Transgenic tobacco plants overexpressing a wheat salt stress root protein (TaSSRP) exhibit enhanced tolerance to heat stress.

BACKGROUND: Heat stress is a detrimental abiotic stress that limits the development of many plant species and is linked to a variety of cellular and physiological problems. Heat stress affects membrane fluidity, which leads to negative effects on cell permeability and ion transport. Research reveals that heat stress causes severe damage to cells and leads to rapid accumulation of reactive oxygen species (ROS), which could cause programmed cell death. METHODS AND RESULTS: This current study aimed to validate the role of Triticum aestivum Salt Stress Root Protein (TaSSRP) in plants' tolerance to heat stress by modulating its expression in tobacco plants. The Relative Water Content (RWC), total chlorophyll content, and Membrane Stability Index (MSI) of the seven distinct transgenic lines (T0 - 2, T0 - 3, T0 - 6, T0 - 8, T0 - 9, T0 - 11, and T0 - 13), increased in response to heat stress. Despite the fact that the same tendency was detected in wild-type (WT) plants, changes in physio-biochemical parameters were greater in transgenic lines than in WT plants. The expression analysis revealed that the transgene TaSSRP expressed from 1.00 to 1.809 folds in different lines in the transgenic tobacco plants. The gene TaSSRP offered resistance to heat stress in Nicotiana tabacum, according to the results of the study. CONCLUSION: These findings could help to improve our knowledge and understanding of the mechanism underlying thermotolerance in wheat, and the novel identified gene TaSSRP could be used in generating wheat varieties with enhanced tolerance to heat stress.

Host-induced RNA interference targeting the neuromotor gene FMRFamide-like peptide-14 (Mi-flp14) perturbs Meloidogyne incognita parasitic success in eggplant.

KEY MESSAGE: The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

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.

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-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.

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.

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.

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.

Amenability of Maruca vitrata (Lepidoptera: Crambidae) to gene silencing through exogenous administration and host-delivered dsRNA in pigeonpea (Cajanus cajan L.).

Insect pests are one of the major biotic stresses limiting yield in commercially important crops. The lepidopteran polyphagous spotted pod borer, Maruca vitrata causes significant economic losses in legumes including pigeonpea. RNA interference (RNAi)-based gene silencing has emerged as one of the potential biotechnological tools for crop improvement. We report in this paper, RNAi in M. vitrata through exogenous administration of dsRNA with sequence specificity to three functionally important genes, Alpha-amylase (α-amylase), Chymotrypsin-like serine protease (CTLP) and Tropomyosin (TPM) into the larval haemolymph and their host-delivered RNAi in pigeonpea. Significant decline in the expression of selected genes supported by over-expression of DICER and generation of siRNA indicated the occurrence of RNAi in the dsRNA-injected larvae. Additionally, the onset of RNAi in the herbivore was demonstrated in pigeonpea, one of the prominent hosts, by host-delivered dsRNA. Transgenics in pigeonpea (cv. Pusa 992), a highly recalcitrant crop, were developed through a shoot apical meristem-targeted in planta transformation strategy and evaluated. Plant level bioassays in transgenic events characterized and selected at molecular level showed mortality of M. vitrata larvae as well as reduced feeding when compared to wild-type. Furthermore, molecular evidence for down regulation of target genes in the insects that fed on transgenic plants authenticated RNAi. Considering the variability of gene silencing in lepidopteran pests, this study provided corroborative proof for the possibility of gene silencing in M. vitrata through both the strategies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01133-3.

CRISPR-Cas9 directed genome engineering for enhancing salt stress tolerance in rice.

Crop productivity in rice is harshly limited due to high concentration of salt in the soil. To understand the intricacies of the mechanism it is important to unravel the key pathways operating inside the plant cell. Emerging state-of-the art technologies have provided the tools to discover the key components inside the plant cell for salt tolerance. Among the molecular entities, transcription factors and/or other important components of sensing and signaling cascades have been the attractive targets and the role of NHX and SOS1 transporters amply described. Not only marker assisted programs but also transgenic approaches by using reverse genetic strategies (knockout or knockdown) or overexpression have been extensively used to engineer rice crop. CRISPR/Cas is an attractive paradigm and provides the feasibility for manipulating several genes simultaneously. Here, in this review we highlight some of the molecular entities that could be potentially targeted for generating rice amenable to sustain growth under high salinity conditions by employing CRISPR/Cas. We also try to address key questions for rice salt stress tolerance other than what is already known.

