Anti-microbial and cytotoxic activity of ZzAMP, a serine protease inhibitor (SPI) with nutraceutical potential from rhizomes of medicinal plant, Zingiber zerumbet.

Serine protease Inhibitors (SPIs) abundantly reported in plant storage organs constitute important candidate molecules for antimicrobial and anticancer therapeutics. Our earlier studies had identified antimicrobial protein/or peptides (AMP) from Zingiber zerumbet rhizomes designated ZzAMP inhibiting serine protease (SP) of necrotrophic phytopathogen, Pythium myriotylum. Considering the high ethno-medicinal applications of Z. zerumbet rhizomes, present study evaluated the anti-bacterial, anti-oxidant and cytotoxic properties of ZzAMP. Though ZzAMP displayed low radical scavenging activity (IC50 1000 µg/ml), it exhibited considerable anti-bacterial activity towards the nosocomial pathogen Klebsiella pneumonia (93%), which produced maximal extracellular protease (30.6 ± 1.47 U/ml) amongst the pathogens screened. Evaluation of cytotoxic activity of ZzAMP revealed decrease in viability of cancer cell line, HeLa (IC50 115.09 µg/ml) compared to normal cells, L929 (IC50 299.95 µg/ml). Present experiments showing antimicrobial and cytotoxic activities of ZzAMP with minimal damage to normal cells are indicative of its potential as a promising nutraceutical protein.

Polyphenol oxidase (PPO) arm of catecholamine pathway catalyzes the conversion of L-tyrosine to L-DOPA in Mucuna pruriens (L.) DC var. pruriens: An integrated pathway analysis using field grown and in vitro callus cultures.

Mucuna pruriens (L.) DC var. pruriens is the natural source for L-DOPA, precursor of the neurotransmitter dopamine, used widely in the treatment of Parkinson's disease. However, L-DOPA synthesis in plants is mediated either by Catecholamine (CA) pathway or alternate pathway catalyzed by Cytochrome P450 (CYP450) class of enzymes. Interestingly, the CA pathway itself can be initiated either by tyrosine hydroxylase (TH) or polyphenol oxidase (PPO). The CA pathway mediated synthesis of L-DOPA has not yet been proved in M. pruriens albeit strong indications. Therefore, the present investigation is focused on metabolite analysis of major intermediates of CA pathway up to the formation of dopamine and expression analysis of the selected genes, in different tissues and callus cultures. The four major intermediates, L-tyrosine, tyramine, L-DOPA and dopamine, were detected using NMR spectroscopy and quantified by HPLC in the callus cultures and in different tissues of the field plant, respectively. The various stages of leaf tissue were also analyzed for metabolite profiling. The relative amount of intermediates detected during the ontogeny of leaf indicates that PPO mediated conversion of L-tyrosine to dopamine through L-DOPA is relatively higher compared to dopamine production from tyramine. Among the two possible enzymes, activity of PPO was 6.5-fold more than TH in metabolically active young leaves compared to intermediate leaves. The gene expression profiles comprising upstream genes of L-tyrosine synthesis and downstream up to dopamine synthesis shows strong correlation with L-DOPA synthesis. The study validates CA pathway mediated synthesis of L-DOPA with PPO as candidate enzyme, in M. pruriens.

Evaluation of DNA Methylation Changes by CRED-RA Analysis Following Prednisone Treatment of Endophyte, Fusarium oxysporum.

Endophytes that represent a sub-set of plant resident microbes are a reservoir of bioactive metabolites. Many of the secondary metabolite biosynthetic gene clusters of endophytes are silent under axenic culture conditions. Epigenetic reprogramming of such cryptic pathways is possible by use of small molecule modulators like prednisone. Methylation changes induced by prednisone, a hypomethylating epigenetic modulator were studied in endophytic Fusarium oxysporum. CRED-RA analysis following exposure to non-cytotoxic dose (300 µM) revealed prednisone as effective in inducing non-methylation and semi-methylation pattern while inhibiting full-methylation of the genome. Effectiveness of prednisone as a DNA methyl transferase inhibitor can be explored in future to study alterations in secondary metabolite gene expression profile in endophytic F. oxysporum.

