus aureus toxaemia in mouse model.

Staphylococcus aureus is a serious pathogen unleashing its virulence through several classes of exotoxins such as hemolysins and enterotoxins. In this study, we designed a novel multi-antigen subunit vaccine which can induce innate, humoral and cellular immune responses. Alpha hemolysin, enterotoxins A and B were selected as protective antigens for combining into a triple antigen chimeric protein (HAB). Immunoinformatics analysis predicted HAB protein as a suitable vaccine candidate for inducing both humoral and cellular immune responses. Tertiary structure of the HAB protein was predicted and validated through computational approaches. Docking studies were performed between the HAB protein and mice TLR2 receptor. Furthermore, we constructed and generated recombinant HAB (r-HAB) protein in E. coli and studied its toxicity, immunogenicity and protective efficacy in a mouse model. Triple antigen chimeric protein (r-HAB) was found to be highly immunogenic in mouse as the anti-r-HAB hyperimmune serum was strongly reactive to all three native exotoxins on Western blot. In vitro toxin neutralization assay using anti-r-HAB antibodies demonstrated > 75% neutralization of toxins on RAW 264.7 cell line. Active immunization with r-HAB toxoid gave ~ 83% protection against 2 × lethal dosage of secreted exotoxins. The protection was mediated by induction of strong antibody responses that neutralized the toxins. Passive immunization with anti-r-HAB antibodies gave ~ 50% protection from lethal challenge. In conclusion, in vitro and in vivo testing of r-HAB found the molecule to be nontoxic, highly immunogenic and induced excellent protection towards native toxins in actively immunized and partial protection to passively immunized mice groups. KEY POINTS: • HAB protein was computationally designed to induce humoral and cellular responses. • r-HAB protein was found to be nontoxic, immunogenic and protective in mouse model. • r-HAB conferred protection against lethal challenge in active and passive immunization.

Quercetin mitigates the deoxynivalenol mycotoxin induced apoptosis in SH-SY5Y cells by modulating the oxidative stress mediators.

Deoxynivalenol (DON) is Fusarium mycotoxin that is frequently found in many cereal-based foods, and its ingestion has a deleterious impact on human health. In this investigation, we studied the mechanism of DON-induced neurotoxicity and followed by cytoprotective efficacy of quercetin (QUE) in contradiction of DON-induced neurotoxicity through assessing the oxidative stress and apoptotic demise in the human neuronal model, i.e. SH-SY5Y cells. DON diminished the proliferation of cells in the manner of dose and time-dependent as revealed by cell viability investigations, i.e. MTT and lactate dehydrogenase assays. Additional studies, such as intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential (MMP), DNA damage, cell cycle, and neuronal biomarkers (amino acid decarboxylase, tyrosine hydroxylase, and brain-derived neurotrophic factor) demonstrated that DON induces apoptotic demise in neuronal cells through oxidative stress intermediaries. On another hand, pre-treatment of neuronal cells with 1 mM of quercetin (QUE) showed decent viability upon exposure to 100 µM of DON. In detailed studies demonstrated that QUE (1 mM) pre-treated cells show strong attenuation efficiency against DON-induced ROS generation, LPO, MMP loss, DNA impairment, cell cycle arrest, and down-regulation of neuronal biomarkers. The consequences of the investigation concluded that QUE mitigates the DON-induced stress viz., decreased ROS production and LPO generation, upholding MMP and DNA integrity and regulation of neuronal biomarker gene expression in SH-SY5Y cells.

Tunable Anticancer Activity of Furoylthiourea-Based RuII -Arene Complexes and Their Mechanism of Action.

Fourteen new RuII -arene (p-cymene/benzene) complexes (C1-C14) have been synthesized by varying the N-terminal substituent in the furoylthiourea ligand and satisfactorily characterized by using analytical and spectroscopic techniques. Electrostatic potential maps predicted that the electronic effect of the substituents was mostly localized, with some influence seen on the labile chloride ligands. The structure-activity relationships of the Ru-p-cymene and Ru-benzene complexes showed opposite trends. All the complexes were found to be highly toxic towards IMR-32 cancer cells, with C5 (Ru-p-cymene complex containing C6 H2 (CH3 )3 as N-terminal substituent) and C13 (Ru-benzene complex containing C6 H4 (CF3 ) as N-terminal substituent) showing the highest activity among each set of complexes, and hence they were chosen for further study. These complexes showed different behavior in aqueous solutions, and were also found to catalytically oxidize glutathione. They also promoted cell death by apoptosis and cell cycle arrest. Furthermore, the complexes showed good binding ability with the receptors Pim-1 kinase and vascular endothelial growth factor receptor 2, commonly overexpressed in cancer cells.

