INTRODUCTION:The legume pod borer, Helicoverpa armigera, is a major biotic constraint to chickpea production, causing substantial global yield losses. The downstream signaling pathways underlying host-insect interactions remain largely unexplored, yet understanding these processes is crucial for developing pest-resilient cultivars. METHODS:A systems biology approach was employed to investigate the leaf proteome of 2 cultivated chickpea genotypes (JG 12 and JG 36) and 1 moderately tolerant wild genotype (ILWC 46) under simulated herbivory. This study aimed to elucidate molecular mechanisms underlying plant defense against insect attack. RESULTS:Proteomic profiling revealed 190-200, 170-210, and 170-200 total protein spots in ILWC 46, JG12, and JG 36, respectively. 76 Differentially Expressed Proteins (DEPs) were visually identified, of which 62 were selected for Mass Spectrometric (MS) analysis. ILWC 46 exhibited 22 DEPs (4 downregulated, 18 upregulated), JG 12 showed 23 DEPs (5 downregulated, 18 upregulated), and JG 36 had 17 DEPs (10 downregulated, 7 upregulated). Five proteins were common to JG 12 and JG 36. DISCUSSION:Genotype-specific proteomic changes suggest variable defense responses: predominant upregulation in ILWC 46 indicates enhanced metabolic activity under stress, whereas JG 36's downregulation implies reduced adaptability. Functional categorization showed modulation of photosynthesis, primary metabolism, and stress-related proteins, highlighting energy reallocation and activation of defense pathways sustaining stress tolerance differences among genotypes. CONCLUSION:This study provides a comprehensive proteomic perspective on chickpea defense responses to herbivory, highlighting candidate proteins and pathways for enhancing insect resistance in both cultivated and wild germplasm.

Abstract Insect herbivory triggers cytosolic proteome reprogramming by activating defense pathways and modulating key metabolic processes. We found that simulated herbivory in pigeon pea ( Cajanus cajan ) induced reactive oxygen species (ROS) production and molecular alterations within 12 hours (h) of post treatment. We compared the leaf proteome profiles of two cultivated genotypes, ICPL 332 (moderately resistant) and ICPL 87 (susceptible), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry (MS). More than 220 protein spots were detected in ICPL 332 and over 200 in ICPL 87. Comparative analysis revealed 75 differentially accumulated proteins (DAPs), of which 40 were consistently reproducible across biological replicates. These included 11 unique to ICPL 87, 9 unique to ICPL 332, and 10 common to both genotypes. Among the shared DAPs, ICPL 332 showed five upregulated and five downregulated, whereas ICPL 87 exhibited only two upregulated and eight downregulated. Functional categorization grouped DAPs into primary metabolism, stress response, and growth and development. Proteins related to primary metabolism were largely downregulated in both genotypes, while stress-associated proteins exhibited substantial downregulation in ICPL 87 compared to ICPL 332. Overall, the results demonstrate proteomic adjustments underlying defense responses in pigeon pea genotypes.

Pigeon pea (Cajanus cajan), widely grown in India, suffers significant yield losses due to pod borers (Helicoverpa armigera and Maruca vitrata). Therefore, studying the host resistance mechanism is pivotal for crop improvement. In this study, we conducted transcriptome analysis on two wild-type (WT) Cajanus scarabaeoides accessions (ICP-15761 and ICP-15738) having high levels of resistance to pod borers and two cultivated C. cajan genotypes, ICPL-332 (moderately resistant) and ICPL-87 (susceptible), following simulated herbivory with H. armigera oral secretions (OS). Differential gene expression analysis identified 3573 and 4677 differentially expressed genes (DEGs) in ICP-15761 and ICP-15738, whereas 4149 and 3639 DEGs were documented in ICPL-332 and ICPL-87, respectively. Genes related to chloroplast biogenesis, photosynthesis, and chlorophyll metabolism exhibited significant differential expression, indicating chloroplast reprogramming under simulated herbivory. Significant upregulation of key defense genes, including chitinases and cysteine proteases, in C. scarabaeoides accessions highlighted robust defense pathway activation. A genotype-specific shift in transcription factors, phytohormones, and calcium signaling-related gene expression was noted. Higher levels of expression of aspartic proteinases and pathogenesis-related proteins in cultivated genotypes suggesting adaptive evolutionary traits. This is a novel insight on molecular mechanism of defense in a wild type, C. scarabaeoides and cultivated genotypes of pigeon pea under simulated herbivory. The information on cytosolic and organellar gene changes in pigeon pea due to H. armigera OS mediated-simulated herbivory may help develop pigeon pea varieties that are resistant to pod borer infestations.