A comprehensive review on the influence of light on signaling cross-talk and molecular communication against phyto-microbiome interactions.

Generally, plant growth, development, and their productivity are mainly affected by their growth rate and also depend on environmental factors such as temperature, pH, humidity, and light. The interaction between plants and pathogens are highly specific. Such specificity is well characterized by plants and pathogenic microbes in the form of a molecular signature such as pattern-recognition receptors (PRRs) and microbes-associated molecular patterns (MAMPs), which in turn trigger systemic acquired immunity in plants. A number of Arabidopsis mutant collections are available to investigate molecular and physiological changes in plants under the presence of different light conditions. Over the past decade(s), several studies have been performed by selecting Arabidopsis thaliana under the influence of red, green, blue, far/far-red, and white light. However, only few phenotypic and molecular based studies represent the modulatory effects in plants under the influence of green and blue lights. Apart from this, red light (RL) actively participates in defense mechanisms against several pathogenic infections. This evolutionary pattern of light sensitizes the pathologist to analyze a series of events in plants during various stress conditions of the natural and/or the artificial environment. This review scrutinizes the literature where red, blue, white, and green light (GL) act as sensory systems that affects physiological parameters in plants. Generally, white and RL are responsible for regulating various defense mechanisms, but, GL also participates in this process with a robust impact! In addition to this, we also focus on the activation of signaling pathways (salicylic acid and jasmonic acid) and their influence on plant immune systems against phytopathogen(s).

Transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein imparts resistance to Maruca vitrata legume pod borer.

KEY MESSAGE: Fertile independent transgenic cowpea lines expressing the BtCry2Aa toxin with increased resistance to the most devastating lepidopteran insect pest, Maruca pod borer has been developed for the first time. Cowpea is a staple legume important for food and nutritional security in sub-Saharan Africa and Asia, where its production is limited by the key pest, legume pod borer (Maruca vitrata). Cowpea varieties resistant to M. vitrata are not known, hence, development of Maruca pod borer resistance cowpea through genetic engineering is a promising approach to improve its production. In the present study, transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein were developed for the first time using Agrobacterium tumefaciens-mediated transformation of cotyledonary explants. T0 plants recovered from Agrobacterium cocultured explants on medium containing 120 mgl-1 of kanamycin were identified on the basis of the presence of transgenes by PCR, their integration into genome by Southern hybridization and expression of their transcripts by semi quantitative PCR (sqRT-PCR) and quantitative Real-time-PCR (qRT-PCR) and protein by Western blot analysis. The transformation efficiency obtained was 3.47% with 11 independent T0 transgenic lines. The bioefficacy of Cry2Aa protein expressed in randomly selected four T0 plant's leaves and pods was evaluated by feeding Maruca pod borer demonstrated a significant lower damage and a high level of Maruca mortality (more than 90%) for all these Bt lines. The inheritance of transgenes from T0 to T1 progeny plants was demonstrated by PCR analysis. The transgenic plants generated in this study can be used in cowpea breeding program for durable and sustainable legume pod borer resistance.

Alteration of proteome in germinating seedlings of piegonpea (Cajanus cajan) after salt stress.

