Genome-wide identification and characterization of DIRIGENT gene family (CcDIR) in pigeonpea (Cajanus cajan L.) provide insights on their spatial expression pattern and relevance to stress response.

This study is a thorough characterization of pigeonpea dirigent gene (CcDIR) family, an important component of the lignin biosynthesis pathway. Genome-wide analysis identified 25 CcDIR genes followed by a range of analytical approaches employed to unravel their structural and functional characteristics. Structural examination revealed a classic single exon and no intron arrangement in CcDIRs contributing to our understanding on evolutionary dynamics. Phylogenetic analysis elucidated evolutionary relationships among CcDIR genes with six DIR sub-families, while motif distribution analysis displayed and highlighted ten conserved protein motifs in CcDIRs. Promoter analyses of all the dirigent genes detected 18 stress responsive cis-acting elements offering insights into transcriptional regulation. While spatial expression analyses across six plant tissues showed preferential expression of CcDIR genes, exposure to salt (CcDIR2 and CcDIR9) and herbivory (CcDIR1, CcDIR2, CcDIR3 and CcDIR11), demonstrated potential roles of specific DIRs in plant defense. Interestingly, increased gene expression during herbivory, also correlated with increased lignin content authenticating the specific response. Furthermore, exogenous application of stress hormones, SA and MeJA on leaves significantly induced the expression of CcDIRs that responded to herbivory. Taken together, these findings contribute to a comprehensive understanding of CcDIR genes impacting development and stress response in the important legume pigeonpea.

Developmental stage-specific proteome analysis of the legume pod borer Maruca vitrata provides insights on relevant proteins.

The spotted pod borer, Maruca vitrata (Lepidoptera: Crambidae) is a destructive insect pest that inflicts significant productivity losses on important leguminous crops. Unravelling insect proteomes is vital to comprehend their fundamental molecular mechanisms. This research delved into the proteome profiles of four distinct stages -three larval and pupa of M. vitrata, utilizing LC-MS/MS label-free quantification-based methods. Employing comprehensive proteome analysis with fractionated datasets, we mapped 75 % of 3459 Drosophila protein orthologues out of which 2695 were identified across all developmental stages while, 137 and 94 were exclusive to larval and pupal stages respectively. Cluster analysis of 2248 protein orthologues derived from MaxQuant quantitative dataset depicted six clusters based on expression pattern similarity across stages. Consequently, gene ontology and protein-protein interaction network analyses using STRING database identified cluster 1 (58 proteins) and cluster 6 (25 proteins) associated with insect immune system and lipid metabolism. Furthermore, qRT-PCR-based expression analyses of ten selected proteins-coding genes authenticated the proteome data. Subsequently, functional validation of these chosen genes through gene silencing reduced their transcript abundance accompanied by a marked increase in mortality among dsRNA-injected larvae. Overall, this is a pioneering study to effectively develop a proteome atlas of M. vitrata as a potential resource for crop protection programs.

Unravelling the Genetic Basis of Moisture Deficit Stress Tolerance in Wheat for Seedling Vigour-Related Traits and Root Traits Using Genome-Wide Association Study.

A key abiotic stress that negatively affects seed germination, plant development, and crop yield is moisture deficit stress. Achieving higher vigour and uniform germination under stress conditions is essential for crop establishment and productivity and to enhance the yield. Hence, revealing wheat's capacity to withstand moisture deficit stress during seed germination and early growth stages is fundamental in improving its overall performance. However, the genetic regulation of moisture deficit stress tolerance during the seed germination phase remains largely unexplored. In this study, a total of 193 wheat genotypes were subjected to simulated moisture deficit stress using PEG-6000 (-0.4 MPa) during the seed germination stage. The induced moisture deficit stress significantly reduced various seedling-vigour-related traits. The genetic regions linked to these traits were found using a genome-wide association study (GWAS). The analysis identified 235 MTAs with a significance -log10(p) value of >4. After applying the Bonferroni correction, the study identified 47 unique single nucleotide polymorphisms (SNPs) that are linked to candidate genes important for the trait of interest. The current study emphasises the effectiveness of genome-wide association studies (GWAS) in identifying promising candidate genes, improving wheat seedling vigour and root traits, and offering essential information for the development of wheat cultivars tolerant to moisture deficit stress.

Identification, characterization, and comprehensive expression profiling of floral master regulators in pigeon pea (Cajanus cajan [L.] Millspaugh).

