Dynamic Evolution of NLR Genes in Dalbergioids.

Dalbergioid is a large group within the family Fabaceae that consists of diverse plant species distributed in distinct biogeographic realms. Here, we have performed a comprehensive study to understand the evolution of the nucleotide-binding leucine-rich repeats (NLRs) gene family in Dalbergioids. The evolution of gene families in this group is affected by a common whole genome duplication that occurred approximately 58 million years ago, followed by diploidization that often leads to contraction. Our study suggests that since diploidization, the NLRome of all groups of Dalbergioids is expanding in a clade-specific manner with fewer exceptions. Phylogenetic analysis and classification of NLRs revealed that they belong to seven subgroups. Specific subgroups have expanded in a species-specific manner, leading to divergent evolution. Among the Dalbergia clade, the expansion of NLRome in six species of the genus Dalbergia was observed, with the exception of Dalbergia odorifera, where a recent contraction of NLRome occurred. Similarly, members of the Pterocarpus clade genus Arachis revealed a large-scale expansion in the diploid species. In addition, the asymmetric expansion of NLRome was observed in wild and domesticated tetraploids after recent duplications in the genus Arachis. Our analysis strongly suggests that whole genome duplication followed by tandem duplication after divergence from a common ancestor of Dalbergioids is the major cause of NLRome expansion. To the best of our knowledge, this is the first ever study to provide insight toward the evolution of NLR genes in this important tribe. In addition, accurate identification and characterization of NLR genes is a substantial contribution to the repertoire of resistances among members of the Dalbergioids species.

Genome-wide association studies of seedling quantitative trait loci against salt tolerance in wheat.

Salinity is one of the significant factors in decreasing wheat yield and quality. To counter this, it is necessary to develop salt-tolerant wheat varieties through conventional and advanced molecular techniques. The current study identified quantitative trait loci in response to salt stress among worldwide landraces and improved varieties of wheat at the seedling stage. A total of 125 landraces and wheat varieties were subjected to salt treatment (50, 100, and 150 mM) with control. Morphological seedling traits, i.e., shoot length, root length, and fresh and dry shoot and root weights for salinity tolerance were observed to assess salt tolerance and genetic analysis using SNP data through DArT-seq. The results showed that, at the seedling stage, 150 mM NaCl treatment decreased shoot length, root length, and fresh and dry weights of the shoot and root. The root length and dry root weight were the most affected traits at the seedling stage. Effective 4417 SNPs encompassing all the chromosomes of the wheat genome with marker density, i.e., 37%, fall in genome B, genome D (32%), and genome A (31%). Five loci were found on four chromosomes 6B, 6D, 7A, and 7D, showing strong associations with the root length, fresh shoot weight, fresh root weight, and dry root weight at the p < 0.03 significance level. The positive correlation was found among all morphological traits under study.

Genome-Wide Characterization of Glutathione Peroxidase (GPX) Gene Family in Rapeseed (Brassica napus L.) Revealed Their Role in Multiple Abiotic Stress Response and Hormone Signaling.

Plant glutathione peroxidases (GPXs) are the main enzymes in the antioxidant defense system that sustain H2O2 homeostasis and normalize plant reaction to abiotic stress conditions. To understand the major roles of the GPX gene family in rapeseed (Brassica napus L.), for the first time, a genome-wide study identified 25 BnGPX genes in the rapeseed genome. The phylogenetic analysis discovered that GPX genes were grouped into four major groups (Group I-Group IV) from rapeseed and three closely interrelated plant species. The universal investigation uncovered that the BnGPXs gene experienced segmental duplications and positive selection pressure. Gene structure and motifs examination recommended that most of the BnGPX genes demonstrated a comparatively well-maintained exon-intron and motifs arrangement within the identical group. Likewise, we recognized five hormones-, four stress-, and numerous light-reactive cis-elements in the promoters of BnGPXs. Five putative bna-miRNAs from two families were also prophesied, targeting six BnGPXs genes. Gene ontology annotation results proved the main role of BnGPXs in antioxidant defense systems, ROS, and response to stress stimulus. Several BnGPXs genes revealed boosted expression profiles in many developmental tissues/organs, i.e., root, seed, leaf, stem, flower, and silique. The qRT-PCR based expression profiling exhibited that two genes (BnGPX21 and BnGPX23) were suggestively up-regulated against different hormones (ABA, IAA, and MeJA) and abiotic stress (salinity, cold, waterlogging, and drought) treatments. In short, our discoveries provide a basis for additional functional studies on the BnGPX genes in future rapeseed breeding programs.