Multi-Biofunctional Properties of Phytofabricated Selenium Nanoparticles From Carica papaya Fruit Extract: Antioxidant, Antimicrobial, Antimycotoxin, Anticancer, and Biocompatibility.

The present study focused on phytofabrication of selenium nanoparticles (SeNPs) from Carica papaya extract and exploration of their multi-biofunctional features. Total phenolics and flavonoids of C. papaya fruit extract were determined as 23.30 ± 1.88 mg gallic acid equivalents and 19.21 ± 0.44 mg quercetin equivalents per gram, respectively, which suggested that C. papaya fruit extract could be a competitive reducing and stabilizing agent during phytofabrication of nanoparticles. UV-Vis and FTIR spectroscopy showed the formation of SeNPs from sodium selenite, which could be related to the reducing and stabilizing activities of C. papaya fruit extract. The SeNPs were found to be stable with a Zeta potential of -32 mV. The average hydrodynamic size of SeNPs was found as 159 nm by dynamic light scattering. The SeNPs showed a broader XRD pattern with no sharp Bragg's peaks and found to be amorphous. SEM showed that SeNPs were spherical in shape and EDX pattern showed that SeNPs were made up of Se (71.81%), C (11.41%), and O (14.88%). The HR-TEM picture showed that SeNPs were spherical in morphology and have a size range of 101-137 nm. The SeNPs exhibited potent antioxidant activity and their EC50 values (effective concentration required to inhibit 50% of radicals) were 45.65 ± 2.01 and 43.06 ± 3.80 µg/ml in DPPH and ABTS assays, respectively. The antimicrobial action of SeNPs was found as a broad spectrum and suppressed microbial pathogens in ascending order: fungi > Gram-positive bacteria > Gram-negative bacteria. The SeNPs have been demonstrated to reduce the growth and ochratoxin A (OTA) of mycotoxigenic Aspergillus ochraceus and Penicillium verrucosum at 40 µg/ml in broth culture, which is noteworthy. The SeNPs reduced cancer cell proliferation (RAW 264.7, Caco-2, MCF-7, and IMR-32) more preferentially than normal cells (Vero), found to be highly biocompatible. Lower doses of SeNPs (up to 50 µg/ml) were shown to be less toxic and did not cause death in Danio rerio (zebrafish) embryos, implying that lower doses of SeNPs could be beneficial for biological purposes. The present study concluded that phytofabricated SeNPs have multiple biofunctional properties, including antioxidant, antimicrobial, antimycotoxin, and anticancer activities, as well as high biocompatibility.

Inhibitory effect of C. zeylanicum, C. longa, O. basilicum, Z. officinale, and C. martini essential oils on growth and ochratoxin A content of A. ochraceous and P. verrucosum in maize grains.

In the study, antifungal and ochratoxin A (OTA) production inhibitory activities of essential oils (EOs) of Cinnamomum zeylanicum, Curcuma longa, Ocimum basilicum, Zingiber officinale, and Cymbopogon martini were reported on Aspergillus ochraceus and Penicillium verrucosum. EOs were obtained by hydrodistillation and GC-MS technique was chosen to deduce their chemical profile. Major chemical compounds in EOs of C. zeylanicum, C. longa, O. basilicum, Z. officinale, and C. martini were (E)-cinnamaldehyde (35.81 %), ar-turmerone (46.13 %), eugenol (36.58 %), geranyl proprionate (18.93 %), and geranyl acetate (14.88 %), respectively. The EOs shown potent antioxidant activity by DPPH and ABTS assays. The EOs presented superlative antifungal activity against P. verrucosum related to A. ochraceus. The C. zeylanicum and C. martini EOs shown superlative antifungal activity related to other EOs. The C. zeylanicum and C. martini EOs completely inhibited the growth and OTA production of P. verrucosum and A. ochraceous at 1500 and 2500 µg/g in maize grains, respectively.

Biofabrication of zinc oxide nanoparticles from Melia azedarach and its potential in controlling soybean seed-borne phytopathogenic fungi.