Pigeonpea (Cajanus cajan) is an important crop in semi-arid regions and a significant source of dietary proteins in India. The plant is sensitive to salinity stress, which adversely affects its productivity. Based on the dosage-dependent influence of salinity stress on the growth and ion contents in the young seedlings of pigeonpea, a comparative proteome analysis of control and salt stressed (150 mM NaCl) plants was conducted using 7 days-old seedlings. Among various amino acids, serine, aspartate and asparagine were the amino acids that showed increment in the root, whereas serine, aspartate and phenylalanine showed an upward trend in shoots under salt stress. Furthermore, a label-free and gel-free comparative Q-Tof, Liquid Chromatography-Mass spectrometry (LC-MS) revealed total of 118 differentially abundant proteins in roots and shoots with and without salt stress conditions. Proteins related to DNA-binding with one finger (Dof) transcription factor family and glycine betaine (GB) biosynthesis were differentially expressed in the shoot and root of the salinity-stressed seedlings. Exogenous application of choline on GB accumulation under salt stress showed the increase of GB pathway in C. cajan. Gene expression analysis for differentially abundant proteins revealed the higher induction of ethanolamine kinase (CcEthKin), choline-phosphate cytidylyltransferase 1-like (CcChoPh), serine hydroxymethyltransferase (CcSHMT) and Dof protein (CcDof29). The results indicate the importance of, choline precursor, serine biosynthetic pathways and glycine betaine synthesis in salinity stress tolerance. The glycine betaine protects plant from cellular damages and acts as osmoticum under stress condition. Protein interaction network (PIN) analysis demonstrated that 61% of the differentially expressed proteins exhibited positive interactions and 10% of them formed the center of the PIN. Further, The PIN analysis also highlighted the potential roles of the cytochrome c oxidases in sensing and signaling cascades governing salinity stress responses in pigeonpea. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01116-w.

Novel biotechnological strategies to combat biotic stresses: polygalacturonase inhibitor (PGIP) proteins as a promising comprehensive option.

Global climate change and combinatorial environmental stresses pose grave challenges to food security and agricultural sustainability. This calls for diverse and futuristic approaches for the development of crops with increased resilience to natural vagaries. Though innumerable strategies involving diverse genes/pathways are being deciphered in plants to aid stress mitigation, the hunt is still on. Furthermore, strategies that work to alleviate a combination of stresses are always pertinent. In this review, we discuss polygalacturonase inhibitor (PGIP) proteins as a plausible option to mitigate multiple biotic stresses. These are ubiquitous cell wall proteins that inhibit the pectin-depolymerizing activity of cell wall loosening enzymes, polygalacturonases (PGs). While plant PGs are those responsible for developmental activities like fruit ripening, pollen tube elongation, etc., PGs from various biotic stress factors like insects, fungal and bacterial pathogens aid in invasion by reducing the plant cell wall rigidity. To counteract, plants secrete PGIPs, which inhibit the pectin hydrolyzing activity of PGs from the attacking pests and pathogens. Multiple approaches in diverse crop species have demonstrated PGIP-based protection against pathogens and insect pests. Additionally, effectual interaction between PGs-PGIP is an important aspect for successful utilization of this approach. Molecular strategies leading to improved PG-PGIPs interaction is a highlight to demonstrate the use of PGIPs as an amenable stress mitigation approach. The review focuses on a comprehensive update on phylogeny of PGIPs, natural variation of resistance as well as their emerging translational utility towards mitigation of various biotic stresses.

Unraveling the proteomic changes involved in the resistance response of Cajanus platycarpus to herbivory by Helicoverpa armigera.

The pigeonpea wild relative Cajanus platycarpus is resistant to Helicoverpa armigera, one of the major pests responsible for yield losses in Cajanus cajan. Deciphering the molecular mechanism underlying host plant resistance is pertinent to identify proteins that aid in the mitigation of the insect pest. The present study adopted comparative proteomics as a tool to interpret the resistance mechanism(s) in C. platycarpus vis-à-vis C. cajan during continued herbivory (up to 96 h). Over-representation analysis of the differentially expressed proteins implicated a multi-dimensional resistance response accomplished by both physical and chemical barriers in C. platycarpus. While the chemical basis for resistance was depicted by the upregulation of proteins playing a rate limiting role in the phenylpropanoid pathway, the physical basis was provided by the regulation of proteins involved in microtubule assembly and synthesis of lignins. Upregulation of proteins in the polyamine pathway indicated the role of metabolite conjugates to be negatively affecting herbivore growth. Reallocation of resources and diversion of metabolic flux to support the production of secondary metabolites could be the probable approach in the wild relative against herbivory. Our study provided deeper insights into the pod borer resistance mechanism in C. platycarpus for utility in crop improvement. KEY POINTS: • Pod borer resistance in Cajanus platycarpus is multi-dimensional. • Pod borer resistance has been arbitrated to cell wall rigidity and secondary metabolites. • Phenylpropanoid pathway derivatives apparently shaped the plant chemical defense against pod borer.

