Institutional Publications

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    Soybean cultivar resistant to mungbean yellow mosaic India virus infection induces viral RNA degradation earlier than the susceptible cultivar
    (Elsevier B.V., 2009) Yadav, Rajiv Kumar; Shukla, Rakesh Kumar; Chattopadhyay, Debasis
    Yellow mosaic disease caused by whitefly-transmitted bipartite Geminiviruses is one of the major con- straints on productivity of a number of pulse crops. We have cloned the bipartite genome of Mungbean Yellow Mosaic India Virus isolated from infected Soybean. We report here that agroinfection of Soybean seedlings with a single uncut recombinant binary plasmid containing tandem dimers of both DNA A and DNA B resulted in 100% infectivity in susceptible varieties. To understand the mechanism of natural resistance in a Soybean variety, we compared the abundance of the viral RNAs in a resistant and a susceptible variety at the early time points after agroinfection. Whilst the resistant variety displayed synthesis but rapid degradation of the early viral RNAs; the degradation in the susceptible variety was delayed resulting in accumulation of those transcripts later in infection. Accumulation of the late viral transcripts and DNA replication were detectable only in the susceptible variety. This indicates that rapid degradation of the early viral transcripts, possibly through siRNA mechanism, is one of the probable mechanisms of natural resistance against geminivirus.
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    Evidence for inhibitory interaction of hyaluronan binding protein 1 with S. pneumoniae hyaluronidase
    (The American Society for Biochemistry and Molecular Biology, Inc., 2009) Yadav, Gitanjali; Prasad, Ramachandra L. A.; Jha, Babal Kant; Rai, Vivek; Bhakuni, Vinod; Datta, Kasturi
    Bacterial hyaluronan lyase enzymes are the major virulence factors that enable greater microbial ingress by cleaving hyaluronan (HA) polymers present predominantly in extracellular space of vertebrates. Based on the premise that effective inhibitors may bind to and stabilize HA thereby protecting it from degradation, here we investigated inhibitory activity of human hyaluronan-binding protein 1 (HABP1) on bacterial hyaluronidase because it is highly specific to HA and localized on the cell surface. Biochemical characterization revealed that HABP1 is a competitive inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of 22 uM. This is thus the first report of an endogenous protein inhibitor that may be used dur- ing natural antibacterial defense. Our findings also support a novel multipronged mechanism for the high efficacy of HABP1- mediated inhibition based on structural modeling of enzyme, substrate, and inhibitor. Evidence from docking simulations and contact interface interactions showed that the inherent charge asymmetry of HABP1 plays a key role in the inhibitory activity. This novel role of HABP1 may pave the way for peptide inhibitors as alternatives to synthetic chemicals in antibacterial research.
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    Towards prediction of metabolic products of polyketide synthases: An in silico analysis
    (PLOS, 2009) Yadav, Gitanjali; Gokhale, Rajesh S.; Mohanty, Debasisa
    Sequence data arising from an increasing number of partial and complete genome projects is revealing the presence of the polyketide synthase (PKS) family of genes not only in microbes and fungi but also in plants and other eukaryotes. PKSs are huge multifunctional megasynthases that use a variety of biosynthetic paradigms to generate enormously diverse arrays of polyketide products that posses several pharmaceutically important properties. The remarkable conservation of these gene clusters across organisms offers abundant scope for obtaining novel insights into PKS biosynthetic code by computational analysis. We have carried out a comprehensive in silico analysis of modular and iterative gene clusters to test whether chemical structures of the secondary metabolites can be predicted from PKS protein sequences. Here, we report the success of our method and demonstrate the feasibility of deciphering the putative metabolic products of uncharacterized PKS clusters found in newly sequenced genomes. Profile Hidden Markov Model analysis has revealed distinct sequence features that can distinguish modular PKS proteins from their iterative counterparts. For iterative PKS proteins, structural models of iterative ketosynthase (KS) domains have revealed novel correlations between the size of the polyketide products and volume of the active site pocket. Furthermore, we have identified key residues in the substrate binding pocket that control the number of chain extensions in iterative PKSs. For modular PKS proteins, we describe for the first time an automated method based on crucial intermolecular contacts that can distinguish the correct biosynthetic order of substrate channeling from a large number of non-cognate combinatorial possibilities. Taken together, our in silico analysis provides valuable clues for formulating rules for predicting polyketide products of iterative as well as modular PKS clusters. These results have promising potential for discovery of novel natural products by genome mining and rational design of novel natural products.
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    Role of CIPK6 in root growth and auxin transport
    (Landes Bioscience, 2009) Tripathi, Vineeta; Syed, Nazia; Laxmi, Ashverya; Chattopadhyay, Debasis
    In our recent publication,1 we have shown that a T-DNA insertion in Arabidopsis CIPK6 gene encoding a CBL-interacting protein kinase caused reduction in expression of the gene and emergence of lateral roots. The change in phenotype in the mutant line was likely due to reduction in shoot-to-root acro- petal and the root tip basipetal auxin transport. Here we report identification of a homozygous knockout line of AtCIPK6 (atcipk6) with no detectable expression of the gene in normal growth condition. The knockout line exhibited considerable decrease in growth rate of the taproot as well as in emergence of lateral roots. The mutant line also showed reduction in the root tip basipetal and shoot-to-root acropetal auxin transport. Relative rate of auxin transport and the root phenotype of the atcipk6 closely matched with those of pgp4-1, an Arabidopsis line mutated in PGP4. This gene encodes an ABC integral membrane transporter, which functions in polar auxin transport. These observations strengthen our earlier proposal that CIPK6 is probably involved in polar auxin transport and indicate that it may function through the PGP4 auxin transporter.
