Publications of NIPGR Scientists
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Item 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, DebasisYellow 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.Item Role of CIPK6 in root growth and auxin transport(Landes Bioscience, 2009) Tripathi, Vineeta; Syed, Nazia; Laxmi, Ashverya; Chattopadhyay, DebasisIn 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.Item 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, DebasisCalcineurin 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.Item 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, DebasisWe 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.Item PLecDom: A program for identification and analysis of plant lectin domains(Oxford University Press, 2009) Shridhar, Smriti; Chattopadhyay, Debasis; Yadav, GitanjaliPLecDom 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.Item Isolation and sequence analysis of DREB2A homologues in three cereal and two legume species(Elsevier B.V., 2009) Nayak, Spurthi N.; Balaji, Jayashree; Upadhyaya, Hari D.; Hash, C. Tom; Kavi Kishor, P.B.; Chattopadhyay, Debasis; Rodriquez, Lina Marıa; Blair, Matthew W.; Baum, Michael; McNally, Kenneth; This, Dominique; Hoisington, David A.; Varshney, Rajeev K.The transcription factor, DREB2A, is one of the promising candidate genes involved in dehydration tolerance in crop plants. In order to isolate DREB2A homologues across cereals (rice, barley and sorghum) and legumes (common bean and chickpea), specific or degenerate primers were used. Gene/phylogenetic trees were constructed using a non-redundant set of 19 DREB1A and 27 DREB2A amino acid sequences and were combined with taxonomic/species tree to prepare reconciled phylogenetic trees. In total, 86 degenerate primers were designed for different clades and 295 degenerate primer combinations were used to amplify DREB homologues in targeted crop species. Successful amplification of DREB2A was obtained in case of sorghum. In parallel, gene-specific primers were used to amplify DREB2A homologues in rice, barley, common bean and chickpea. Seven to eight diverse genotypes from targeted species were used for sequence analysis at DREB2A locus identified/isolated. A maximum of eight SNPs were found in the common bean DREB2A, indicating two distinct haplotypes, three SNPs with five haplotypes were observed in barley whereas a single SNP was observed in rice, sorghum and chickpea. Parsimony based phylogenetic tree revealed distinct clustering of cereals and legumes. Furthermore, alignment of corresponding amino acid sequences showed conservation of AP2 domain across the targeted species.Item CaZF, a plant transcription factor functions through and parallel to HOG and calcineurin pathways in Saccharomyces cerevisiae to provide osmotolerance(PLOS, 2009) Jain, Deepti; Roy, Nilanjan; Chattopadhyay, DebasisSalt-sensitive yeast mutants were deployed to characterize a gene encoding a C2H2 zinc finger protein (CaZF) that is differentially expressed in a drought-tolerant variety of chickpea (Cicer arietinum) and provides salinity-tolerance in transgenic tobacco. In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca(2+)/calmodulin-regulated protein phosphatase 2B. In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways. CaZF partially suppresses salt-hypersensitive phenotypes of hog1, can and hog1can mutants and in conjunction, stimulates HOG and CAN pathway genes with subsequent accumulation of glycerol in absence of Hog1p and CAN. CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast. Transactivation and salt tolerance assays of CaZF deletion mutants showed that other than the transactivation domain a C-terminal domain composed of acidic and basic amino acids is also required for its function. Altogether, results from this study suggests that CaZF is a potential plant salt-tolerance determinant and also provide evidence that in budding yeast expression of HOG and CAN pathway genes can be stimulated in absence of their regulatory enzymes to provide osmotolerance.Item Two divergent genes encoding L-myo-inositol 1-phosphate synthase1 (CaMIPS1) and 2 (CaMIPS2) are differentially expressed in chickpea(Wiley-Blackwell, 2008) Kaur, Harmeet; Shukla, Rakesh Kumar; Yadav, Gitanjali; Chattopadhyay, Debasis; Majee, ManojL-myo-inositol 1-phosphate synthase (MIPS; EC5.5.1.4) catalyses the rate-limiting step in inositol biosynthetic pathway, and is extremely widespread in living organisms including plants. Several plants possess multiple copies of MIPS gene(s) indicating a possibility of differential expression of each gene to perform distinct physiological functions. To explore this, two MIPS genes (CaMIPS1 and CaMIPS2) were isolated from a drought-tolerant plant chickpea. Both genes are extremely divergent in respect to their introns, at the same time retaining 85% identity to their exons and functionally complementing inositol auxotroph Schizosaccharomyces pombe. Expression analysis showed both genes were expressed in all organs except seed, where only CaMIPS2 transcript was detected. Under environmental stresses, only CaMIPS2 was induced whereas CaMIPS1 expression remained same, which could be explained by the divergence of their 5' upstream regulatory sequences. Remarkably, both gene products exhibited similar biochemical characteristics; however, CaMIPS2 retained higher activity than CaMIPS1 at a high temperature and salt concentration. Furthermore, functional expression of CaMIPS2 in S. pombe results better growth response than CaMIPS1 under stress environment. Taken together, our results suggest that CaMIPS1 and CaMIPS2 are differentially expressed in chickpea to play discrete though overlapping roles in plant; however CaMIPS2 is likely to be evolved through gene duplication to function under environmental stresses.Item Expression of CAP2, an AP2-family transcription factor from chickpea enhances growth and tolerance to dehydration and salt tress in transgenic tobacco(American Society of Plant Biologists, 2006) Shukla, Rakesh K.; Raha, Sumita; Tripathi, Vineeta; Chattopadhyay, DebasisThe APETALA2 (AP2) domain defines a large family of DNA-binding proteins that play important roles in plant morphology, development, and stress response. We describe isolation and characterization of a gene (CAP2) from chickpea (Cicer arietinum) encoding a novel AP2-family transcription factor. Recombinant CAP2 protein bound specifically to C-repeat/dehydration- responsive element in gel-shift assay and transactivated reporter genes in yeast (Saccharomyces cerevisiae) one-hybrid assay. CAP2 appeared to be a single/low copy intronless gene, and the protein product localized in the nucleus. Transcript level of CAP2 increased by dehydration and by treatment with sodium chloride, abscisic acid, and auxin, but not by treatment with low temperature, salicylic acid, and jasmonic acid. The 35S promoter-driven expression of CAP2 in tobacco (Nicotiana tabacum) caused drastic increase in the leaf cell size, and, thereby, in leaf surface area and number of lateral roots. Transgenic plants demonstrated more tolerance to dehydration and salt stress than the wild-type plants. Transgenic plants expressed higher steady-state transcript levels of abiotic stress-response genes NtERD10B and NtERD10C and auxin-response genes IAA4.2 and IAA2.5. Taken together, our results indicated a mutual interrelation between plant growth-development and abiotic stress- response pathways and a probable involvement of CAP2 in both the signaling pathways.Item Criteria for the choice of plant species for genome sequencing, to advance plant biology and agriculture(Indian Academy of Sciences, 2006) Kumar, Sushil; Chattopadhyay, Debasis; Prasad, ManojInformation from genome sequences is essential to understand the organization of genomes, chromosomes, genes, regulatory and repetitive elements present in plant genomes and also to understand the evolutionary significance of variations observed in genome compositions. Understandably, the plant genome sequencing effort has been growing in recent years, since the plant genome organization is not fully understood. The genome sequences of Arabidopsis thaliana and rice have been completed and a draft sequence of the poplar genome (http://genome.jgi- psf.org/Poptr1/Poptr1.home.html) has also been completed.