Advances in "Omics" Approaches for Improving Toxic Metals/Metalloids Tolerance in Plants.

Food safety has emerged as a high-urgency matter for sustainable agricultural production. Toxic metal contamination of soil and water significantly affects agricultural productivity, which is further aggravated by extreme anthropogenic activities and modern agricultural practices, leaving food safety and human health at risk. In addition to reducing crop production, increased metals/metalloids toxicity also disturbs plants' demand and supply equilibrium. Counterbalancing toxic metals/metalloids toxicity demands a better understanding of the complex mechanisms at physiological, biochemical, molecular, cellular, and plant level that may result in increased crop productivity. Consequently, plants have established different internal defense mechanisms to cope with the adverse effects of toxic metals/metalloids. Nevertheless, these internal defense mechanisms are not adequate to overwhelm the metals/metalloids toxicity. Plants produce several secondary messengers to trigger cell signaling, activating the numerous transcriptional responses correlated with plant defense. Therefore, the recent advances in omics approaches such as genomics, transcriptomics, proteomics, metabolomics, ionomics, miRNAomics, and phenomics have enabled the characterization of molecular regulators associated with toxic metal tolerance, which can be deployed for developing toxic metal tolerant plants. This review highlights various response strategies adopted by plants to tolerate toxic metals/metalloids toxicity, including physiological, biochemical, and molecular responses. A seven-(omics)-based design is summarized with scientific clues to reveal the stress-responsive genes, proteins, metabolites, miRNAs, trace elements, stress-inducible phenotypes, and metabolic pathways that could potentially help plants to cope up with metals/metalloids toxicity in the face of fluctuating environmental conditions. Finally, some bottlenecks and future directions have also been highlighted, which could enable sustainable agricultural production.

In-silico pharmacophoric and molecular docking-based drug discovery against the Main Protease (Mpro) of SARS-CoV-2, a causative agent COVID-19.

COVID-19 (Coronavirus Disease 2019) caused by a novel 'SARS-CoV-2' virus resulted in public health emergencies across the world. An effective vaccine to cure this virus is not yet available, thus requires concerted efforts at various scales. In this study, we employed Computer-Aided Drug Design (CADD) based approach to identify the drug-like compounds - inhibiting the replication of the main protease (Mpro) of SARS-CoV-2. Our database search using an online tool "ZINC pharmer" retrieved ~1500 compounds based on pharmacophore features. Lipinski's rule was applied to further evaluate the drug-like compounds, followed by molecular docking-based screening, and the selection of screening ligand complex with Mpro based on S-score (higher than reference inhibitor) and root-mean-square deviation (RMSD) value (less than reference inhibitor) using AutoDock 4.2. Resultantly, ~200 compounds were identified having strong interaction with Mpro of SARS-CoV-2. After evaluating their binding energy using the AutoDock 4.2 software, three compounds (ZINC20291569, ZINC90403206, ZINC95480156) were identified that showed highest binding energy with Mpro of SARS-CoV-2 and strong inhibition effect than the N3 (reference inhibitor). A good binding energy, drug likeness and effective pharmacokinetic parameters suggest that these candidates have greater potential to stop the replication of SARS-CoV-2, hence might lead to the cure of COVID-19.

In silico Characterization of a Candidate Protein from Aphid Gelling Saliva with Potential for Aphid Control in Plants.

BACKGROUND: Sheath or gelling saliva, secreted during feeding by aphids, is a hard material that supports the piercing mouthparts and remains in the plant after feeding. Solidification or gelling of the saliva might be due to the composition of amino acids in the constituent proteins, many of which probably interact with plant defenses. OBJECTIVE: The complete complement of proteins in the gelling saliva are still unknown, although one sheath protein (SHP) has previously been identified as a potential candidate protein to control aphid feeding, but its structure and its physiochemical role remains obscure. The current study provides structural information and biochemical properties of the aphid sheath protein. METHODS: The Sheath protein encoding gene was amplified from cDNA of the pea aphid (Acyrthosiphon pisum) through PCR using specific gene primers. Sequence was in silico characterized by using EXPASY, Berkeley Drosophila Genome Project (BDGP) Neural Network Promoter Prediction, BioEdit, Mega7, ProtParam, Phyre server, 3D LigandSite SMART, MEME and GSDS programs, available online. RESULTS: BLASTp analysis revealed that the sequenced gene was identical (100%) to the sequence from Acyrthosiphon pisum, with 87% identity to Metpolophium dirhodum and 84% identity to Sitobion avenae. Phylogenetically monocot feeders such as M. dirhodum and S. avenae are in a sister taxa to dicot feeders. In silico analysis of the sequence revealed that sheath protein has a molecular weight of 144 kDa and 50% of the protein is composed of only six amino acids, i.e., threonine, serine, aspartic acid, glutamic acid, isoleucine and tyrosine. The computed IP value revealed that sheath protein is acidic in nature. Ligand binding sites for sheath protein were predicted on residues 1123 and 1125 (isoleucine and glutamine, respectively). Metallic heterogens are also present in sheath protein that are iron, zinc and magnesium, respectively. CONCLUSION: It is conceivable that variation in the salivary gene sequences may reveal important biological information of relevance to the insect-plant interaction. Further exploration of insect salivary proteins, their composition and structure will provide powerful information, especially when these proteins are interacting with plant proteins, and specific information about the sheath protein, which is interacting with plants at a molecular/cellular level, will be important to progress strategies aimed specifically against sucking pests such as aphids.

