Differential Multi-cellularity Is Required for the Adaptation for Bacillus licheniformis to Withstand Heavy Metals Toxicity.

Bacillus licheniformis is a multi-metal tolerant bacteria, isolated from the paddy rhizospheric soil sample. Upon the multiple metal toxicity, B. licheniformis altered their phenotypic/morphogenesis. Here we examined the effects of cadmium (Cd2+), chromium (Cr2+), and mercury (Hg2+) on the morphogenesis of B. licheniformis in comparison to control. We found that the ability of bacteria to grow effectively in presence of cadmium and chromium comes at a cost of acquiring cell density-driven mobility and reformation of filamentous to donut shape respectively. In particular, when bacteria grown on mercury it showed the bacteriostatic strategy to resist mercury. Furthermore, the findings suggest a large variation in the production of exo-polysaccharides (EPS) and suggest the possible role of EPS in gaining resistance to cadmium and chromium. Together this study identifies previously unknown characteristics of B. licheniformis to participate in bioremediation and provides the first evidence on positive effects of bacterial morphogenesis and the involvement of EPS in bacteria to resisting metal toxicity.

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor ß-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction.

Identification of Biomarkers for Resistance to Fusarium oxysporum f. sp. cubense Infection and in Silico Studies in Musa paradisiaca Cultivar Puttabale through Proteomic Approach.

Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the major disease constraints of banana production. Previously, we reported the disease resistance Musa paradisiaca cv. puttabale clones developed from Ethylmethanesulfonate and Foc culture filtrate against Foc inoculation. Here, the same resistant clones and susceptible clones were used for the study of protein accumulation against Foc inoculation by two-dimensional gel electrophoresis (2-DE), their expression pattern and an in silico approach. The present investigation revealed mass-spectrometry identified 16 proteins that were over accumulated and 5 proteins that were under accumulated as compared to the control. The polyphosphoinositide binding protein ssh2p (PBPssh2p) and Indoleacetic acid-induced-like (IAA) protein showed significant up-regulation and down-regulation. The docking of the pathogenesis-related protein (PR) with the fungal protein endopolygalacturonase (PG) exemplify the three ionic interactions and seven hydrophobic residues that tends to good interaction at the active site of PG with free energy of assembly dissociation (1.5 kcal/mol). The protein-ligand docking of the Peptide methionine sulfoxide reductase chloroplastic-like protein (PMSRc) with the ligand ß-1,3 glucan showed minimum binding energy (-6.48 kcal/mol) and docking energy (-8.2 kcal/mol) with an interaction of nine amino-acid residues. These explorations accelerate the research in designing the host pathogen interaction studies for the better management of diseases.

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

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 ß-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.

Immuno-affinity purification of PglPGIP1, a polygalacturonase-inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen.

Polygalacturonase-inhibitor proteins (PGIPs) are important plant defense proteins which modulate the activity of microbial polygalacturonases (PGs) leading to elicitor accumulation. Very few studies have been carried out towards understanding the role of PGIPs in monocot host defense. Hence, present study was taken up to characterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native glycosylated PGIP (PglPGIP1) of ~43 kDa and pI 5.9 was immunopurified from pearl millet. Comparative inhibition studies involving PglPGIP1 and its non-glycosylated form (rPglPGIP1; recombinant pearl millet PGIP produced in Escherichia coli) against two PGs, PG-II isoform from Aspergillus niger (AnPGII) and PG-III isoform from Fusarium moniliforme, showed both PGIPs to inhibit only AnPGII. The protein glycosylation was found to impact only the pH and temperature stability of PGIP, with the native form showing relatively higher stability to pH and temperature changes. Temporal accumulation of both PglPGIP1 protein (western blot and ELISA) and transcripts (real time PCR) in resistant and susceptible pearl millet cultivars showed significant Sclerospora graminicola-induced accumulation only in the incompatible interaction. Further, confocal PGIP immunolocalization results showed a very intense immuno-decoration with highest fluorescent intensities observed at the outer epidermal layer and vascular bundles in resistant cultivar only. This is the first native PGIP isolated from millets and the results indicate a role for PglPGIP1 in host defense. This could further be exploited in devising pearl millet cultivars with better pathogen resistance.

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

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.

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. ß-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.

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.