Plant-herbivore interactions create a link between the organisms in various environments, including freshwater, marine, and terrestrial ecosystems. These connections are the essential link that generates animal biomass from simple sunlight in everything from simple organisms to complex organisms. Thus, a comprehensive study of the ecology and evolution of almost every ecosystem depends on these relationships. Metabolomics can be adopted to gain more insights and to study this plant-herbivore interaction in depth. Metabolomics, through the production of a profile of small molecules obtained from cellular metabolism, offers knowledge regarding numerous processes of plant physiology by showing the complex networks formed through various biochemical events in the cell. This chapter provides a guidance to studies of the current changes in the food chain, thus helping us to understand the environment better.

Defensins are small, cysteine-rich peptides involved in plant defense, though their insecticidal properties remain largely unexplored. Previously, based on transcriptome we identified a defensin gene in black gram in response to bruchid (Callosobruchus maculatus) infestation. In the present study, we cloned and sequenced full-length cDNAs of defensin genes from multiple legumes and conducted phylogenetic analyses. Two sequence variants were identified, exhibiting 95-98% homology with a previously reported insecticidal defensin gene (Accession no. AF326687). Variant 1 (DefV1) was present in black gram, pea, cowpea, and common bean, whereas variant 2 (DefV2) was identified in mung bean, chickpea, and pigeon pea. Computational analysis, including molecular docking, visualization, and molecular dynamics (MD) simulations, demonstrated enhanced interactions between DefV1 and bruchid α-amylase, suggesting a "Cork in the Bottle" inhibitory mechanism. Additionally, insect bioassays using artificial seeds supplemented with DefV1 showed no adult emergence. These findings highlight black gram defensin as a promising insecticidal agent and a potential candidate for genetic improvement of bruchid resistance in legumes.

Rice cultivation in Northeast India (NEI) primarily relies on rainfed conditions, making it susceptible to severe drought spells that promote the onset of brown spot disease (BSD) caused by Bipolaris oryzae. This study investigates the response of prevalent rice cultivars of NEI to the combined stress of drought and B. oryzae infection. Morphological, physiological, biochemical, and molecular changes were recorded post-stress imposition. Qualitative assessment of reactive oxygen species through DAB (3,3-diaminobenzidine) assay confirmed the elicitation of plant defense responses. Based on drought scoring system and biochemical analyses, the cultivars were categorized into susceptible (Shasharang and Bahadur), moderately susceptible (Gitesh and Ranjit), and moderately tolerant (Kapilee and Mahsuri) groups. Antioxidant enzyme accumulation (catalase, guaiacol peroxidase) and osmolyte (proline) levels increased in all stressed plants, with drought-tolerant cultivars exhibiting higher enzyme activities, indicating stress mitigation efforts. Nevertheless, electrolyte leakage and lipid peroxidation rates increased in all stressed conditions, though variations were observed among stress types. Based on findings from a previous transcriptomic study, a total of nine genes were chosen for quantitative real-time PCR analysis. Among these, OsEBP89 appeared as a potential negative regulatory gene, demonstrating substantial upregulation in the susceptible cultivars at both 48 and 72 h post-treatment (hpt). This finding suggests that OsEBP89 may play a role in conferring drought-induced susceptibility to BSD in the rice cultivars being investigated.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.