Present study, report the biofabrication of zinc oxide nanoparticles from aqueous leaf extract of Melia azedarach (MaZnO-NPs) through solution combustion method and their novel application in preventing the growth of seed-borne fungal pathogens of soybean (Cladosporium cladosporioides and Fusarium oxysporum). The standard blotter method was employed to isolate fungi and was identified through molecular techniques. The characterization of MaZnO-NPs was carried out by UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FT-IR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) equipped with Energy Dispersive Spectroscopy (EDS) and Transmission Electron Microscopy (TEM). The physicochemical characterization confirmed the particles were of high purity and nano size (30-40 nm) with a hexagonal shape. The synthesized MaZnO-NPs inhibited the growth of C. cladosporioides and F. oxysporum in a dose dependent manner. Biomass, ergosterol, lipid peroxidation, intracellular reactive oxygen species and membrane integrity determination upon MaZnO-NPs treatment offered significant activities there by confirming the mechanism of action against the test pathogens. In conclusion, due to the effectiveness of MaZnO-NPs in controlling the growth of C. cladosporioides and F. oxysporum, the synthesized MaZnO-NPs provides insight towards their potential application in agriculture and food industries.

Application of real sample analysis and biosensing: Synthesis of new naphthyl derived chemosensor for detection of Al3+ ions.

A new chemosensor (NANH) based on naphthyl moiety was synthesized with good selectivity and sensitivity towards Al3+ ions via the inhibition by operating through dual mechanisms like photo-induced electron transfer (PET) and excited-state intramolecular proton transfer (ESIPT). The synthesized NANH was validated by various techniques such as 1H, 13C NMR and mass spectrum. While prominent fluorescent enhancement was observed from the NANH upon binding with Al3+ ions, however, other metal ions have not responded in the emission spectrum. Detection limit and association constant of NANH for Al3+ were calculated as 1.2 × 10-7 M and 4.09 × 104 M-1 by using fluorescence titration method. Binding ratio (1:1) of NANH with Al3+ ions were proved by Job's plot and DFT studies. Furthermore, aluminium in variety of water samples was determined, and NANH could be used for biosensing of Al3+ in living cells.

Single Nucleotide Polymorphisms in Starch Biosynthetic Genes Associated With Increased Resistant Starch Concentration in Rice Mutant.

Resistant Starch (RS), plays a crucial role in human health and nutrition by controlling glucose metabolism. RS or dietary fibre content in rice is low because it goes through a variety of process before it is ready for cooking and consumption. Hence, this study was carried out to develop a rice mutant with increased RS. The rice mutant (γ278) with increased RS was developed by utilizing gamma (γ) rays as a mutagen. Mutant γ278 was characterized for mutations in the starch biosynthetic genes viz., GBSSI, SSI, SSIIa, SSIIIa, SBEIa, and SBEIIb to reveal the functional mutations/variations led to high RS content in rice. A total of 31 sequence variants/mutations in six genes were identified. We report the discovery of three deleterious mutation/variants each in GBSSI, SSIIa, and SSIIIa with the potential to increase RS content in rice. Further, wild × mutant crosses were made to develop an F2 population to study the effect of combination of deleterious mutations. The SNP (GBSSI:ssIIa:ssIIIa) combination responsible for high RS content in F2 population was identified and recorded highest amylose content (AC) (26.18%) and RS (8.68%) content. In conclusion, this marker combination will be highly useful to develop a rice variety with increased RS.

Fusarium oxysporum f. sp. lycopersici causal agent of vascular wilt disease of tomato: Biology to diversity- A review.

Tomato (Lycopersicon esculentum) is one of the widely grown vegetables worldwide. Fusarium oxysporum f. sp. lycopersici (FOL) is the significant contributory pathogen of tomato vascular wilt. The initial symptoms of the disease appear in the lower leaves gradually, trail by wilting of the plants. It has been reported that FOL penetrates the tomato plant, colonizing and leaving the vascular tissue dark brown, and this discoloration extends to the apex, leading to the plants wilting, collapsing and dying. Therefore, it has been widely accepted that wilting caused by this fungus is the result of a combination of various physiological activities, including the accumulation of fungal mycelia in and around xylem, mycotoxin production, inactivation of host defense, and the production of tyloses; however, wilting symptoms are variable. Therefore, the selection of molecular markers may be a more effective means of screening tomato races. Several studies on the detection of FOL have been carried out and have suggested the potency of the technique for diagnosing FOL. This review focuses on biology and variability of FOL, understanding and presenting a holistic picture of the vascular wilt disease of tomato in relation to disease model, biology, virulence. We conclude that genomic and proteomic approachesare greater tools for identification of informative candidates involved in pathogenicity, which can be considered as one of the approaches in managing the disease.