Resistance status of Helicoverpa armigera against Bt cotton in Pakistan.

Transgenic cotton expressing the toxin Cry1Ac from Bacillus thuringiensis L. (Bt) is widely cultivated in Pakistan after its formal approval in 2010. The exposure of the local target pests to the Cry1Ac endotoxin for this duration might have changed the baseline susceptibility. To probe the status of resistance in one of the main target pests, Helicoverpa armigera, field-collected larvae were reared in the lab for conducting leaf fed bioassays. Twenty-six cotton accessions collected from farmers, including 25 Bt-cotton and one non-Bt, were tested to quantify the level of Cry1Ac, an insecticidal crystalline protein (ICP), in leaves of lower, middle and upper canopies of plants. The concentration of ICP was tested through Enzyme-linked Immunosorbent Assay and found significantly variable (P < 0.01) between and within accessions. The highest mean expression was observed in Accession-2 and Accession-4, while the lowest in Accession-21 and Accession-19. Among fresh leaf tissues from different parts of the plant, the highest mean expression was recorded at 60 days after sowing in upper canopy leaves of cotton accessions, which decreased in lower parts of the plant with the lowest mean expression in lower canopy leaves. Laboratory bioassays, to calculate lethal dose, for H. armigera showed that LD50 and LD95 were 0.62 µg/g and 1.59 µg/g of fresh tissue weight, respectively. A strong positive correlation also exists between the levels of Cry1Ac protein and insect mortality (r = 0.84). These findings suggested the future risk of cultivation of Bt cotton, carrying single Cry1Ac gene, in Pakistan, as resistance surging in H. armigera against Cry protein. These results may also have significant implications for the resistance management in Bt crops, especially cotton, in future.

What are farmers really planting? Measuring the presence and effectiveness of Bt cotton in Pakistan.

Genetically modified, insect-resistant Bacillus thuringiensis (Bt) cotton is cultivated extensively in Pakistan. Past studies, however, have raised concerns about the prevalence of Bt cotton varieties possessing weak or nonperforming insect-resistance traits conferred by the cry gene. We examine this issue using data drawn from a representative sample of cotton-growing households that were surveyed in six agroclimatic zones spanning 28 districts in Pakistan in 2013, as well as measurements of Cry protein levels in cotton tissue samples collected from the sampled households' main fields. The resultant dataset combines information from 593 sampled households with corresponding plant tissue diagnostics from 70 days after sowing, as well as information from 589 sampled households with corresponding diagnostics from 120 days after sowing. Our analysis indicates that 11 percent of farmers believed they were cultivating Bt cotton when, in fact, the Cry toxin was not present in the tested tissue at 70 days after sowing (i.e., a Type I error). The analysis further indicates that 5 percent of farmers believed they were cultivating non-Bt cotton when, in fact, the Cry toxin was present in the tested tissue (i.e., a Type II error). In addition, 17 percent of all sampled farmers were uncertain whether or not they were cultivating Bt cotton. Overall, 33 percent of farmers either did not know or were mistaken in their beliefs about the presence of the cry gene in the cotton they cultivated. Results also indicate that toxic protein levels in the plant tissue samples occurred below threshold levels for lethality in a significant percentage of cases, although these measurements may also be affected by factors related to tissue sample collection, handling, storage, and testing procedures. Nonetheless, results strongly suggest wide variability both in farmers' beliefs and in gene expression. Such variability has implications for policy and regulation in Pakistan's transgenic cotton seed market.