Pigeon pea is an important protein-rich pulse crop. Identification of flowering master regulators in pigeon pea is highly imperative as indeterminacy and late flowering are impediments towards yield improvement. A genome-wide analysis was performed to explore flowering orthologous groups in pigeon pea. Among the 412 floral orthologs identified in pigeon pea, 148 genes belong to the meristem identity, photoperiod-responsive, and circadian clock-associated ortholog groups. Our comparative genomics study revealed purifying selection pressures (ka/ks) on floral orthologs, and duplication patterns and evolution through synteny with other model species. Phylogenetic analysis of floral genes substantiated a connection between pigeon pea plant architecture and flowering time as all the PEBP domain-containing genes belong to meristem identity floral networks of pigeon pea. Expression profiling of eleven major orthologs in contrasting determinate and indeterminate genotypes indicated that these orthologs might be involved in flowering regulation. Expression of floral inducer, FT, and floral repressor, TFL1, was non-comparable in indeterminate genotypes across all the developmental stages of pigeon pea. However, dynamic FT/TFL1 expression ratio detected in all tissues of both the genotypes suggested their role in floral transition. One TFL1 ortholog having high sequence conserveness across pigeon pea genotypes showed differential expression indicating genotype-dependent regulation of this ortholog. Presence of conserved 6mA-methylation patterns in light-responsive elements and in other cis-regulatory elements of FT and TFL1 across different plant genotypes indicated possible involvement of epigenetic regulation in flowering.

Phylogenomic Analysis of micro-RNA Involved in Juvenile to Flowering-Stage Transition in Photophilic Rice and Its Sister Species.

Vegetative to reproductive phase transition in phototropic plants is an important developmental process and is sequentially mediated by the expression of micro-RNA MIR172. To obtain insight into the evolution, adaptation, and function of MIR172 in photophilic rice and its wild relatives, we analyzed the genescape of a 100 kb segment harboring MIR172 homologs from 11 genomes. The expression analysis of MIR172 revealed its incremental accumulation from the 2-leaf to 10-leaf stage, with maximum expression coinciding with the flag-leaf stage in rice. Nonetheless, the microsynteny analysis of MIR172s revealed collinearity within the genus Oryza, but a loss of synteny was observed in (i) MIR172A in O. barthii (AA) and O. glaberima (AA); (ii) MIR172B in O. brachyantha (FF); and (iii) MIR172C in O. punctata (BB). Phylogenetic analysis of precursor sequences/region of MIR172 revealed a distinct tri-modal clade of evolution. The genomic information generated in this investigation through comparative analysis of MIRNA, suggests mature MIR172s to have evolved in a disruptive and conservative mode amongst all Oryza species with a common origin of descent. Further, the phylogenomic delineation provided an insight into the adaptation and molecular evolution of MIR172 to changing environmental conditions (biotic and abiotic) of phototropic rice through natural selection and the opportunity to harness untapped genomic regions from rice wild relatives (RWR).

Host-Delivered RNA Interference for Durable Pest Resistance in Plants: Advanced Methods, Challenges, and Applications.

Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effective plant protection strategy to attain durable insect-pest resistance in crops. Development of transgenic plant using novel strategies to achieve durable resistance against the target insect.

Drought and Oxidative Stress in Flax (Linum usitatissimum L.) Entails Harnessing Non-Canonical Reference Gene for Precise Quantification of qRT-PCR Gene Expression.

Flax (Linum usitatissimum L.) is a self-pollinating, annual, diploid crop grown for multi-utility purposes for its quality oil, shining bast fiber, and industrial solvent. Being a cool (Rabi) season crop, it is affected by unprecedented climatic changes such as high temperature, drought, and associated oxidative stress that, globally, impede its growth, production, and productivity. To precisely assess the imperative changes that are inflicted by drought and associated oxidative stress, gene expression profiling of predominant drought-responsive genes (AREB, DREB/CBF, and ARR) was carried out by qRT-PCR. Nevertheless, for normalization/quantification of data obtained from qRT-PCR results, a stable reference gene is mandatory. Here, we evaluated a panel of four reference genes (Actin, EF1a, ETIF5A, and UBQ) and assessed their suitability as stable reference genes for the normalization of gene expression data obtained during drought-induced oxidative stress in flax. Taking together, from the canonical expression of the proposed reference genes in three different genotypes, we report that EF1a as a stand-alone and EF1a and ETIF5A in tandem are suitable reference genes to be used for the real-time visualization of cellular impact of drought and oxidative stress on flax.

