Publications of NIPGR Scientists

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Author: search.filters.author.Singh, Ajeet

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    The landscape of fusion transcripts in plants: a new insight into genome complexity
    (BioMed Central Ltd, 2024) Chitkara, Pragya; Singh, Ajeet; Gangwar, Rashmi; Bhardwaj, Rohan; Zahra, Shafaque; Arora, Simran; Hamid, Fiza; Arya, Ajay; Sahu, Namrata; Chakraborty, Srija; Ramesh, Madhulika; Kumar, Shailesh
    Background Fusion transcripts (FTs), generated by the fusion of genes at the DNA level or RNA-level splicing events significantly contribute to transcriptome diversity. FTs are usually considered unique features of neoplasia and serve as biomarkers and therapeutic targets for multiple cancers. The latest findings show the presence of FTs in normal human physiology. Several discrete reports mentioned the presence of fusion transcripts in planta, has important roles in stress responses, morphological alterations, or traits (e.g. seed size, etc.). Results In this study, we identified 169,197 fusion transcripts in 2795 transcriptome datasets of Arabidopsis thaliana, Cicer arietinum, and Oryza sativa by using a combination of tools, and confirmed the translational activity of 150 fusion transcripts through proteomic datasets. Analysis of the FT junction sequences and their association with epigenetic factors, as revealed by ChIP-Seq datasets, demonstrated an organised process of fusion formation at the DNA level. We investigated the possible impact of three-dimensional chromatin conformation on intra-chromosomal fusion events by leveraging the Hi-C datasets with the incidence of fusion transcripts. We further utilised the longread RNA-Seq datasets to validate the most reoccurring fusion transcripts in each plant species followed by further authentication through RT-PCR and Sanger sequencing. Conclusions Our findings suggest that a significant portion of fusion events may be attributed to alternative splicing during transcription, accounting for numerous fusion events without a proportional increase in the number of RNA pairs. Even non-nuclear DNA transcripts from mitochondria and chloroplasts can participate in intra- and inter-chromosomal fusion formation. Genes in close spatial proximity are more prone to undergoing fusion formation, especially in intra-chromosomal FTs. Most of the fusion transcripts may not undergo translation and serve as long non-coding RNAs. The low validation rate of FTs in plants indicated that the fusion transcripts are expressed at very low levels, like in the case of humans. FTs often originate from parental genes involved in essential biological processes, suggesting their relevance across diverse tissues and stress conditions. This study presents a comprehensive repository of fusion transcripts, offering valuable insights into their roles in vital physiological processes and stress responses.
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    Comprehensive profiling of rRNA-derived small RNAs in Arabidopsis thaliana using rsRNAfinder pipeline
    (Elsevier B.V., 2024) Kalakoti, Garima; Vivek, AT; Kamboj, Anshul; Singh, Ajeet; Chakraborty, Srija; Kumar, Shailesh
    Ribosomal RNA (rRNA) gives rise to non-random small RNA fragments known as ribosomal-derived small RNAs (rsRNAs), which despite their biological importance, have been relatively understudied in comparison to other short non-coding RNAs. There exists a compelling necessity to develop a methodology for the identification, categorization, and quantification of rsRNAs from small RNA sequencing (sRNA-seq) data sets, considering the unique characteristics of ribosomal RNA (rRNA). To bridge this gap, we introduce 'rsRNAfinder' a specialized pipeline designed within the Snakemake framework. This analytical approach enables robust identification of rsRNAs using sRNA-seq datasets from Arabidopsis thaliana. Our methodology constitutes an integrated bioinformatic pipeline designed for different kinds of analysis.1.sRNA-seq data analysis: It performs in-depth analysis of reference-aligned sRNA-seq data, facilitating rsRNA annotation and quantification.2.Parametric reporting: Our pipeline provides comprehensive reports encompassing key parameters such as rsRNA size distributions, strandedness, genomic origin, and source rRNA origin.3.Illustrative validation: We have demonstrated the utility of our approach by conducting comprehensive rsRNA annotation in Arabidopsis thaliana. This validation reveals unique rsRNAs originating from all rRNA types, each of them distinguished by distinct identity, abundance, and length.