Agrobacterium tumefaciens-mediated in planta transformation strategy for development of transgenics in cotton (Gossypium hirsutum L.) with GFP as a visual marker.

Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8-6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.

Comparative transcriptome analyses provide novel insights into the differential response of Pigeonpea (Cajanus cajan L.) and its wild relative (Cajanus platycarpus (Benth.) Maesen) to herbivory by Helicoverpa armigera (Hübner).

KEY MESSAGE: Deeper insights into the resistance response of Cajanus platycarpus were obtained based on comparative transcriptomics under Helicoverpa armigera infestation. Devastation by pod borer, Helicoverpa armigera is one of the major factors for stagnated productivity in Pigeonpea. Despite possessing a multitude of desirable traits including pod borer resistance, wild relatives of Cajanus spp. have remained under-utilized due to linkage drag and cross-incompatibility. Discovery and deployment of genes from them can provide means to tackle key pests like H. armigera. Transcriptomic differences between Cajanus platycarpus and Cajanus cajan during different time points (0, 18, 38, 96 h) of pod borer infestation were elucidated in this study. For the first ever time, we demonstrated captivating variations in their response; C. platycarpus apparently being reasonably agile with effectual transcriptomic reprogramming to deter the insect. Deeper insights into the differential response were obtained by identification of significant GO-terms related to herbivory followed by combined KEGG and ontology analyses. C. platycarpus portrayed a multilevel response with cardinal involvement of SAR, redox homeostasis and reconfiguration of primary metabolites leading to a comprehensive defense response. The credibility of RNA-seq analyses was ascertained by transient expression of selected putative insect resistance genes from C. platycarpus viz., chitinase (CHI4), Alpha-amylase/subtilisin inhibitor (IAAS) and Flavonoid 3_5 hydroxylase (C75A1) in Nicotiana benthamiana followed by efficacy analysis against H. armigera. qPCR validated results of the study provided innovative insights and useful leads for development of durable pod borer resistance.

Allele-specific analysis of single parent backcross population identifies HOX10 transcription factor as a candidate gene regulating rice root growth.

Understanding the molecular and physiological mechanisms of trait diversity is crucial for crop improvement to achieve drought adaptation. Root traits such as high biomass and/or deep rootedness are undoubtedly important drought adaptive traits. The major aim of this investigation was to functionally characterize a set of ethyl methane sulfonate-induced rice mutants for root traits. We report the identification of a high-root biomass mutant through a novel screening strategy for yield and Δ13 C measurements. The high-root mutant (392-9-1) thus identified, had a 66% higher root biomass compared to wild-type (Nagina-22). Better maintenance of leaf turgor and carbon assimilation rates resulted in lower drought susceptibility index in 392-9-1. Targeted resequencing revealed three non-synonymous single nucleotide variations in 392-9-1 for the genes HOX10, CITRATE SYNTHASE and ZEAXANTHIN EPOXIDASE. Segregation pattern of phenotype and mutant alleles in a single parent backcross F2 population revealed a typical 3:1 segregation for each of the mutant alleles. The number of F2 progeny with root biomass equal to or greater than that of 392-9-1 represented approximately one-third of the population indicating a major role played by HOX10 gene in regulating root growth in rice. Allele-specific Sanger sequencing in contrasting F2 progenies confirmed the co-segregation of HOX10 allele with the root biomass. The non-synonymous mutations in the other two genes did not reveal any specific pattern of co-segregation with root phenotype, indicating a strong role of HOX10, an upstream transcription factor, in regulating root biomass in rice.

Comprehensive in silico allergenicity assessment of novel protein engineered chimeric Cry proteins for safe deployment in crops.

BACKGROUND: Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. RESULTS: In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. CONCLUSIONS: The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.