Amelioration of cold-induced sweetening in potato by RNAi mediated silencing of StUGPase encoding UDP-glucose pyrophosphorylase.

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Cloning and Molecular Characterization of the phlD Gene Involved in the Biosynthesis of "Phloroglucinol", a Compound with Antibiotic Properties from Plant Growth Promoting Bacteria Pseudomonas spp.

phlD is a novel kind of polyketide synthase involved in the biosynthesis of non-volatile metabolite phloroglucinol by iteratively condensing and cyclizing three molecules of malonyl-CoA as substrate. Phloroglucinol or 2,4-diacetylphloroglucinol (DAPG) is an ecologically important rhizospheric antibiotic produced by pseudomonads; it exhibits broad spectrum anti-bacterial and anti-fungal properties, leading to disease suppression in the rhizosphere. Additionally, DAPG triggers systemic resistance in plants, stimulates root exudation, as well as induces phyto-enhancing activities in other rhizobacteria. Here, we report the cloning and analysis of the phlD gene from soil-borne gram-negative bacteria-Pseudomonas. The full-length phlD gene (from 1078 nucleotides) was successfully cloned and the structural details of the PHLD protein were analyzed in-depth via a three-dimensional topology and a refined three-dimensional model for the PHLD protein was predicted. Additionally, the stereochemical properties of the PHLD protein were analyzed by the Ramachandran plot, based on which, 94.3% of residues fell in the favored region and 5.7% in the allowed region. The generated model was validated by secondary structure prediction using PDBsum. The present study aimed to clone and characterize the DAPG-producing phlD gene to be deployed in the development of broad-spectrum biopesticides for the biocontrol of rhizospheric pathogens.

The plant specialized metabolite epicatechin- 3-gallate (EC3G) perturbs lipid metabolism and attenuates fat accumulation in pigeonpea pod borer, Helicoverpa armigera.

Control of pod borer Helicoverpa armigera, a notorious polyphagous pest requires paramount attention with focus on environment-friendly management approaches. Overproduction of catechins (epigallocatechin-EGC and epicatechin-3-gallate-EC3G) in the pod borer-resistant pigeonpea wild relative, Cajanus platycarpus during continued herbivory prodded us to assess their underlying molecular effect on H. armigera. Significant reduction in larval and pupal growth parameters was observed when reared on artificial diet incorporated with 100 ppm EC3G vis a vis 100 ppm EGC and EGC + EC3G. Comparative RNAseq analyses of larvae that fed on normal and EC3G-incorporated diet revealed 62 differentially expressed genes dominated by detoxification and lipid metabolism. While lipase and fatty acid-binding protein 2-like were up-regulated, delta9-FADS-like involved in fatty acid synthesis was downregulated, indicating effect of EC3G on fat metabolism. Validation of RNAseq data by qPCR; midgut glutathione-S-transferase and esterase assays depicted increased lipolysis and reduced lipogenesis in EC3G-fed larvae. Additionally, differential accumulation of stearic acid and oleic acid in EC3G-fed and control larvae/adults ascertained perturbation in lipogenesis. Supported by modelling, molecular docking and simulations, we demonstrate the possible involvement of the insect adipokinetic hormone receptor (AKHR) in the EC3G-mediated response. The study demonstrates plant specialized metabolite EC3G as a potential candidate for H. armigera control.

Cajanus platycarpus Flavonoid 3'5' Hydroxylase_2 (CpF3'5'H_2) Confers Resistance to Helicoverpa armigera by Modulating Total Polyphenols and Flavonoids in Transgenic Tobacco.

Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3'5' hydroxylase_2 (F3'5'H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3'5'H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3'5'H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3'5'H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3'5'H_2. The relevance of CpF3'5'H_2 in H. armigera resistance was further validated in CpF3'5'H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3'5'H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3'5'H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs.

RNAi-based knockdown of candidate gut receptor genes altered the susceptibility of Spodoptera frugiperda and S. litura larvae to a chimeric toxin Cry1AcF.