Abstract Background Bacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34′39.11''N and Longitude: 93°10′16.04''E). Results The whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein. Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences. Conclusion This is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

A multitude of climatic factors, such as temperature, drought, salinity, and heavy metal toxicity, affect agricultural production. In this respect, to avert a drop in agricultural productivity resulting from climate change, plant breeding innovations and genetic engineering strategies to boost abiotic stress tolerance are important. Biotechnologists have been able to tweak any DNA sequence with excellent accuracy via the latest discoveries in first-generation genome editing techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). These strategies, however, are expensive and laborious, since they require complex steps involving protein engineering. Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas strategy as a second-generation genome editing system has gained great interest over the last few years as an effective method to create heritable genetic mutations. Using the CRISPR/Cas strategy, a handful of studies have been reported to date on improving abiotic stress resilience in crop plants. In addition, the development of more specialized forms of Cas9 proteins such as Cpf1, dCas9 greatly enhances the flexibility of the CRISPR/Cas system. This chapter focuses on the perspective and promising applications, recent advances, and potential challenges of targeted genome engineering for increased plant tolerance to abiotic stress and productivity.

Precise genome engineering approaches could be perceived as a second paradigm for targeted trait improvement in crop plants, with the potential to overcome the constraints imposed by conventional CRISPR/Cas technology. The likelihood of reduced agricultural production due to highly turbulent climatic conditions increases as the global population expands. The second paradigm of stress-resilient crops with enhanced tolerance and increased productivity against various stresses is paramount to support global production and consumption equilibrium. Although traditional breeding approaches have substantially increased crop production and yield, effective strategies are anticipated to restore crop productivity even further in meeting the world’s increasing food demands. CRISPR/Cas, which originated in prokaryotes, has surfaced as a coveted genome editing tool in recent decades, reshaping plant molecular biology in unprecedented ways and paving the way for engineering stress-tolerant crops. CRISPR/Cas is distinguished by its efficiency, high target specificity, and modularity, enables precise genetic modification of crop plants, allowing for the creation of allelic variations in the germplasm and the development of novel and more productive agricultural practices. Additionally, a slew of advanced biotechnologies premised on the CRISPR/Cas methodologies have augmented fundamental research and plant synthetic biology toolkits. Here, we describe gene editing tools, including CRISPR/Cas and its imitative tools, such as base and prime editing, multiplex genome editing, chromosome engineering followed by their implications in crop genetic improvement. Further, we comprehensively discuss the latest developments of CRISPR/Cas technology including CRISPR-mediated gene drive, tissue-specific genome editing, dCas9 mediated epigenetic modification and programmed self-elimination of transgenes in plants. Finally, we highlight the applicability and scope of advanced CRISPR-based techniques in crop genetic improvement.

With the increasing volume of the world population, there is a relatively increased demand for food production. Conventional breeding methods no more remain viable to overcome the increase in food demand. The field of biotechnology and molecular biology has opened up new horizons of science by revolutionizing the agriculture and farming methods. It has greatly facilitated our understanding of the molecular processes underlying the stress tolerance mechanism of plants, by providing new breakthroughs. Advancements in the fields of genomics, stress biology, and bioinformatics can help in the development of stress-tolerant crops. Simultaneously, advanced molecular biology techniques have provided us with the most attractive tools for typical laboratory approaches. In this chapter, we discuss the advanced molecular approaches and novel biotechnological tools for sustainable crop improvement. A number of techniques including plant tissue culture, mutagenesis, and transformation have been presented. Advanced functional genomics studies give a better understanding of the plant genome and its modification. RNA interference, CRISPR/Cas, next-generation sequencing, and nanotechnology have become new promising techniques for improving crops according to future need. These are viable options to obtain improved genotypes that can survive under changing climate. In this chapter, we also focus on the applications of molecular biology for crop improvement, such as allele mining, gene pyramiding, linkage and association mapping, genetic engineering (GE), molecular breeding (MB), marker-assisted backcrossing (MABC), marker-assisted recurrent selection (MARS), genome-wide selection (GWS), and next-generation sequencing (NGS). Lastly, we also highlight a field case study considered as one of the few examples of trait improvement in plants.