Molecular Mechanism of T-2 Toxin-Induced Cerebral Edema by Aquaporin-4 Blocking and Permeation.

The present study aimed to reveal the molecular mechanism of T-2 toxin-induced cerebral edema by aquaporin-4 (AQP4) blocking and permeation. AQP4 is a class of aquaporin channels that is mainly expressed in the brain, and its structural changes lead to life-threatening complications such as cardio-respiratory arrest, nephritis, and irreversible brain damage. We employed molecular dynamics simulation, text mining, and in vitro and in vivo analysis to study the structural and functional changes induced by the T-2 toxin on AQP4. The action of the toxin leads to disrupted permeation of water and permeation coefficients are found to be affected, from the native (2.49 ± 0.02 × 10-14 cm3/s) to toxin-treated AQP4 (7.68 ± 0.15 × 10-14 cm3/s) channels. Furthermore, the T-2 toxin forms strong electrostatic interactions at the binding site and pushes the key residues (Ala210, Phe77, Arg216, and His201) outward at the selectivity filter. Also, the role of a histidine residue in the AQP4 channel was identified by alchemical transformation and umbrella sampling methods. Alchemical free-energy perturbation energy for H201A ↔ A201H, which was found to be 3.07 ± 0.18 kJ/mol, indicates the structural importance of the histidine residue at 201. In addition, histopathology and expression of AQP4 in the Mus musculus brain tissues show the damaged and altered expression of the protein. Text mining reveals the co-occurrence of genes/proteins associated with the AQP4 expression and T-2 toxin-induced cell apoptosis, which leads to cerebral edema.

Synthesis and Anticancer Activity of [RuCl2(η6-arene)(aroylthiourea)] Complexes-High Activity against the Human Neuroblastoma (IMR-32) Cancer Cell Line.

Eight new organometallic Ru(II)-arene complexes of the type [RuCl2(η6-arene)(η1-S-aroylthiourea)] (arene = p-cymene or benzene) were synthesized in order to evaluate the effect of the arene moiety and the substituent of the aroylthiourea ligand on the cytotoxicity of the complexes. The ligands (L1 and L2) and complexes (1-8) were characterized using analytical and spectroscopic (UV-visible, infrared, 1H NMR, 13C NMR, and mass) methods. The structure of the ligands (L1 and L2) and complexes (1 and 3-6) was obtained from single-crystal X-ray diffraction studies. The cytotoxicity of the complexes was evaluated against four different cancer cell lines: MCF-7 (breast), COLO 205 (colon), A549 (lung), and IMR-32 (neuroblastoma). All the complexes showed good cytotoxicity and the highest was in the IMR-32 cell line, which articulates the specificity of these complexes toward the IMR-32 cancer cell line. The complexes 5, 7, and 8 exhibited remarkable cytotoxicity in the entire cancer cell lines tested, which was comparable with the standard drug, cisplatin. The anticancer mechanism of the complexes 3 and 7 in IMR-32 cells was evaluated by bright-field microscopy, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), DNA damage, and caspase-3 analyses. The cells treated with the complexes showed upregulated caspase-3 compared to the control, and it was found that ROS and MMP were dose-dependent on analysis. Also, bright-field microscopy and 4',6-diamidino-2-phenylindole (DAPI) staining have correspondingly shown cellular membrane blebbing and DNA damage, which were morphological hallmarks of apoptosis. The study concluded that the complexes promoted the oxidative stress-mediated apoptotic death of the cancer cells through the generation of intracellular ROS, depletion of MMP, and damage of the nuclear material.

Biofabrication of Zinc Oxide Nanoparticles With Syzygium aromaticum Flower Buds Extract and Finding Its Novel Application in Controlling the Growth and Mycotoxins of Fusarium graminearum.