Background: A multitude of Cry toxins (secreted by Bacillus thuringiensis or Bt) has been deployed globally either via transgenic mean or bio-pesticidal formulations in order to manage insect pests. However, Bt resistance development in insects is emerging as a major concern. To avoid this problem, multiple gene pyramiding or protein-engineered chimeric toxin-based strategy has been analyzed. Methods: In the present study, one such chimeric toxin Cry1AcF (contain the swapped domains of Cry1Ac and Cry1F) was used to investigate its in vivo pathogenesis process in lepidopteran pests Spodoptera frugiperda and S. litura. A number of biochemical and molecular analysis were performed. Results: Oral ingestion of Cry1AcF caused greater toxicity in S. frugiperda than S. litura with larvae displaying increased hemolymph melanization. Histopathology of the midgut transverse sections exhibited Cry1AcF-induced extensive gut damage in both the test insects followed by cytotoxicity in terms of reduced hemocyte numbers and viability. Elevated hemolymph phenoloxidase activity indicated the immune-stimulatory nature of Cry1AcF. In order to analyze the role of gut receptor proteins in Cry1AcF intoxication in test insects, we performed RNAi-mediated silencing using bacterially-expressed dsRNAs of individual receptor-encoding genes including CAD, ABCC2, ALP1 and APN. Target-specific induced downregulation of receptor mRNAs differentially altered the insect susceptibility to Cry1AcF toxin in our study. The susceptibility of ALP1 and APN dsRNA pre-treated S. frugiperda was considerably decreased when treated with Cry1AcF in LD50 and LD90 doses, whereas susceptibility of CAD and ABCC2 dsRNA pre-treated S. litura was significantly reduced when ingested with Cry1AcF in different doses. CAD/ABCC2-silenced S. frugiperda and ALP1/APN-silenced S. litura were vulnerable to Cry1AcF alike of control larvae. In conclusion, our results indicate ALP1/APN and CAD/ABCC2 as the functional receptor for Cry1AcF toxicity in S. frugiperda and S. litura, respectively.

A novel tasi RNA-based micro RNA-induced gene silencing strategy to tackle multiple pests and pathogens in cotton (Gossypium hirsutum L.).

MAIN CONCLUSION: This study demonstrates the combinatorial management of multiple pests through a trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy. Transgenic cotton events demonstrated improved efficacy against cotton leaf curl disease, cotton leaf hopper and root-knot nematode. Cotton (Gossypium hirsutum L.), an important commercial crop grown worldwide is confronted by several pests and pathogens, thus reiterating interventions for their management. In this study, we report, the utility of a novel Arabidopsis miRNA173-directed trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy for the simultaneous management of cotton leaf curl disease (CLCuD), cotton leaf hopper (CLH; Amrasca biguttula biguttula) and root-knot nematode (RKN, Meloidogyne incognita). Cotton transgenics were developed with the MIGS construct targeting a total of 7 genes by an apical meristem-targeted in planta transformation strategy. Stable transgenics were selected using stringent selection pressure, molecular characterization and stress-specific bio-efficacy studies. We identified 8 superior events with 50-100% resistance against CLCuD, while reduction in the root-knot nematode multiplication factor in the range of 35-75% confirmed resistance to RKN. These transgenic cotton events were also detrimental to the growth and development of CLH, as only 43.3-62.5% of nymphs could survive. Based on the corroborating evidences obtained by all the bioefficacy analyses, 3 events viz., L-75-1, E-27-11, E-27-7 were found to be consistent in tackling the target pests. To the best of our knowledge, this report is the first of its kind demonstrating the possibility of combinatorial management of pests/diseases in cotton using MIGS approach. These identified events demonstrate immense utility of the strategy towards combinatorial stress management in cotton improvement programs.

Amenability of an Agrobacterium tumefaciens-mediated shoot apical meristem-targeted in planta transformation strategy in Mango (Mangifera indica L.).

Mango (Mangifera indica L.) is one of the most popular tropical fruits in the world owing to its rich taste, flavor, color, production volume and diverse end usage. Conventional mango breeding practices are unable to withstand the demand for improved varieties as it is time consuming and requires heavy investment. However, problems associated with traditional plant breeding can be curtailed through genetic transformation. Nevertheless, major limitation of transgenic development has been its recalcitrant nature toward tissue culture practices involving latent microbial infection, phenol exudation, etc. This opens wide scope for tissue culture-independent in planta transformation approaches These strategies have proved to be easy to execute and cost effective in producing large number of transformants. One such apical meristem targeted in planta approach was successfully exploited to demonstrate its utility in transforming a tree species. Mango variety Amrapali was transformed with two visual marker gene vectors GFP::hptII in pCAMBIA1302 and GUS::nptII in pCAMBIA2301 individually, to demonstrate its amenability. Preliminary confirmations identified 65.0% of GFP and 57.14% of GUS plants to be transformed. Further, molecular characterization of these primary transformants demonstrated transgene integration at genomic and transcript level in some of the plants. This established protocol holds good for functional gene validation and knock in/out studies and aid in mango improvement programs.

