Kukkundoor Ramachandra Kini

Production of Unusual Dispiro Metabolites in Pestalotiopsis virgatula Endophyte Cultures: HPLC-SPE-NMR, Electronic Circular Dichroism, and Time-Dependent Density-Functional Computation Study

The endophytic fungus Pestalotiopsis virgatula, derived from the plant Terminalia chebula and previously found to produce a large excess of a single metabolite when grown in the minimal M1D medium, was induced to produce a variety of unusual metabolites by growing in potato dextrose broth medium. Analysis of the fermentation medium extract was performed using an HPLC-PDA-MS-SPE-NMR hyphenated system, which led to the identification of a total of eight metabolites (1-8), six of which are new. Most of the metabolites are structurally related and are derivatives of benzo[c]oxepin, rare among natural products. This includes dispiro derivatives 7 and 8 (pestalospiranes A and B), having a novel 1,9,11,18-tetraoxadispiro[6.2.6.2]octadecane skeleton. Relative and absolute configurations of the latter were determined by a combination of NOESY spectroscopy and electronic circular dichroism spectroscopy supported by time-dependent density-functional theory calculations (B3LYP/TZVP level). This work demonstrates that a largely complete structure elucidation of numerous metabolites present in a raw fermentation medium extract can be performed by the HPLC-SPE-NMR technique using only a small amount of the extract, even with unstable metabolites that are difficult to isolate by traditional methods.

HPLC-SPE-NMR identification of a novel metabolite containing the benzo[c]oxepin skeleton from the endophytic fungus Pestalotiopsis virgatula culture.

HPLC-SPE-NMR analysis of a crude extract of fermentation broth of cultured PESTALOTIOPSIS VIRGATULA isolate TC-320 from TERMINALIA CHEBULA Retz. (Combretaceae) disclosed the presence of a simple but unprecedented low-molecular-weight metabolite, 9-hydroxybenzo[ C]oxepin-3[1 H]-one, subsequently isolated by a targeted purification procedure.

Proteomic analysis of elicitation of downy mildew disease resistance in pearl millet by seed priming with β-aminobutyric acid and Pseudomonas fluorescens.

UNLABELLED Downy mildew is one of the severe diseases of pearl millet, globally affecting its commercial production. Priming of seeds of a susceptible cultivar of pearl millet with β-aminobutyric acid (BABA) and Pseudomonas fluorescens has reduced the downy mildew disease incidence level under field studies. In the current study, proteomic approach was used to elucidate the poorly studied resistance mechanism in these elicitor primed pearl millet seeds in response to Sclerospora graminicola infection. 2DE-MS/MS based proteomic approach revealed that majority of the 63 differentially accumulated (p≤0.05) proteins associated with energy and metabolism followed by stress and defense category. Multivariate statistics disclosed that infection caused by the pathogen rather than elicitor treatment had a major influence on the dynamics of protein abundance. Mechanism of priming mediated by BABA and P. fluorescens were different from each other as evident by the protein abundance profile of hierarchical clustering analysis. Over-representation of proteins pertaining to glucose metabolism suggests that seed priming ensures plant protection against disease without compromising its normal growth and development. In addition the study forms a basis for future investigation by functional analysis of these differentially accumulated proteins to further unravel the resistance mechanism of elicitor primed plant against the S. graminicola. BIOLOGICAL SIGNIFICANCE The study is based on the comparative proteomic analysis between BABA and P. fluorescens mediated resistance in pearl millet, in response to downy mildew causing biotroph - S. graminicola. To our knowledge, this article is the first to report on seedling proteome of pearl millet whose genome is not yet sequenced. In addition, the study also provides clue for the plausible antagonistic cross-talk that might exist between jasmonic acid signaling and salicylic acid signaling in SAR and ISR mediated resistance by BABA and P. fluorescens against the downy mildew pathogen. Furthermore, pearl millet seedling proteome being perturbed by pathogen inoculation was more apparent than that caused by elicitor treatment, as revealed by multivariate statistics like PCA. Analysis by gene enrichment tools further revealed that the glucose metabolism pathway was majorly being affected in our study. This could be attributed to the essential balance that is being maintained in energy diversion towards stress and normal physiological process due to the priming effect of the elicitors against biotic stress.