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    Long non-coding RNA and microRNA landscape of two major domesticated cotton species
    (Elsevier B.V., 2023) Singh, Ajeet; AT, Vivek; Gupta, Kanika; Sharma, Shruti; Kumar, Shailesh
    Allotetraploid cotton plants Gossypium hirsutum and Gossypium barbadense have been widely cultivated for their natural, renewable textile fibres. Even though ncRNAs in domesticated cotton species have been extensively studied, systematic identification and annotation of lncRNAs and miRNAs expressed in various tissues and developmental stages under various biological contexts are limited. This influences the comprehension of their functions and future research on these cotton species. Here, we report high confidence lncRNAs and miRNA collection from G. hirsutum accession and G. barbadense accession using large-scale RNA-seq and small RNA-seq datasets incorporated into a user-friendly database, CoNCRAtlas. This database provides a wide range and depth of lncRNA and miRNA annotation based on the systematic integration of extensive annotations such as expression patterns derived from transcriptome data analysis in thousands of samples, as well as multi-omics annotations. We assume this comprehensive resource will accelerate evolutionary and functional studies in ncRNAs and inform future breeding programs for cotton improvement. CoNCRAtlas is accessible at http://www.nipgr.ac.in/CoNCRAtlas/.
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    Morphophysiological and transcriptome analysis reveal that reprogramming of metabolism, phytohormones and root development pathways governs the potassium (K+) deficiency response in two contrasting chickpea cultivars
    (Frontiers Media S.A., 2023) Ankit, Ankit; Singh, Ajeet; Kumar, Shailesh; Singh, Amarjeet
    Potassium (K+) is an essential macronutrient for plant growth and development. K+ deficiency hampers important plant processes, such as enzyme activation, protein synthesis, photosynthesis and stomata movement. Molecular mechanism of K+ deficiency tolerance has been partly understood in model plants Arabidopsis, but its knowledge in legume crop chickpea is missing. Here, morphophysiological analysis revealed that among five high yielding desi chickpea cultivars, PUSA362 shows stunted plant growth, reduced primary root growth and low K+ content under K+ deficiency. In contrast, PUSA372 had negligible effect on these parameters suggesting that PUSA362 is K+ deficiency sensitive and PUSA372 is a K+ deficiency tolerant chickpea cultivar. RNA-seq based transcriptome analysis under K+ deficiency revealed a total of 820 differential expressed genes (DEG's) in PUSA362 and 682 DEGs in PUSA372. These DEGs belongs to different functional categories, such as plant metabolism, signal transduction components, transcription factors, ion/nutrient transporters, phytohormone biosynthesis and signalling, and root growth and development. RNA-seq expression of randomly selected 16 DEGs was validated by RT-qPCR. Out of 16 genes, 13 showed expression pattern similar to RNA-seq expression, that verified the RNA-seq expression data. Total 258 and 159 genes were exclusively up-regulated, and 386 and 347 genes were down-regulated, respectively in PUSA362 and PUSA372. 14 DEGs showed contrasting expression pattern as they were up-regulated in PUSA362 and down-regulated in PUSA372. These include somatic embryogenesis receptor-like kinase 1, thaumatin-like protein, ferric reduction oxidase 2 and transcription factor bHLH93. Nine genes which were down-regulated in PUSA362 found to be up-regulated in PUSA372, including glutathione S-transferase like, putative calmodulin-like 19, high affinity nitrate transporter 2.4 and ERF17-like protein. Some important carbohydrate metabolism related genes, like fructose-1,6-bisphosphatase and sucrose synthase, and root growth related Expansin gene were exclusively down-regulated, while an ethylene biosynthesis gene 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1) was up-regulated in PUSA362. Interplay of these and several other genes related to hormones (auxin, cytokinin, GA etc.), signal transduction components (like CBLs and CIPKs), ion transporters and transcription factors might underlie the contrasting response of two chickpea cultivars to K+ deficiency. In future, some of these key genes will be utilized in genetic engineering and breeding programs for developing chickpea cultivars with improved K+ use efficiency (KUE) and K+ deficiency tolerance traits.