Reactive oxygen species in plants: an invincible fulcrum for biotic stress mitigation.

Climate change-associated environmental vagaries have amplified the incidence of pests and pathogens on plants, thus imparting the increased quest for management strategies. Plants respond to stresses through intricate signaling networks that regulate diverse cellular mechanisms. Reactive oxygen species (ROS) are cardinal towards the maintenance of normal plant activities as well as improving stress management. Plants that exhibit a fine balance between ROS levels and its management apparently mitigate stresses better. There have been very many compendiums on signaling and management of ROS during several abiotic stresses. However, expansion of knowledge related to ROS induction and homeostasis during biotic stresses is pertinent. Hence, considering its importance, we provide insights in this review on how plants signal and manage ROS upon an oxidative burst during their interaction with pathogens and herbivores. Substantial degree of molecular changes and pivotal roles of ROS have been detected during phyto-pathogen/herbivore interactions, opening novel platforms to understand signaling/management of events under varied biotic stresses. It is interesting to know that, though plants react to biotic stresses through oxidative burst, receptors and elicitors involved in the signal transduction differ across stresses. The review provides explicit details about the specific signaling of ROS production in plants under pathogen and herbivore attack. Furthermore, we also provide an update about tackling the accumulated ROS under biotic stresses as another pivotal step. ROS signaling and homeostasis can be exploited as critical players and a fulcrum to tackle biotic stresses, thus paving the way for futuristic combinatorial stress management strategies. KEY POINTS: • The review is a comprehension of redox signaling and management in plants during herbivory and pathogen infection • Reactive oxygen species (ROS) is an important factor during normal plant activities as well as in their response to stresses. Diverse modes of ROS signaling and management have been observed during both biotic stresses independently • Exploration of plant biology in multi-stress resistant plants like the crop wild relatives could pave the way for combinatorial management of stress for a better tomorrow.

RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin.

BACKGROUND: Due to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella. RESULTS: Present study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein-protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process. CONCLUSION: Since the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.

Deciphering of Pod Borer [Helicoverpa armigera (Hübner)] Resistance in Cajanus platycarpus (Benth.) Offers Novel Insights on the Reprogramming and Role of Flavonoid Biosynthesis Pathway

Management of pod borer, Helicoverpa armigera in pigeonpea (Cajanus cajan L.), an important legume crop, has been a pertinent endeavor globally. As with other crops, wild relatives of pigeonpea are bestowed with various resistance traits that include the ability to deter the H. armigera. Understanding the molecular basis of pod borer resistance could provide useful leads for the management of this notorious herbivore. Earlier studies by our group in deciphering the resistance response to herbivory through multiomics approaches in the pigeonpea wild relative, Cajanus platycarpus, divulged the involvement of the flavonoid biosynthesis pathway, speculating an active chemical response of the wild relative to herbivory. The present study is a deeper understanding of the chemical basis of pod borer (H. armigera) resistance in, C. platycarpus, with focus on the flavonoid biosynthesis pathway. To substantiate, quantification of transcripts in H. armigera-challenged C. platycarpus (8 h, 24 h, 48 h, 96 h) showed dynamic upregulation (up to 11-fold) of pivotal pathway genes such as chalcone synthase, dihydroflavonol-4-reductase, flavonoid-3'5'-hydroxylase, flavonol synthase, leucoanthocyanidin reductase, and anthocyanidin synthase. Targeted LC-MS analyses demonstrated a concomitant increase (up to 4-fold) in naringenin, kaempferol, quercetin, delphinidin, cyanidin, epigallocatechin, and epicatechin-3-gallate. Interestingly, H. armigera diet overlaid with the over-produced flavonoids (100 ppm) showed deleterious effects on growth leading to a prolonged larval period demonstrating noteworthy coherence between over-accumulation of pathway transcripts/metabolites. The study depicts novel evidence for the directed metabolic reprogramming of the flavonoid biosynthesis pathway in the wild relative to pod borer; plant metabolic potential is worth exploiting for pest management.

