Publications of NIPGR Scientists
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Item BAR domain is essential for early endosomal trafficking and dynamics in Ascochyta rabiei(Springer Nature Publishing AG, 2023) Shree, Ankita; Sinha, Manisha; Verma, Praveen K.Ascochyta blight disease is a devastating disease caused by the fungal pathogen Ascochyta rabiei that threatens chickpea production around the globe. Endocytic mechanism has a significant role in fungal growth and virulence. The underlying biology of biogenesis of central component of endocytosis viz Rab5 vesicles, is not completely understood. The involvement of F-BAR domain containing protein (ArF-BAR) in various cellular processes that collectively make ArF-BAR as an important virulence determinant. Here, we report that ArF-BAR is involved in biogenesis and motility of early endosome. In the absence of ArF-BAR gene (Δarf-bar), fungal mutants exhibited reduced number of EGFP coated ArRab5 vesicles, along with the considerable reduction in their dynamics. Here, we show that ArF-BAR interacts with clathrin light chain (ArCLC), specifically with its F-BAR domain. These findings suggests the novel role of ArF-BAR in biogenesis and dynamics of early endosome. Additionally, ArF-BAR is involved in clathrin-mediated mechanism of endocytosis which is required for host infection and disease development. Identification of this pathway offers new impending targets for disease intervention in plants.Item Genome-wide identification, expression, and characterization of CaLysM-RLKs in chickpea root nodule symbiosis(Elsevier B.V., 2022) Singh, Jawahar; Verma, Praveen K.Legumes establish a nitrogen-fixing symbiosis with rhizobia which depends on the exchange of chemical signals between the two partners. Successful interaction between legumes and rhizobia depends on the perception of Nod factors (NFs) by the receptor-like kinases. A few cell surface receptors have been identified that perceive the rhizobial NFs and activate the downstream signaling pathway for nodulation in model legumes like Lotus japonicus and Medicago truncatula. However, crop legume such as chickpea lacks detailed analysis of LysM-RLKs genes for NFs perception. Here, we identified and characterized CaLysM-RLK genes and analyzed their gene structure, motif composition, chromosomal localization, phylogenetic relationship with Arabidopsis, Oryza sativa, and Medicago truncatula. To understand the spatiotemporal expression patterns, we performed transcript profiling of CaLysM-RLKs genes at a different time interval of the chickpea-rhizobia symbiotic interaction that revealed CaNFP and CaLYK3 can act as candidate genes for NF perception. Down-regulation of CaNFP and CaLYK3 in chickpea by agrobacterium rhizogenes mediated hairy root sysytem leads to signifificant reduction in nodule numbers. This study will be useful for improving root nodule symbiosis in chickpea.Item Functional characterization of genes involved in legume nodulation using hairy root cultures(Springer Nature Publishing AG, 2020) Singh, Jawahar; Kumar, Kamal; Verma, Praveen K.Legumes, the second most important crop to humans possess unique ability to fix atmospheric nitrogen, making them one of the major contributors to sustainable agriculture. In legumes, molecular characterization of genes by stable transformation is difficult due to their recalcitrant nature to the whole-plant regeneration in desired varieties. The Agrobacterium rhizogenes-mediated generation of transgenic hairy roots or composite plants may facilitate a rapid and convenient alternative to study nodule biology. Functional analysis of genes involved in legume nodulation has been proven as successful for model legumes, viz., Medicago truncatula and Lotus japonicus, using transgenic hairy roots. Besides sharing some common features of nodulation among legumes, the symbiotic signaling is a complex and specific process. Here, we describe an improved protocol for hairy root transformation of a legume crop chickpea (Cicer arietinum L.) and the method to study nodulation to uncover the signaling components. Using the described protocol, transgenic hairy roots were generated in chickpea and selected based on the red fluorescence protein (RFP) microscopy. This protocol can be extended to other underutilized legumes.Item Comparative structural modelling of a monothiol GRX from chickpea: insight in iron-sulfur cluster assembly(Elsevier, 2012) Yadav, Saurabh; Kushwaha, Hemant Ritturaj; Kumar, Kamal; Verma, Praveen K.Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537 bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX([29/30/32])KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster.Item Differential transcript accumulation in chickpea during early phases of compatible interaction with a necrotrophic fungus Ascochyta rabiei(Springer Science, 2012) Jaiswal, Purnima; Cheruku, Jyothi Reddy; Kumar, Kamal; Yadav, Saurabh; Singh, Archana; Kumari, Pragati; Dube, Sunil Chandra; Upadhyaya, Kailash C.; Verma, Praveen K.The initial phases of the disease establishment are very crucial for the compatible interactions. Pathogens must overcome the responses generated by the host for the onset of disease invasion. The compatible interaction is inadequately represented in plant-pathogen interaction studies. To gain broader insight into the early responses elicited by chickpea blight fungus Ascochyta rabiei during compatible interaction; we isolated early responsive genes of chickpea using PCR based suppression subtractive hybridization (SSH) strategy. We obtained ~250 unique genes after homology search and redundancy elimination. Based on their potential cellular functions, these genes were broadly classified into eleven different categories viz. stress, signaling, gene regulation, cellular metabolism and genes of unknown functions. Present study revealed few unexpected genes which have a possible role in induced immunity and disease progression. We employed macroarray, northern blot, real-time PCR and cluster analysis to develop transcript profiles. Most of the genes analyzed were early induced and were transcriptionally upregulated upon 24 h post inoculation. Our approach has rendered the isolation of early responsive genes involved in signaling and regulation of metabolic changes upon fungal infection. The information obtained will help to dissect the molecular mechanisms during compatible chickpea-Ascochyta interactions.Item A highly efficient Agrobacterium mediated transformation system of chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root colonization(Elsevier, 2012) Islam, Md. Nazrul; Nizam, Shadab; Verma, Praveen K.The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicer arietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated transformation (ATMT) has served as a resourceful tool for plant-pathogen interaction studies and offers a number of advantages over conventional transformation systems. Here, we developed a highly efficient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutive expression of red fluorescent protein (DsRed-Express) was used to study developmental stages and host-pathogen interactions. Southern hybridisation was performed to confirm the transformation event and the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants showed single copy integrations at random positions. Microscopic studies revealed significant levels of fluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with the transformed Foc showed rapid colonisation. These studies will allow us to develop strategies to determine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics for the characterisation of involved genes at the molecular level either by insertional mutagenesis or gene knock-out.Item Differential transcript accumulation in Cicer arietinum L. in response to a chewing Insect Helicoverpa armigera and defense regulators correlate with reduced insect performance(Oxford University Press, 2008) Singh, Archana; Singh, Indrakant Kumar; Verma, Praveen K.Monitoring transcriptional reorganization triggered in response to a particular stress is an essential first step for the functional analysis of genes involved in the process. To characterize Cicer arietinum L. defence responses against Helicoverpa armigera feeding, transcript patterns elicited by both herbivore and mechanical wounding were profiled and compared, and the application of defence regulators was assessed. A combination of approaches was employed to develop transcript profiles, including suppression subtractive hybridization (SSH), macroarray, northern blot, and cluster analysis. Of the 63 unique genes isolated, 29 genes expressed differentially when Helicoverpa feeding and wounding responses were compared. Comparative macroarray analyses revealed that most of the Helicoverpa-induced transcripts were methyl jasmonate (MeJA) and ethylene (ET) regulated. The effects of mild insect infestation and the exogenous application of signalling compounds on larval feeding behaviour were also monitored. Bioassays were performed to measure dispersal percentage and growth of larvae on elicited plants. Larvae released on elicited plants had decreased larval performance, demonstrating the central role of induced plant defence against herbivory. Similarly, wounding and exogenous application of MeJA and ET also affected larval growth and feeding behaviour. Our results demonstrated that Helicoverpa attack up-regulated large transcriptional changes and induced chickpea defence responses. Therefore, the results of this study advance the understanding of non-model plant–insect interactions on a broader scale.