Genome-wide characterization and comparative analysis of the OSCA gene family and identification of its potential stress-responsive members in legumes.

Cicer arietinum, Cajanus cajan, Vigna radiata, and Phaseolus vulgaris are economically important legume crops with high nutritional value. They are negatively impacted globally by different biotic and abiotic stresses. Hyperosmolality-gated calcium-permeable channels (OSCA) have been characterized as osmosensors in Arabidopsis thaliana but have not previously reported in legumes. This study provides a genome-wide identification, characterization, and comparative analysis of OSCA genes in legumes. Our study identified and characterized 13 OSCA genes in C. cajan, V. radiata, P. vulgaris, and 12 in C. arietinum, classified into four distinct clades. We found evidence to suggest that the OSCAs might be involved in the interaction between hormone signalling pathways and stress signalling pathways. Furthermore, they play a major role in plant growth and development. The expression levels of the OSCAs vary under different stress conditions in a tissue-specific manner. Our study can be used to develop a detailed understanding of stress regulatory mechanisms of the OSCA gene family in legumes.

tncRNA Toolkit: A pipeline for convenient identification of RNA (tRNA)-derived non-coding RNAs.

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.

PtRNAdb: a web resource of plant tRNA genes from a wide range of plant species.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03255-7.

PtncRNAdb: plant transfer RNA-derived non-coding RNAs (tncRNAs) database.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03174-7.

In Silico Methods for the Identification of Viral-Derived Small Interfering RNAs (vsiRNAs) and Their Application in Plant Genomics.

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.

Transfer RNA-derived non-coding RNAs (tncRNAs): Hidden regulation of plants' transcriptional regulatory circuits.

The emergence of distinct classes of non-coding RNAs has led to better insights into the eukaryotic gene regulatory networks. Amongst them, the existence of transfer RNA (tRNA)-derived non-coding RNAs (tncRNAs) demands exploration in the plant kingdom. We have designed a methodology to uncover the entire perspective of tncRNAome in plants. Using this pipeline, we have identified diverse tncRNAs with a size ranging from 14 to 50 nucleotides (nt) by utilizing 2448 small RNA-seq samples from six angiosperms, and studied their various features, including length, codon-usage, cleavage pattern, and modified tRNA nucleosides. Codon-dependent generation of tncRNAs suggests that the tRNA cleavage is highly specific rather than random tRNA degradation. The nucleotide composition analysis of tncRNA cleavage positions indicates that they are generated through precise endoribonucleolytic cleavage machinery. Certain nucleoside modifications detected on tncRNAs were found to be conserved across the plants, and hence may influence tRNA cleavage, as well as tncRNA functions. Pathway enrichment analysis revealed that common tncRNA targets are majorly enriched during metabolic and developmental processes. Further distinct tissue-specific tncRNA clusters highlight their role in plant development. Significant number of tncRNAs differentially expressed under abiotic and biotic stresses highlights their potential role in stress resistance. In summary, this study has developed a platform that will help in the understanding of tncRNAs and their involvement in growth, development, and response to various stresses. The workflow, software package, and results are freely available at http://nipgr.ac.in/tncRNA.

From current knowledge to best practice: A primer on viral diagnostics using deep sequencing of virus-derived small interfering RNAs (vsiRNAs) in infected plants.

Plants have evolved many defense strategies for combating viral infections. One major surveillance strategy adopted by them is manipulating viral sequences to generate distinct small RNA products via Dicer-like enzymes (DCL), and thereby restricting virus multiplication through the RNA interference (RNAi) mechanism. The power of high-throughput sequencing technologies, with diverse computational tools to handle small RNA sequencing (sRNA-Seq) data, bestows unprecedented opportunities to answer fundamental questions in plant virology. Here, we present some basic concepts of virus-derived, small interfering RNA (vsiRNA) biogenesis in plants, optimization strategies, caveats, and best practices for efficient discovery and diagnosis of known as well as novel plant viruses/viroids using deep sequencing of small RNA (sRNA) pools.

AtFusionDB: a database of fusion transcripts in Arabidopsis thaliana.