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    CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plant
    (Wiley-Blackwell, 2009) Tripathi, Vineeta; Parasuraman, Boominathan; Laxmi, Ashverya; Chattopadhyay, Debasis
    Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) mediate plant responses to a variety of external stresses. Here we report that Arabidopsis CIPK6 is also required for the growth and development of plants. Phenotype of tobacco plants ectopically expressing a homologous gene (CaCIPK6) from the leguminous plant chickpea (Cicer arietinum) indicated its functional conservation. A lesion in AtCIPK6 significantly reduced shoot-to-root and root basipetal auxin transport, and the plants exhibited developmental defects such as fused cotyledons, swollen hypocotyls and compromised lateral root formation, in conjunction with reduced expression of a number of genes involved in auxin transport and abiotic stress response. The Arabidopsis mutant was more sensitive to salt stress compared to wild-type, while overexpression of a constitutively active mutant of CaCIPK6 promoted salt tolerance in transgenic tobacco. Furthermore, tobacco seedlings expressing the constitutively active mutant of CaCIPK6 showed a developed root system, increased basipetal auxin transport and hypersensitivity to auxin. Our results provide evidence for involvement of a CIPK in auxin transport and consequently in root development, as well as in the salt-stress response, by regulating the expression of genes.
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    Characterization and phylogenetic analysis of environmental stress responsive SAP gene family encoding A20/AN1 zinc-finger proteins in tomato
    (Springer, 2009) Solanke, Amolkumar U.; Sharma, Manoj K.; Tyagi, Akhilesh K.; Sharma, Arun Kumar
    Characterization of genes responsive to stress is important for eVorts on improving stress tolerance of plants. To address components involved in stress tolerance of tomato (Solanum lycopersicum), a stress-responsive gene family encoding A20/AN1 zinc Wnger proteins was characterized. In the present study, 13 members of this gene family were cloned from tomato cultivar Pusa Ruby and named as Stress Associated Protein (SAP) genes. Out of 13 genes, 12 have been mapped on their respective chromo- somes. Expression of these genes in response to cold, heat, salt, desiccation, wounding, abscisic acid, oxidative and submergence stresses was analysed. All tomato SAP genes were found to be responsive to one or other type of environ- mental stress. The phylogenetic analysis of these genes, along with their orthologs from Solanaceae species sug- gests the presence of a common set of SAP genes in the studied Solanaceae species. The present study characterizes a SAP gene family, which encodes A20/AN1 zinc Wnger containing proteins from tomato for the Wrst time. Genes showing high expression in response to a particular stress can be exploited for improving stress tolerance of tomato and other Solanaceae members.
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    CAP2 enhances germination of transgenic tobacco seeds at high temperature and promotes heat stress tolerance in yeast
    (Wiley-Blackwell, 2009) Shukla, Rakesh Kumar; Tripathi, Vineeta; Jain, Deepti; Yadav, Rajiv Kumar; Chattopadhyay, Debasis
    We reported earlier that ectopic expression of CAP2, a single AP2 domain containing transcription activator from chickpea (Cicer arietinum) in tobacco improves growth and development, and tolerance to dehydration and salt stress, of the transgenic plants. Here, we report that, in addition, the CAP2-transgenic tobacco seeds also exhibit higher germination efficiency at high temperature and show higher expression levels of genes for tobacco heat shock proteins and a heat shock factor. CAP2 was able to activate the 5¢-upstream activating sequence of tobacco heat shock factor. Surprisingly, expression of CAP2 cDNA in Saccharomyces cerevisiae also enhanced heat tolerance, with increased expression of the gene for yeast heat shock factor 1 (Hsf1) and its target, the gene for yeast heat shock protein 104 (Hsp104). Sequence analysis of the Hsf1 promoter revealed the presence of a dehydration-responsive element ⁄ C-repeat-like element (DRE/ CRE). Recombinant CAP2 protein bound to the DRE/CRE in the Hsf1 promoter in a gel shift assay and transactivated the Hsf1 promoter–His reporter construct. The full-length CAP2 protein was required to provide thermotolerance in yeast. If these findings are taken together, our results suggest that CAP2 is involved in the heat stress response and provides an example of functioning of a plant transcription factor in yeast, highlighting the strong evolutionary conservation of the stress response mechanism.
