Browsing by Author "Purayannur, Savithri"

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An alternative approach in Gateway ® cloning when the bacterial antibiotic selection cassettes of the entry clone and destination vector are the same
(Springer, 2013) Kumar, Kamal; Yadav, Saurabh; Purayannur, Savithri; Verma, Praveen K.
The Gateway(®) recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.
Ascochyta rabiei: A threat to global chickpea production
(John Wiley & Sons, 2022) Singh, Ritu; Kumar, Kamal; Purayannur, Savithri; Chen, Weidong; Verma, Praveen K.
The necrotrophic fungus Ascochyta rabiei causes Ascochyta blight (AB) disease in chickpea. A. rabiei infects all aerial parts of the plant, which results in severe yield loss. At present, AB disease occurs in most chickpea-growing countries. Globally increased incidences of A. rabiei infection and the emergence of new aggressive isolates directed the interest of researchers toward understanding the evolution of pathogenic determinants in this fungus. In this review, we summarize the molecular and genetic studies of the pathogen along with approaches that are helping in combating the disease. Possible areas of future research are also suggested.
Genetic engineering to improve biotic stress tolerance in plants
(Springer Nature, 2017) Purayannur, Savithri; Kumar, Kamal; Verma, Praveen K.
Genetic engineering of plants for resistance is an effective method to counter pathogens and pests owing to the specificity and efficiency of the technology. The genes that have been used to genetically engineer resistance are as diverse as the diseases they act against. In cases where gene-for-gene resistance coded by resistance (R) genes exists, engineering resistance in plants becomes a straight path. Different classes of R genes have been engineered to provide resistance against viruses, bacteria, filamentous phytopathogens, and nematodes. Where the resistance mechanism is not R gene mediated, myriad of other mechanisms have been tried. These include the use of genes coding for antimicrobial compounds against bacterial and filamentous pathogens. The cloning of transcription factors, receptor genes, proteases, and genes involved in the systemic acquired resistance (SAR) has also been found to be effective. RNA silencing against specific genes involved in pathogenicity has proved to be an efficacious strategy against viruses and nematodes. Posttranscriptional silencing of genes coding for viral coat proteins has been successful, both scientifically and commercially. The most extensively used technology till date has been the introduction of cry genes from the bacterium Bacillus thuringiensis into plants to render them resistant against insect pests. Advances in molecular biology have paved the way for new strategies, the phenomenon of host-induced gene silencing (HIGS) being an interesting example. Amidst all the hue and cry raised against genetic modification of crops, it is necessary to highlight the scientific principles involved so as to make full use of a technology that could very well solve the problem of food shortage.
Phylogenomic analysis of MKKs and MAPKs from 16 legumes and detection of interacting pairs in chickpea divulge MAPK signalling modules
(Nature Publishing Group, 2017) Purayannur, Savithri; Kumar, Kamal; Kaladhar, Vemula Chandra; Verma, Praveen K.
The mitogen-activated protein kinase (MAPK)-mediated phosphorylation cascade is a vital component of plant cellular signalling. Despite this, MAPK signalling cascade is less characterized in crop legumes. To fill this void, we present here a comprehensive phylogeny of MAPK kinases (MKKs) and MAPKs identified from 16 legume species belonging to genistoid (Lupinus angustifolius), dalbergioid (Arachis spp.), phaseoloid (Glycine max, Cajanus cajan, Phaseolus vulgaris, and Vigna spp.), and galegoid (Cicer arietinum, Lotus japonicus, Medicago truncatula, Pisum sativum, Trifolium spp., and Vicia faba) clades. Using the genes of the diploid crop chickpea (C. arietinum), an exhaustive interaction analysis was performed between MKKs and MAPKs by split-ubiquitin based yeast two-hybrid (Y2H). Twenty seven interactions of varying strengths were identified between chickpea MKKs and MAPKs. These interactions were verified in planta by bimolecular fluorescence complementation (BiFC). As a first report in plants, four intra-molecular interactions of weak strength were identified within chickpea MKKs. Additionally; two TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors of class I were identified as novel down-stream interacting partners of seven MAPKs. We propose that this highly reliable MAPK interaction network, presented here for chickpea, can be utilized as a reference for legumes and thus will help in deciphering their role in legume-specific events.
WRKY domain-encoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s)
(Oxford University Press, 2016) Kumar, Kamal; Srivastava, Vikas; Purayannur, Savithri; Kaladhar, V. Chandra; Cheruvu, Purnima Jaiswal; Verma, Praveen K.
The WRKY genes have been identified as important transcriptional modulators predominantly during the environmental stresses, but they also play critical role at various stages of plant life cycle. We report the identification of WRKY domain (WD)-encoding genes from galegoid clade legumes chickpea (Cicer arietinum L.) and barrel medic (Medicago truncatula). In total, 78 and 98 WD-encoding genes were found in chickpea and barrel medic, respectively. Comparative analysis suggests the presence of both conserved and unique WRKYs, and expansion of WRKY family in M. truncatula primarily by tandem duplication. Exclusively found in galegoid legumes, CaWRKY16 and its orthologues encode for a novel protein having a transmembrane and partial Exo70 domains flanking a group-III WD. Genomic region of galegoids, having CaWRKY16, is more dynamic when compared with millettioids. In onion cells, fused CaWRKY16-EYFP showed punctate fluorescent signals in cytoplasm. The chickpea WRKY group-III genes were further characterized for their transcript level modulation during pathogenic stress and treatments of abscisic acid, jasmonic acid, and salicylic acid (SA) by real-time PCR. Differential regulation of genes was observed during Ascochyta rabiei infection and SA treatment. Characterization of A. rabiei and SA inducible gene CaWRKY50 showed that it localizes to plant nucleus, binds to W-box, and have a C-terminal transactivation domain. Overexpression of CaWRKY50 in tobacco plants resulted in early flowering and senescence. The in-depth comparative account presented here for two legume WRKY genes will be of great utility in hastening functional characterization of crop legume WRKYs and will also help in characterization of Exo70Js.