Sequence and Bioinformatic Analysis of Family 1 Glycoside Hydrolase (GH) 1 Gene from the Oomycete Pythium myriotylum Drechsler.

The oomycetous phytopathogen Pythium myriotylum secretes cellulases for growth/nutrition of the necrotroph. Cellulases are multi-enzyme system classified into different glycoside hydrolase (GH) families. The present study deals with identification and characterization of GH gene sequence from P. myriotylum by a PCR strategy using consensus primers. Cloning of the full-length gene sequence using genome walker strategy resulted in identification of 1230-bp P. myriotylum GH gene sequence, designated as PmGH1. Analysis revealed that PmGH1 encodes a predicted cytoplasmic 421 amino acid protein with an apparent molecular weight of 46.77 kDa and a theoretical pI of 8.11. Tertiary structure of the deduced amino acid sequence showed typical (α/ß)8 barrel folding of family 1 GHs. Sequence characterization of PmGH1 identified the conserved active site residues, viz., Glu 181 and Glu 399, that function as acid-base catalyst and catalytically active nucleophile, respectively. Binding sites for N-acetyl-D-glucosamine (NAG) were revealed in the PmGH1 3D structure with Glu181 and Glu399 positioned on either side to form a catalytic pair. Phylogenetic analysis indicated a closer affiliation of PmGH1 with sequences of GH1 family. Results presented are first attempts providing novel insights into the evolutionary and functional perspectives of the identified P. myriotylum GH.

Purification and biochemical characterization of an extracellular endoglucanase from the necrotrophic oomycete, Pythium myriotylum Dreschler.

An extracellular endoglucanase (EG) that catalyzes the hydrolysis of carboxy-methyl cellulose (CMC) as substrate was purified to homogeneity from the soft-rot causing oomycete P. myriotylum with maximum EG production observed in presence of 1% (w/v) sucrose. The enzyme designated PmEG was observed to be monomeric with a molecular weight of 78 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal activity of PmEG was determined at pH 5.0 and 25 °C with stability observed at pH extending over acidic to alkaline ranges viz., 3.0-10.0 and thermal stability upto 75 °C for 1 h. Optimal PmEG activity was obtained by addition of metal ions viz., Ca(2+) , Fe(3+) , Zn(2+) , Cu(2+) , Al(3+) , and also in presence of DTT and ß-mercaptoethanol while it was inhibited by Cr(2+) . Various organic solvents, surfactants, and the oxidant, H2 O2 had little/no effect on PmEG activity reflecting its robustness and potential commercial significance. Kinetic constants of PmEG, Km and Vmax were determined as 1.1 mM and 407 µmol min(-1) mg(-1) protein, respectively. Glucose was observed to cause mixed non-competitive inhibition of PmEG.

Metabolic profiling of Zingiber zerumbet following Pythium myriotylum infection: investigations on the defensive role of the principal secondary metabolite, zerumbone.

Induced biosynthesis of bioactive secondary metabolites constitutes one of the mechanisms of plant basal innate immunity to fungal infection. Metabolic changes were studied in rhizomes of Zingiber zerumbet, a wild congener of ginger, after infection with soft rot-causative necrotrophic phytopathogen, Pythium myriotylum, by gas chromatography-mass spectrometry (GC-MS) analysis. Infection triggered a considerable alteration in the relative content of zerumbone and α-caryophyllene (humulene) with enhancement in zerumbone content (81.59%) and that of α-caryophyllene (11.91%) compared to 9.97 and 1.11%, respectively, in uninfected rhizomes. While zerumbone is the principal secondary metabolite in Z. zerumbet, α-caryophyllene is its immediate precursor. Principal component analysis (PCA) identified the correlations between metabolite changes in Z. zerumbet rhizomes and P. myriotylum infection. Radial diffusion assay with zerumbone indicated a concentration-dependent P. myriotylum growth inhibition with 93.75% inhibition observed at 700 µg and 50% maximal effective concentration (EC50) value of 206 µg. Scanning electron microscopy (SEM) analysis revealed that the mechanistic basis of zerumbone's antagonistic action on P. myriotylum growth involved the induction of aberrant morphology including severe hyphal deformities and membrane disruption. Results are discussed highlighting the critical role played by sesquiterpenoid zerumbone in affording resistance in Z. zerumbet and could expedite the development of appropriate strategies for biocontrol of Pythium spp., thus reducing the usage of broad-spectrum fungicides.