Involvement of mitogen-activated protein kinase signalling in pearl millet–downy mildew interaction

Mitogen-activated protein kinase (MAPK) cascade-mediated signalling is essential in the establishment of resistance towards pathogens. The present study compared MAPK activities in a compatible and incompatible interaction between pearl millet [Pennisetum glaucum (L.) R. Br.] and downy mildew pathogen Sclerospora graminicola. Differential expression was observed with rapid and increased activation of MAPKs, PgMPK1 (48kDa) and PgMPK2 (44kDa), in the incompatible interaction; with a weak activity of only PgMPK1 in the compatible interaction. Immunoblot analysis showed PgMPK1 and PgMPK2 to be orthologs of salicylic acid-induced protein kinase and wound-induced protein kinase, respectively. Immunocytochemical analysis revealed pathogen-induced accumulation and nuclear localisation of PgMPKs only in the incompatible interaction with highest signals in the vascular tissues. Maximum PgMPKs activation correlated with the activation of several defence-related enzymes. In addition, inhibition of MAPK-activation by kinase cascade inhibitors correlated with the suppression of defence-related enzyme activities and pathogen-induced H2O2 accumulation. Treatment of pearl millet seedlings with abiotic and biotic elicitors led to a strong early induction of only PgMPK1. β-Amino butyric acid and H2O2 were found to be best activators of PgMPK1. These results suggest that in pearl millet MAPK signalling is involved in mediating several defence mechanisms in response to pathogen infection.

Antioxidant and hepatoprotective effects of Solanum xanthocarpum leaf extracts against CCl4-induced liver injury in rats

Abstract Context: Solanum xanthocarpum Schard. and Wendl. (Solanaceae) has been used in traditional Indian medicines for its antioxidant, anti-inflammatory, and antiasthmatic properties. Objective: The present study demonstrates the antioxidant and hepatoprotective effects of S. xanthocarpum. On the basis of in vitro antioxidant properties, the active fraction from column chromatography of the methanol extract of S. xanthocarpum leaves (SXAF) was chosen as the potent fraction and used for hepatoprotective studies in rats. Materials and methods: The antioxidant activity was evaluated by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and reducing power assays. Rats were pre-treated with 100 and 200 mg/kg b.w. of SXAF for 14 d with a single dose of CCl4 in the last day. Hepatoprotective properties were determined by serum biochemical enzymes, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), antioxidant enzymes (SOD, CAT, GSH, and GST), and histopathology studies. Results: SXAF exhibited significant antioxidant activity in scavenging free radicals with IC50 values of 11.72 µg (DPPH) and 17.99 µg (ABTS). Rats pre-treated with SXAF demonstrated significantly reduced levels of serum LDH (1.7-fold), ALP (1.6-fold), and AST (1.8-fold). Similarly, multiple dose SXAF administration at 200 mg/kg b.w. demonstrated significantly enhanced levels of SOD (1.78 ± 0.13), CAT (34.63 ± 1.98), GST (231.64 ± 14.28), and GSH (8.23 ± 0.48) in liver homogenates. Histopathological examination showed lowered liver damage in SXAF-treated groups. Discussion and conclusion: These results demonstrate that SXAF possesses potent antioxidant properties as well as hepatoprotective effects against CCl4-induced hepatotoxicity.

Inhibition of TMV multiplication by siRNA constructs against TOM1 and TOM3 genes of Capsicum annuum

The host proteins TOM1 and TOM3 associated with tonoplast membrane are shown to be required for efficient multiplication of Tobamoviruses. In this study, homologous of TOM1 and TOM3 genes were identified in pepper (Capsicum annuum) using specific primers. Their gene sequences have similarity to Nicotiana tabacum NtTOM1 and NtTOM3. Sequence alignment showed that CaTOM1 and CaTOM3 are closely related to TOM1 and TOM3 of N. tabacum and Solanum lycopersicum with 90% and 70% nucleotide sequence identities, respectively. RNA interference approach was used to suppress the TOM1 and TOM3 gene expression which in turn prevented Tobacco mosaic virus replication in tobacco. Nicotiana plants agro-infiltrated with siRNA constructs of TOM1 or TOM3 showed no mosaic or necrotic infection symptoms upon inoculation with TMV. The results indicated that silencing of TOM1 and TOM3 of pepper using the siRNA constructs is an efficient method for generating TMV-resistant plants.

Changes in peroxidase activity in sunflower during infection by necrotrophic pathogen Alternaria helianthi