Relevance of methionine sulfoxide reductase(s) (MSR) as candidate proteins in redox homeostasis-mediated resistance response to Helicoverpa armigera (Hübner) in the pigeonpea wild relative Cajanus platycarpus (Benth.) Maesen.

Pod borer, Helicoverpa armigera, a polyphagus herbivore causes extensive economic losses to crops, including pigeonpea. Exploitation of pod borer resistance in wild relatives is pertinent due to the absence of resistance sources in cultivated pigeonpea and crossing-incompatibility with the resistant wild relatives. We present leads obtained in deeper understanding of pod borer resistance mechanism in Cajanus platycarpus, a pigeonpea wild relative. Surge in cellular ROS during herbivory leads to redox-PTMs (post translational modifications) of methionine-rich proteins including antioxidant enzymes, causing oxidative damage. Plants then officiate methionine sulfoxide reductases (MSRs), that maintain the redox status of methionine and hence homeostasis. We demonstrate functionality of MSRs (MSRA and MSRB) in the resistance response of the wild relative to pod borer. Among 5 MSRA and 3 MSRB genes, CpMSRA2 and CpMSRB1 were herbivore-responsive based on expression during herbivory. Clues about the stress-responsiveness were obtained upon analyses of cis-elements and co-expressing genes. Apparently, the wild relative followed a non-canonical mode of redox management, as divulged by antioxidant enzymes and the scavenging capacity. Differential lipid peroxidation as an early response provided evidences for an effective redox management in the wild relative. This is the first report signifying redox homeostasis in the resistance response towards herbivory.

Exploring Genomic Variations in Nematode-Resistant Mutant Rice Lines.

Rice (Oryza sativa) production is seriously affected by the root-knot nematode Meloidogyne graminicola, which has emerged as a menace in upland and irrigated rice cultivation systems. Previously, activation tagging in rice was utilized to identify candidate gene(s) conferring resistance against M. graminicola. T-DNA insertional mutants were developed in a rice landrace (acc. JBT 36/14), and four mutant lines showed nematode resistance. Whole-genome sequencing of JBT 36/14 was done along with the four nematode resistance mutant lines to identify the structural genetic variations that might be contributing to M. graminicola resistance. Sequencing on Illumina NovaSeq 6000 platform identified 482,234 genetic variations in JBT 36/14 including 448,989 SNPs and 33,245 InDels compared to reference indica genome. In addition, 293,238-553,648 unique SNPs and 32,395-65,572 unique InDels were found in the four mutant lines compared to their JBT 36/14 background, of which 93,224 SNPs and 8,170 InDels were common between all the mutant lines. Functional annotation of genes containing these structural variations showed that the majority of them were involved in metabolism and growth. Trait analysis revealed that most of these genes were involved in morphological traits, physiological traits and stress resistance. Additionally, several families of transcription factors, such as FAR1, bHLH, and NAC, and putative susceptibility (S) genes, showed the presence of SNPs and InDels. Our results indicate that subject to further genetic validations, these structural genetic variations may be involved in conferring nematode resistance to the rice mutant lines.

Molecular characterization and functional analysis of Cry toxin receptor-like genes from the model insect Galleria mellonella.

Crystal (Cry) toxins produced from the soil bacterium, Bacillus thuringiensis (Bt), have gained worldwide attention for long due to their insecticidal potential. A number of receptor proteins located on the epithelial cells of the larval midgut were shown to be crucial for Cry intoxication in different insect pests belonging to order Lepidoptera, Diptera and Coleoptera. A beehive pest, Galleria mellonella, serves as an excellent insect model for biochemical research. However, information on the Cry receptor-like genes in G. mellonella is limited. In the present study, the full-length sequences of four putative Cry receptor genes (ABC transporter, alkaline phosphatase, aminopeptidase N and cadherin) were cloned from G. mellonella. All these receptor genes were substantially upregulated in the midgut tissue of fourth-instar G. mellonella larvae upon early exposure (6 h) to a sub-lethal dose of Cry1AcF toxin. Oral and independent delivery of bacterially-expressed dsRNAs corresponding to four receptor genes in G. mellonella suppressed the transcription of target receptors which in turn significantly reduced the larval sensitivity to Cry1AcF toxin. As the laboratory populations of G. mellonella develop Bt resistance in a relatively short time, molecular characterization of Cry receptor genes in G. mellonella performed in the present study may provide some useful information for future research related to the genetic basis of Bt resistance in the model insect.