Fusion transcripts are chimeric RNAs generated as a result of fusion either at DNA or RNA level. These novel transcripts have been extensively studied in the case of human cancers but still remain underexamined in plants. In this study, we introduce the first plant-specific database of fusion transcripts named AtFusionDB (http://www.nipgr.res.in/AtFusionDB). This is a comprehensive database that contains the detailed information about fusion transcripts identified in model plant Arabidopsis thaliana. A total of 82 969 fusion transcript entries generated from 17 181 different genes of A. thaliana are available in this database. Apart from the basic information consisting of the Ensembl gene names, official gene name, tissue type, EricScore, fusion type, AtFusionDB ID and sample ID (e.g. Sequence Read Archive ID), additional information like UniProt, gene coordinates (together with the function of parental genes), junction sequence, expression level of both parent genes and fusion transcript may be of high utility to the user. Two different types of search modules viz. 'Simple Search' and 'Advanced Search' in addition to the 'Browse' option with data download facility are provided in this database. Three different modules for mapping and alignment of the query sequences viz. BLASTN, SW Align and Mapping are incorporated in AtFusionDB. This database is a head start for exploring the complex and unexplored domain of gene/transcript fusion in plants.

PVsiRNAdb: a database for plant exclusive virus-derived small interfering RNAs.

Ribonucleic acids (RNA) interference mechanism has been proved to be an important regulator of both transcriptional and post-transcription controls of gene expression during biotic and abiotic stresses in plants. Virus-derived small interfering RNAs (vsiRNAs) are established components of the RNA silencing mechanism for incurring anti-viral resistance in plants. Some databases like siRNAdb, HIVsirDB and VIRsiRNAdb are available online pertaining to siRNAs as well as vsiRNAs generated during viral infection in humans; however, currently there is a lack of repository for plant exclusive vsiRNAs. We have developed `PVsiRNAdb (http://www.nipgr.res.in/PVsiRNAdb)', a manually curated plant-exclusive database harboring information related to vsiRNAs found in different virus-infected plants collected by exhaustive data mining of published literature so far. This database contains a total of 322 214 entries and 282 549 unique sequences of vsiRNAs. In PVsiRNAdb, detailed and comprehensive information is available for each vsiRNA sequence. Apart from the core information consisting of plant, tissue, virus name and vsiRNA sequence, additional information of each vsiRNAs (map position, length, coordinates, strand information and predicted structure) may be of high utility to the user. Different types of search and browse modules with three different tools namely BLAST, Smith-Waterman Align and Mapping are provided at PVsiRNAdb. Thus, this database being one of its kind will surely be of much use to molecular biologists for exploring the complex viral genetics and genomics, viral-host interactions and beneficial to the scientific community and can prove to be very advantageous in the field of agriculture for producing viral resistance transgenic crops.

PtRFdb: a database for plant transfer RNA-derived fragments.

Transfer RNA-derived fragments (tRFs) represent a novel class of small RNAs (sRNAs) generated through endonucleolytic cleavage of both mature and precursor transfer RNAs (tRNAs). These 14-28 nt length tRFs that have been extensively studied in animal kingdom are to be explored in plants. In this study, we introduce a database of plant tRFs named PtRFdb (www.nipgr.res.in/PtRFdb), for the scientific community. We analyzed a total of 1344 sRNA sequencing datasets of 10 different plant species and identified a total of 5607 unique tRFs (758 tRF-1, 2269 tRF-3 and 2580 tRF-5), represented by 487 765 entries. In PtRFdb, detailed and comprehensive information is available for each tRF entry. Apart from the core information consisting of the tRF type, anticodon, source organism, tissue, sequence and the genomic location; additional information like PubMed identifier (PMID), Sample accession number (GSM), sequence length and frequency relevant to the tRFs may be of high utility to the user. Two different types of search modules (Basic Search and Advanced Search), sequence similarity search (by BLAST) and Browse option with data download facility for each search is provided in this database. We believe that PtRFdb is a unique database of its kind and it will be beneficial in the validation and further characterization of plant tRFs.Database URL: http://www.nipgr.res.in/PtRFdb/.