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    tncRNA Toolkit: A pipeline for convenient identification of RNA (tRNA)-derived non-coding RNAs
    (Elsevier B.V., 2023) Zahra, Shafaque; Singh, Ajeet; Kumar, Shailesh
    Insights into the eukaryotic gene regulation networks have improved due to the advent of diverse classes of non-coding RNAs. The transfer RNA (tRNA)-derived non-coding RNAs or tncRNAs is a novel class of non-coding RNAs, shown to regulate gene expression at transcription and translation levels. Here, we present a pipeline 'tncRNA Toolkit' for accurately identifying tncRNAs using small RNA sequencing (sRNA-seq) data. Previously, we identified tncRNA in six major angiosperms by utilizing our pipeline and highlighted the significant points regarding their generation and functions. The 'tncRNA Toolkit' is available at the URL: http://www.nipgr.ac.in/tncRNA. The scripts are written in bash and Python3 programming languages. The program can be efficiently run as a standalone command-line tool and installed in any Linux-based Operating System (OS). The user can run this program by providing the input of sRNA-seq data and genome file.The various features of the 'tncRNA Toolkit' are as follows:•Major tncRNA classes identified by this tool include tRF-5, tRF-3, tRF-1, 5'tRH, 3'tRH, and leader tRF. Also, it categorizes miscellaneous tncRNAs as other tRF.•It provides the following information for each identified tncRNA viz. tncRNA class, raw and normalized read count (RPM), read length, progenitor tRNA information (amino acid, anticodon, locus, strand), tncRNA sequence, and tRNA modification sites.•We hope to facilitate quick and reliable tncRNA identification, which will boost the exploration of this novel class of non-coding RNAs and their relevance in the living world, including plants.
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    PtRNAdb: a web resource of plant tRNA genes from a wide range of plant species
    (Springer Nature Publishing AG, 2022) Singh, Ajeet; Zahra, Shafaque; Das, Durdam; Kumar, Shailesh
    tRNA, as well as their derived products such as short interspersed nuclear elements (SINEs), pseudogenes, and transfer RNA (tRNA)-derived fragments (tRFs), have now been shown to be vital for cellular life, functioning, and adaptation during different stress conditions in all diverse life forms. In this study, we have developed PtRNAdb (www.nipgr.ac.in/PtRNAdb), a plant-exclusive tRNA database containing 113,849 tRNA gene sequences from phylogenetically diverse plant species. We have analyzed a total of 106 nuclear, 89 plastidial, and 38 mitochondrial genomes of plants by the tRNAscan-SE software package, and after careful curation of the output data, we integrated the data and developed this database. The information about the tRNA gene sequences obtained was further enriched with a consensus sequence-based study of tRNA genes based on their isoacceptors and isodecoders. We have also built covariance models based on the isoacceptors and isodecoders of all the tRNA sequences using the infernal tool. The user can also perform BLAST not only against PtRNAdb entries but also against all the tRNA sequences stored in the PlantRNA database and annotated tRNA genes across the plant kingdom available at NCBI. This resource is believed to be of high utility for plant researchers as well as molecular biologists to carry out further exploration of the plant tRNAome on a wider spectrum, as well as for performing comparative and evolutionary studies related to tRNAs, and their derivatives across all domains of life. Database URL: http://www.nipgr.ac.in/PtRNAdb/
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    PTPAMP: prediction tool for plant-derived antimicrobial peptides
    (Springer Nature Publishing AG, 2023) Jaiswal, Mohini; Singh, Ajeet; Kumar, Shailesh
    The emergence of antimicrobial peptides (AMPs) as a potential alternative to conventional antibiotics has led to the development of efficient computational methods for predicting AMPs. Among all organisms, the presence of multiple genes encoding AMPs in plants demands the development of a plant-based prediction tool. To this end, we developed models based on multiple peptide features like amino acid composition, dipeptide composition, and physicochemical attributes for predicting plant-derived AMPs. The selected compositional models are integrated into a web server termed PTPAMP. The designed web server is capable of classifying a query peptide sequence into four functional activities, i.e., antimicrobial (AMP), antibacterial (ABP), antifungal (AFP), and antiviral (AVP). Our models achieved an average area under the curve of 0.95, 0.91, 0.85, and 0.88 for AMP, ABP, AFP, and AVP, respectively, on benchmark datasets, which were ~ 6.75% higher than the state-of-the-art methods. Moreover, our analysis indicates the abundance of cysteine residues in plant-derived AMPs and the distribution of other residues like G, S, K, and R, which differ as per the peptide structural family. Finally, we have developed a user-friendly web server, available at the URL: http://www.nipgr.ac.in/PTPAMP/. We expect the substantial input of this predictor for high-throughput identification of plant-derived AMPs followed by additional insights into their functions.