Characterization of major hydrolytic enzymes secreted by Pythium myriotylum, causative agent for soft rot disease.

Pythium myriotylum, an oomycetous necrotroph is the causal agent of soft rot disease affecting several crops. Successful colonization by necrotrophs depends on their secretion of a diverse array of plant cell wall degrading enzymes (CWDEs). The induction dynamics of CWDEs secreted by P. myriotylum was analysed as little information is available for this pathogen. Activities of CWDEs that included pectinase, cellulase, xylanase and protease were detected using radial diffusion assay and differential staining. In Czapek Dox minimal medium supplemented with respective substrates as carbon source, the increase in CWDE activities was observed till 8 days of incubation after which a gradual decline in enzymatic activities was observed. With sucrose as sole carbon source, all the enzymes studied showed increase in activity with fungal growth while with cell wall material derived from ginger rhizome as sole carbon source, an initial spurt in cellulase, xylanase and pectinase activities was observed 3 days post incubation while protease activity increased from three days of incubation and reached maximum at 13 days of incubation. To further evaluate the role of CWDEs in pathogenicity, UV-induced mutants (pmN14uv1) were generated wherein significant reduction in cellulase, pectinase and protease activities were observed while that of xylanase remained unchanged compared to wild type isolate (RGCBN14). Bioassays indicated changes in infection potential of pmN14uv1 thereby suggesting the crucial role played by P. myriotylum CWDEs in initiating the rotting process. Hence appropriate strategies that target the production/activity of these secretory hydrolytic enzymes will help in reducing disease incidence/pathogen virulence.

Molecular characterization of ZzR1 resistance gene from Zingiber zerumbet with potential for imparting Pythium aphanidermatum resistance in ginger.

Soft rot disease caused by the oomycete Pythium aphanidermatum (Edson) Fitzp. is the most economically significant disease of ginger (Zingiber officinale Rosc.) in tropical countries. All available ginger cultivars are susceptible to this pathogen. However a wild ginger relative viz., Zingiber zerumbet L. Smith, was identified as a potential soft rot resistance donor. In the present study, a putative resistance (R) gene designated, ZzR1 was isolated and characterized from Z. zerumbet using sequence information from Zingiber RGCs identified in our earlier experiments. Analysis of the 2280 bp segment revealed a 2157 bp open reading frame (ORF) encoding a putative cytoplasmically localized protein. The deduced ZzR1 protein shared high homology with other known R-genes belonging to the CC-NBS-LRR (coiled coil-nucleotide binding site-leucine rich repeat) class and had a calculated molecular weight of 84.61kDa. Real-time PCR analysis of ZzR1 transcription in Z. zerumbet following pathogen infection demonstrated activation at 3 hpi thus suggesting an involvement of ZzR1 in Z. zerumbet defense mechanism. Although many R-genes have been characterized from different taxa, none of them will help in the development of resistant ginger cultivars owing to the phenomenon of "Restricted Taxonomic Functionality" (RTF). Thus ZzR1 gene characterized from the resistant wild Zingiber accession represents a valuable genomic resource for ginger improvement programs. This first report on R-gene isolation from the Zingiber secondary gene pool is pivotal in designing strategies for engineering resistance in ginger, which is otherwise not amenable to conventional improvement programs owing to sexual reproduction barriers.