Fusarium graminearum is a leading plant pathogen that causes Fusarium head blight, stalk rot, and Gibberella ear rot diseases in cereals and posing the immense threat to the microbiological safety of the food. Herein, we report the green synthesis of zinc oxide nanoparticles from Syzygium aromaticum (SaZnO NPs) flower bud extract by combustion method and investigated their application for controlling of growth and mycotoxins of F. graminearum. Formation of SaZnO NPs was confirmed by spectroscopic methods. The electron microscopic (SEM and TEM) analysis revealed the formation of triangular and hexagonal shaped SaZnO NPs with size range 30-40 nm. The synthesized SaZnO NPs reduced the growth and production of deoxynivalenol and zearalenone of F. graminearum in broth culture. Further analysis revealed that treatment of mycelia with SaZnO NPs resulted in the accumulation of ROS in the dose-dependent manner. Also, SaZnO NPs treatment enhanced lipid peroxidation, depleted ergosterol content, and caused detrimental damage to the membrane integrity of fungi. Moreover, SEM observations revealed that the presence of diverged micro-morphology (wrinkled, rough and shrank surface) in the macroconidia treated with SaZnO NPs. Taken together, SaZnO NPs may find a potential application in agriculture and food industries due to their potent antifungal activity.

Phytofabrication of Selenium Nanoparticles From Emblica officinalis Fruit Extract and Exploring Its Biopotential Applications: Antioxidant, Antimicrobial, and Biocompatibility.

In the present study, phytofabricated selenium nanoparticles (PF-SeNPs) were prepared from aqueous fruit extract of Emblica officinalis in a facile, green, economic, tactic and eco-friendly way. The aqueous fruit extract of E. officinalis was found to be rich with various secondary metabolites including phenolics (59.18 ± 2.91 mg gallic acid equivalents/g), flavonoids (38.50 ± 2.84 mg catechin equivalents/g), and tannins (44.28 ± 3.09 mg tannic acid equivalents/g) and determined that highly appropriate for the biosynthesis of nanoparticles. The facile phytofabrication of PF-SeNPs was confirmed by UV-visible and FTIR spectroscopic analysis. The XRD pattern and Raman spectroscopy showed that synthesized PF-SeNPs were amorphous in nature. The Zeta potential analysis confirmed that PF-SeNPs were negatively charged (-24.4 mV). The DLS analysis revealed that PF-SeNPs were in nano size and less aggregated with poly-dispersity index of less than 0.2. The SEM images depicted that PF-SeNPs were spherical in shape. The EDX analysis revealed that PF-SeNPs were constituted with Se (61.60%), C (29.96%), and O (4.41%). The HR-TEM analysis determined that PF-SeNPs were in nano size with an average diameter of 15-40 nm. The PF-SeNPs have offered fascinating bio-potential applications, such as antioxidant, antimicrobial and biocompatibility. They have also exhibited dose-dependent free radical scavenging activity, and EC50 was determined as 15.67 ± 1.41 and 18.84 ± 1.02 µg/mL for DPPH and ABTS assays, respectively. The PF-SeNPs has also shown the wide range of antimicrobial activity on foodborne pathogens, and it was found to be highly efficient on fungi followed by Gram-positive and Gram-negative bacteria. The biocompatibility of PF-SeNPs was assessed in N2a cells with much higher IC50 value (dose required to inhibit 50% of cell viability) compared to sodium selenite. Also, mitochondrial membrane potential (MMP) and caspase-3 were much less altered on treatment of PF-SeNPs related to sodium selenite. The cytotoxic studies clearly determined that PF-SeNPs was much less toxic and safer related to sodium selenite. Thus, PF-SeNPs could find suitable application as antioxidant and antimicrobial agent in food, biomedical, and pharmaceutical industry.

Isolation and identification of enterotoxigenic Staphylococcus aureus isolates from Indian food samples: evaluation of in-house developed aptamer linked sandwich ELISA (ALISA) method.