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    PtncRNAdb: plant transfer RNA-derived non-coding RNAs (tncRNAs) database
    (Springer Nature Publishing AG, 2022) Zahra, Shafaque; Bhardwaj, Rohan; Sharma, Shikha; Singh, Ajeet; Kumar, Shailesh
    Specific endonucleolytic cleavage of tRNA molecules leads to the biogenesis of heterogeneously sized fragments called tRNA-derived non-coding RNAs (tncRNAs). The role of tncRNAs is well studied in human processes, and diseases including different types of cancers and other ailments. They are also generated under stress conditions in plants. Considering the potential role of tncRNAs in the plant system, we have developed a user-friendly, open-access web resource, PtncRNAdb (https://nipgr.ac.in/PtncRNAdb). PtncRNAdb consists of 4,809,503 tncRNA entries identified from ~ 2500 single-end small RNA-seq libraries from six plants, viz., Arabidopsis thaliana, Cicer arietinum, Zea mays, Oryza sativa, Medicago truncatula, and Solanum lycopersicum. It is provided with assorted options to search, browse, visualize, interpret, and download tncRNAs data. Users can perform query search using ‘BLASTN’ against PtncRNAdb entries. Highcharts have been included for better statistical PtncRNAdb data readability to the users. Additionally, PtncRNAdb includes ‘DE tncRNAs’ module for differentially expressed tncRNAs under various conditions. Their secondary structure, putative targets, interactive networks of target enrichment, and related publications are also incorporated for further interpretation of their biological functions. PtncRNAdb is an efficient, user-friendly, and exhaustive database, which will aid the ongoing research in plant tncRNAs as well as help in deciphering their role in gene regulation. We hope that it provides a promising platform for researchers to facilitate the understanding of tncRNAs, and their involvement in numerous pathways related to plant development and stress tolerance.
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    In silico methods for the identification of viral-derived small interfering RNAs (vsiRNAs) and their application in plant genomics
    (Springer Nature Publishing AG, 2022) Narayan, Aditya; Zahra, Shafaque; Singh, Ajeet; Kumar, Shailesh
    The current era of high-throughput sequencing (HTS) technology has expedited the detection and diagnosis of viruses and viroids in the living system including plants. HTS data has become vital to study the etiology of the infection caused by both known as well as novel viral elements in planta, and their impact on overall crop health and productivity. Viral-derived small interfering RNAs are generated as a result of defence response by the host via RNAi machinery. They are immensely exploited for performing exhaustive viral investigations in plants using bioinformatics as well as experimental approaches. This chapter briefly presents the basics of virus-derived small interfering RNAs (vsiRNAs) biology in plants and their applications in plant genomics and highlights in silico strategies exploited for virus/viroid detection. It gives a systematic pipeline for vsiRNAs identification using currently available bioinformatics tools and databases. This will surely work as a quick beginner’s recipe for the in silico revelation of plant vsiRNAs as well as virus/viroid diagnosis using high-throughput sequencing data.
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    Microscopic and transcriptomic analyses of Dalbergoid legume peanut reveal a divergent evolution leading to Nod Factor dependent epidermal crack-entry and terminal bacteroid differentiation
    (American Phytopathological Society, 2022) Raul, Bikash; Bhattacharjee, Oindrila; Ghosh, Amit; Upadhyay, Priya; Tembhare, Kunal; Singh, Ajeet; Shaheen, Tarannum; Ghosh, Asim Kumar; Torres-Jerez, Ivone; Krom, Nick; Clevenger, Josh; Udvardi, Michael; Scheffler, Brian E.; Ozias-Akins, Peggy; Sharma, Ravi Datta; Bandyopadhyay, Kaustav; Gaur, Vineet; Kumar, Shailesh; Sinharoy, Senjuti
    Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS come from model legumes that exhibit a root-hair mode of bacterial infection in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step towards understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated a comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen scavenging mechanisms in the Papilionoid family. Finally, absence of cysteine-rich motif-1 containing-NCRs, but the recruitment of defensin like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobial symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.