Green tea or its concentrated extract is coveted for its health promoting catechin-like polyphenols, especially epigallocatechin-3-gallate (EGCG). However, its amicable efficacy is now being doubted considering the recent occurrence of several cases of hepato- and nephrotoxicity, after the ingestion of EGCG-fortified (>= 85-90%) nutritional supplements. Therefore, the current study was carried out to ascertain the effect of green tea leaves extract (GTE), having low EGCG content (73.8%), on liver and kidney functions of male Wistar rats using various in vivo experiments and in vitro radical scavenging activity. In terms of acute toxicity, GTE was observed to be safe when delivered at a dosage of 2000 mg kg(-1) body weight (BW). Oral delivery of GTE for 28 days at a concentration of 200 mg kg(-1) BW/day did not trigger sub-acute toxicity to the liver and kidneys, as per serum biochemical analyses and histopathological examination. In contrast, GTE counteracted the effects of carbon tetrachloride (a potent hepato-degenerative compound) on the liver. Furthermore, increase in high-density lipoprotein-cholesterol with concomitant lowering of serum triglycerides and low-density lipoprotein-cholesterol were noticed in GTE-treated rats. These findings suggest that low EGCG containing GTE, with appreciable antioxidant activity (IC50 = 53.18-71.28 mu g mL(-1)), can serve as a hepatoprotective, hypolipidemic, and hypocholesterolemic ingredient.

Globally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.

Background Black gram [ Vigna mungo (L)] seeds are a rich source of digestible protein and dietary fibre, both for human and animal consumption. However, the quality and quantity of the Vigna seeds are severely affected by bruchid beetles during storage. Therefore, analyses of the expression of the bruchid induced transcript dynamics in black gram pods would be helpful to understand the underlying defense mechanism against bruchid oviposition. Results We used the RNAseq approach to survey the changes in transcript profile in the developing seeds of a moderately resistant cultivar IC-8219 against bruchid oviposition using a susceptible cultivar T-9 as a control. A total of 96,084,600 and 99,532,488 clean reads were generated from eight (4 each) samples of IC-8219 and T-9 cultivar, respectively. Based on the BLASTX search against the NR database, 32,584 CDSs were generated of which 31,817 CDSs were significantly similar to Vigna radiata , a close relative of Vigna mungo . The IC-8219 cultivar had 630 significantly differentially expressed genes (DEGs) of which 304 and 326 genes up and down-regulated, respectively. However, in the T-9 cultivar, only 168 DEGs were identified of which 142 and 26 genes up and down-regulated, respectively. The expression analyses of 10 DEGs by qPCR confirmed the accuracy of the RNA-Seq data. Gene Ontology and KEGG pathway analyses helped us to better understand the role of these DEGs in oviposition mediated defense response of black gram. In both the cultivars, the most significant transcriptomic changes in response to the oviposition were related to the induction of defense response genes, transcription factors, secondary metabolites, enzyme inhibitors, and signal transduction pathways. It appears that the bruchid ovipositioning mediated defense response in black gram is induced by SA signaling pathways and defense genes such as defensin, genes for secondary metabolites, and enzyme inhibitors could be potential candidates for resistance to bruchids. Conclusion We generated a transcript profile of immature black gram pods upon bruchid ovipositioning by de novo assembly and studied the underlying defense mechanism of a moderately resistant cultivar.

Main conclusion In a nutshell, tissue-specific CRISPR/Cas genome editing is the most promising approach for crop improvement which can bypass the hurdle associated with constitutive GE such as off target and pleotropic effects for targeted crop improvement. CRISPR/Cas is a powerful genome-editing tool with a wide range of applications for the genetic improvement of crops. However, the constitutive genome editing of vital genes is often associated with pleiotropic effects on other genes, needless metabolic burden, or interference in the cellular machinery. Tissue-specific genome editing (TSGE), on the other hand, enables researchers to study those genes in specific cells, tissues, or organs without disturbing neighboring groups of cells. Until recently, there was only limited proof of the TSGE concept, where the CRISPR-TSKO tool was successfully used in Arabidopsis, tomato, and cotton, laying a solid foundation for crop improvement. In this review, we have laid out valuable insights into the concept and application of TSGE on relatively unexplored areas such as grain trait improvement under favorable or unfavorable conditions. We also enlisted some of the prominent tissue-specific promoters and described the procedure of their isolation with several TSGE promoter expression systems in detail. Moreover, we highlighted potential negative regulatory genes that could be targeted through TSGE using tissue-specific promoters. In a nutshell, tissue-specific CRISPR/Cas genome editing is the most promising approach for crop improvement which can bypass the hurdle associated with constitutive GE such as off target and pleotropic effects for targeted crop improvement.