Staphylococcus aureus is one of the major food contaminants worldwide, and its enterotoxins are documented as food poisoning and bioterrorism agents. In the present study, an attempt was made to account on the incidences of toxigenic S. aureus and its antibiotic resistance profiles in ready to eat bakery food products from different parts of Southern India (Andhra Pradesh, Karnataka, Kerala, Tamil Nadu, and Telangana). A total of 100 food samples, including milk, cake, cheese and chicken products were assessed for S. aureus and Staphylococcal Enterotoxin B (SEB) by PCR. Among the subjected food samples, a total of 51 isolates belong to genus Staphylococcus and out of that, 34 isolates were S. aureus. Among 34 S. aureus isolates, 14 isolates were found positive for SEB. The PCR results were further co-evaluated with in-house developed aptamer linked immunosorbent assay (ALISA) for the specific and sensitive detection of SEB. The obtained ALISA results were promising and found consistent with PCR analysis. Furthermore, 24%, 47%, 91%, 82%, 59%, and 47% of S. aureus isolates were found resistant to chloramphenicol, methicillin, penicillin, ampicillin, erythromycin, and oxacillin, respectively and concluded as a multidrug resistance (MDR). In conclusion, the present study revealed high presence of toxigenic and MDR resistant S. aureus species among the studied regions of Southern India. The present study cautions the need of stringent food safety regulations in India to control the toxigenic and MDR S. aureus from food sources and to minimize the risks associated with S. aureus.

Assessment of Detoxification Efficacy of Irradiation on Zearalenone Mycotoxin in Various Fruit Juices by Response Surface Methodology and Elucidation of Its in-vitro Toxicity.

Fruits are vital portion of healthy diet owed to rich source of vitamins, minerals, and dietary fibers, which are highly favorable in keeping individual fit. Unfortunately, these days, one-third of fruits were infested with fungi and their toxic metabolites called mycotoxins, which is most annoying and pose significant health risk. Therefore, there is a need to suggest appropriate mitigation strategies to overcome the mycotoxins contamination in fruits. In the present study, detoxification efficiency of irradiation on zearalenone (ZEA) mycotoxin was investigated in distilled water and fruit juices (orange, pineapple, and tomato) applying statistical program response surface methodology (RSM). The independent factors were distinct doses of irradiation and ZEA, and response factor was a percentage of ZEA reduction in content. A central composite design (CCD) consists of 13 experiments were planned applying software program Design expert with distinct doses of irradiation (up to 10 kGy) and ZEA (1-5 µg). The results revealed that independent factors had a positive significant effect on the response factor. The analysis of variance (ANOVA) was followed to fit a proper statistical model and suggested that quadratic model was appropriate. The optimized model concluded that doses of irradiation and ZEA were the determinant factors for detoxification of ZEA in fruit juices. Further, toxicological safety of irradiation mediated detoxified ZEA was assessed in the cell line model by determining the cell viability (MTT and live/dead cell assays), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nuclear damage, and caspase-3 activity. The higher level of live cells and MMP, lower extent of intracellular ROS molecules and caspase-3, and intact nuclear material were noticed in cells treated with irradiation mediated detoxified ZEA related to non-detoxified ZEA. The results confirmed that toxicity of ZEA was decreased with irradiation treatment and detoxification of ZEA by irradiation is safe. The study concluded that irradiation could be a potential post-harvest food processing technique for detoxification of ZEA mycotoxin in fruit juices. However, irradiation of fruit juices with high dose of 10 kGy has minimally altered the quality of fruit juices.

Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation.

Downy mildew of pearl millet caused by the biotrophic oomycete Sclerospora graminicola is the most devastating disease which impairs pearl millet production causing huge yield and monetary losses. Chitosan nanoparticles (CNP) were synthesized from low molecular weight chitosan having higher degree of acetylation was evaluated for their efficacy against downy mildew disease of pearl millet caused by Sclerospora graminicola. Laboratory studies showed that CNP seed treatment significantly enhanced pearl millet seed germination percentage and seedling vigor compared to the control. Seed treatment with CNP induced systemic and durable resistance and showed significant downy mildew protection under greenhouse conditions in comparison to the untreated control. Seed treatment with CNP showed changes in gene expression profiles wherein expression of genes of phenylalanine ammonia lyase, peroxidase, polyphenoloxidase, catalase and superoxide dismutase were highly upregulated. CNP treatment resulted in earlier and higher expression of the pathogenesis related proteins PR1 and PR5. Downy mildew protective effect offered by CNP was found to be modulated by nitric oxide and treatment with CNP along with NO inhibitors cPTIO completely abolished the gene expression of defense enzymes and PR proteins. Further, comparative analysis of CNP with Chitosan revealed that the very small dosage of CNP performed at par with recommended dose of Chitosan for downy mildew management.