Abstract Detailed analysis of threshold levels of resistance in sunflower genotypes to Alternaria helianthi and its relation to peroxidase activity was carried out. Fourteen sunflower genotypes were categorized into resistant, moderately resistant, susceptible and highly susceptible groups based on field reaction on disease rating scale of 0 – 9. Sunflower genotypes MSH-59 and PF-56 were resistant with disease reaction scale of 3, PC-63, PC-64, RHA6D-1 and G-47 were moderately resistant with disease reaction scale of 5, CMS-859, PC-52, AC-48, R-265, CMS-338 and MR-1 were susceptible with a disease reaction scale of 7, Morden and CMS-62 belonged to a highly susceptible category with a disease reaction scale of 8. Time-course study of peroxidase (PO) activity was analysed in resistant (MSH-59) and susceptible (Morden) sunflower genotypes using spectrophotometry. High PO activity was recorded at 2 and 12 h post-inoculation with A. helianthi. At 2 h post-inoculation, resistant genotypes recorded 12.8 units while the susceptible genotype had 2.4 units of PO activity. At 12 h PO activity increased to 39.2 and 4.2 units in resistant and highly susceptible genotypes respectively. Treatment with antioxidant inhibitors sodium azide and sodium metabisulphite at various molar concentrations reduced the pathogen-induced as well as constitutive levels of PO activity. Pre-treatment of resistant sunflower genotypes with inhibitor followed by pathogen inoculation increased the disease reaction when compared to control. Higher PO activities were recorded in sunflower genotypes with high threshold levels of resistance and lesser in susceptible genotype. The study therefore gives strong evidence for the role of PO as an important enzyme of the central defense system against necrotrophic pathogen A. helianthi and could be used as a reliable biomarker for assessing resistance.

Genetic diversity and antimicrobial activity of endophytic Myrothecium spp. isolated from Calophyllum apetalum and Garcinia morella.

Calophyllumapetalum and Garciniamorella, medicinal plants are endemic to Western Ghats, Karnataka, India. Sixteen Myrothecium isolates were obtained from the tissues of bark and twigs of these plants. The purpose of this study was to explore the antimicrobial activity and genetic variability of the endophytic Myrothecium isolates. The antimicrobial activity as well as the genetic diversity of endophytic Myrothecium species was investigated through RAPD, ISSR and ITS sequence analysis. Myrothecium isolates were genotypically compared by RAPD and ISSR techniques, 510 and 189 reproducible polymorphic bands were obtained using 20 RAPD and ten ISSR primers respectively. The isolates grouped into four main clades and subgroups using unweighted pair group method with arithmetic mean cluster analysis. rDNA ITS sequence analysis presented better resolution for characterising the isolates of Myrothecium spp. The clustering patterns of the isolates were almost similar when compared with RAPD and ISSR dendograms. The results signify that RAPD, ISSR and ITS analysis can be employed to distinguish the genetic diversity of the Myrothecium species. The endophytic and pathogenic strains were compared by maximum parsimony, maximum likelihood and neighbour joining methods. One isolate (JX862206) amongst the 16 Myrothecium isolates exhibited potent antibacterial and as well as anti-Candida activity.

Differential expression of sunflower peroxidase isoforms and transcripts during necrotrophic interaction with Alternaria helianthi

Sunflower leaf spot caused by the necrotrophic pathogen Alternaria helianthi is one of the most important biotic constraints in sunflower production. The biochemical basis of resistance to infection by A. helianthi in sunflower is poorly elucidated. Differential activation of early defense responses like increased peroxidase activity predisposes the host plant resistance. In the present study, the active role of peroxidase in resistant and susceptible interactions of sunflower with the leaf spot pathogen was demonstrated using native PAGE analysis and Northern blotting. Differential time-dependent induction of peroxidase isoforms was evident in resistant and susceptible genotypes after pathogen inoculation. In the resistant interaction, three peroxidase (PO) isoforms (PO1, PO2, and PO3) were found to be expressed at 2 and 12 h after inoculation, but in susceptible interaction, PO2 was present at 24 and 48 h and PO3 was found only at 24 h after inoculation. However, in the susceptible genotype, expression intensity was lower than in the resistant interaction at all the time intervals. Molecular expression profiling by Northern blotting using heterologous cDNA probes showed rapid up-regulation of peroxidase at 2 and 12 h in resistant interaction. These results demonstrate that peroxidase isoforms are involved in defense against A. helianthi, and genetic regulation in the form of transcript accumulation is important for expression of disease resistance.

Inhibition of virus infection by transient expression of short hairpin RNA targeting the methyltransferase domain of Tobacco mosaic virus replicase.

One of the most efficient mechanisms by which plants protect themselves from viruses is the specific RNA-dependent silencing pathway termed post-transcriptional gene silencing (PTGS). Inhibition of Tobacco mosaicvirus in tobacco was demonstrated through transient expression of virus-derived short hairpin RNA encoding the methyltransferase domain of TMV replicase corresponding to the nucleotides 630 to 1,510 introduced through agro-infiltration into the leaves of tobacco plants. RT-PCR revealed that TMV infection was absent in hpRNA agro-infiltrated leaves while control leaves showed amplification of TMV. Protein analysis further confirmed the absence of TMV coat protein in hpRNA-containing leaves while it was prominent in the control leaves. Our data demonstrate that expression of hpRNA is an effective and predictable new approach to engineering resistance to TMV.