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    PLecDom: A program for identification and analysis of plant lectin domains
    (Oxford University Press, 2009) Shridhar, Smriti; Chattopadhyay, Debasis; Yadav, Gitanjali
    PLecDom is a program for detection of Plant Lectin Domains in a polypeptide or EST sequence, followed by a classification of the identified domains into known families. The web server is a collection of plant lectin domain families represented by align- ments and profile Hidden Markov Models. PLecDom was developed after a rigorous analysis of evolu- tionary relationships between available sequences of lectin domains with known specificities. Users can test their sequences for potential lectin domains, catalog the identified domains into broad substrate classes, estimate the extent of divergence of new domains with existing homologs, extract domain boundaries and examine flanking sequences for further analysis. The high prediction accuracy of PLecDom combined with the ease with which it handles large scale input, enabled us to apply the program to protein and EST data from 48 plant genome-sequencing projects in various stages of completion. Our results represent a signif- icant enrichment of the currently annotated plant lectins, and highlight potential targets for biochemical characterization. The search algorithm requires input in fasta format and is designed to process simultaneous connection requests from multiple users, such that huge sets of input sequences can be scanned in a matter of seconds. PLecDom is available at http://www.nipgr.res.in/plecdom.html.
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    Polyphasic taxonomic analysis establishes Mycobacterium indicus pranii as a distinct species
    (PLOS, 2009) Saini, Vikram; Raghuvanshi, Saurabh; Talwar, Gursaran P.; Ahmed, Niyaz; Khurana, Jitendra P.; Hasnain, Seyed E.; Tyagi, Akhilesh K.; Tyagi, Anil K.
    Background: Mycobacterium indicus pranii (MIP), popularly known as Mw, is a cultivable, non-pathogenic organism, which, based on its growth and metabolic properties, is classified in Runyon Group IV along with M. fortuitum, M. smegmatis and M. vaccae. The novelty of this bacterium was accredited to its immunological ability to undergo antigen driven blast transformation of leukocytes and delayed hypersensitivity skin test in leprosy patients, a disease endemic in the Indian sub- continent. Consequently, MIP has been extensively evaluated for its biochemical and immunological properties leading to its usage as an immunomodulator in leprosy and tuberculosis patients. However, owing to advances in sequencing and culture techniques, the citing of new strains with almost 100% similarity in the sequences of marker genes like 16S rRNA, has compromised the identity of MIP as a novel species. Hence, to define its precise taxonomic position, we have carried out polyphasic taxonomic studies on MIP that integrate its phenotypic, chemotaxonomic and molecular phylogenetic attributes. Methodology/Principal Findings: The comparative analysis of 16S rRNA sequence of MIP by using BLAST algorithm at NCBI (nr database) revealed a similarity of $99% with M. intracellulare, M. arosiense, M. chimaera, M. seoulense, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis and M. bohemicum. Further analysis with other widely used markers like rpoB and hsp65 could resolve the phylogenetic relationship between MIP and other closely related mycobacteria apart from M. intracellulare and M. chimaera, which shares $99% similarity with corresponding MIP orthologues. Molecular phylogenetic analysis, based on the concatenation of candidate orthologues of 16S rRNA, hsp65 and rpoB, also substantiated its distinctiveness from all the related organisms used in the analysis excluding M. intracellulare and M. chimaera with which it exhibited a close proximity. This necessitated further analysis of MIP with more sensitive and segregating parameters to ascertain its precise taxonomic position as a new species. The analysis of MIP and its comparison with other mycobacterial reference strains based on cellular and biochemical features, growth characteristics and chemotaxonomic studies like FAME profiling confirmed that MIP is uniquely endowed with diverse metabolic attributes that effectively distinguishes it from all the closely related mycobacteria including M. intracellulare and M. chimaera. Conclusion: The results presented in this study coupled with the non-pathogenic nature and different biochemical and immunomodulatory properties of MIP affirm it as a distinct species belonging to M. avium complex (MAC). It is further proposed to use an earlier suggested name Mycobacterium indicus pranii for this newly established mycobacterial species. This study also exemplifies the growing need for a uniform, consensus based broader polyphasic frame work for the purpose of taxonomy and speciation, particularly in the genus Mycobacterium.
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    Rhythmic expression of mitogen activated protein kinase activity in rice
    (Springer, 2009) Rao, Kudupudi Prabhakara; Vani, Gubbala; Kumar, Kundan; Sinha, Alok Krishna
    Mitogen activated protein kinase (MAPK) are known to get activated during various stress signals and transduce the message from the cell membrane to the nucleus for appropriate cellular reorganization. Though, a certain basal activity of MAPK is often observed in the control plants. Prolonged exposure of rice plants to lowered or elevated temperature exhibited a rhythm in the activation of MAPKs. We analyzed existence of a possible endogenous rhythm in the activity of MAPKs in rice plants. The plants growing at constant temperature entrained in 16/8 h day-night cycle showed diurnal rhythm in activity. When the activation of MAPK was tested under continuous conditions by shifting plants to continuous darkness for a period of 72 h, the periodic rhythm persisted and followed a circadian pattern. Analysis of the transcripts of group A, B and C members of MAPKs under above conditions by quantitative real time PCR revealed that the members of group C exhibit periodic rhythm. Our data indicates that the MAP kinase activity in rice follows rhythmic expression in a circadian manner.