Exploring the structural assembly of rice ADP-glucose pyrophosphorylase subunits using MD simulation.

ADP-glucose pyrophosphorylase plays a pivotal role as an allosteric enzyme, essential for starch biosynthesis in plants. The higher plant AGPase comparises of a pair of large and a pair of small subunits to form a heterotetrameric complex. Growing evidence indicates that each subunit plays a distinct role in regulating the underlying mechanism of starch biosynthesis. In the rice genome, there are four large subunit genes (OsL1-L4) and three small subunit genes (OsS1, OsS2a, and OsS2b). While the structural assembly of cytosolic rice AGPase subunits (OsL2:OsS2b) has been elucidated, there is currently no such documented research available for plastidial rice AGPases (OsL1:OsS1). In this study, we employed protein modeling and MD simulation approaches to gain insights into the structural association of plastidial rice AGPase subunits. Our results demonstrate that the heterotetrameric association of OsL1:OsS1 is very similar to that of cytosolic OsL2:OsS2b and potato AGPase heterotetramer (StLS:StSS). Moreover, the yeast-two-hybrid results on OsL1:OsS1, which resemble StLS:StSS, suggest a differential protein assembly for OsL2:OsS2b. Thus, the regulatory and catalytic mechanisms for plastidial AGPases (OsL1:OsS1) could be different in rice culm and developing endosperm compared to those of OsL2:OsS2b, which are predominantly found in rice endosperm.

Mapping of QTLs associated with yield and related traits under reproductive stage drought stress in rice using SNP linkage map.

BACKGROUND: Drought stress is a major constraint for rice production worldwide. Reproductive stage drought stress (RSDS) leads to heavy yield losses in rice. The prospecting of new donor cultivars for identification and introgression of QTLs of major effect (Quantitative trait locus) for drought tolerance is crucial for the development of drought-resilient rice varieties. METHODS AND RESULTS: Our study aimed to map QTLs associated with yield and its related traits under RSDS conditions. A saturated linkage map was constructed using 3417 GBS (Genotyping by sequencing) derived SNP (Single nucleotide polymorphism) markers spanning 1924.136 cM map length with an average marker density of 0.56 cM, in the F3 mapping population raised via cross made between the traditional ahu rice cultivar, Koniahu (drought tolerant) and a high-yielding variety, Disang (drought susceptible). Using the Inclusive composite interval mapping approach, 35 genomic regions governing yield and related traits were identified in pooled data from 198 F3 and F4 segregating lines evaluated for two consecutive seasons under both RSDS and irrigated control conditions. Of the 35 QTLs, 23 QTLs were identified under RSDS with LOD (Logarithm of odds) values ranging between 2.50 and 7.83 and PVE (phenotypic variance explained) values of 2.95-12.42%. Two major QTLs were found to be linked to plant height (qPH1.29) and number of filled grains per panicle (qNOG5.12) under RSDS. Five putative QTLs for grain yield namely, qGY2.00, qGY5.05, qGY6.16, qGY9.19, and qGY10.20 were identified within drought conditions. Fourteen QTL regions having ≤ 10 Mb QTL interval size were further analysed for candidate gene identification and a total of 4146 genes were detected out of these 2263 (54.63%) genes were annotated to at least one gene ontology (GO) term. CONCLUSION: Several QTLs associated with grain yield and yield components and putative candidate genes were identified. The putative QTLs and candidate genes identified could be employed to augment drought resilience in rice after further validation through MAS strategies.

Molecular mapping of drought-responsive QTLs during the reproductive stage of rice using a GBS (genotyping-by-sequencing) based SNP linkage map.

BACKGROUND: In rice, drought stress at reproductive stage drastically reduces yield, which in turn hampers farmer's efforts towards crop production. The majority of the rice varieties have resistance genes against several abiotic and biotic stresses. Therefore, the traditional landraces were studied to identify QTLs/candidate genes associated with drought tolerance. METHODS AND RESULTS: A high-density SNP-based genetic map was constructed using a Genotyping-by-sequencing (GBS) approach. The recombinant inbred lines (RILs) derived from crossing 'Banglami × Ranjit' were used for QTL analysis. A total map length of 1306.424 cM was constructed, which had an average inter-marker distance of 0.281 cM. The phenotypic evaluation of F6 and F7 RILs were performed under drought stress and control conditions. A total of 42 QTLs were identified under drought stress and control conditions for yield component traits explaining 1.95-13.36% of the total phenotypic variance (PVE). Among these, 19 QTLs were identified under drought stress conditions, whereas 23 QTLs were located under control conditions. A total of 4 QTLs explained a PVE ≥ 10% which are considered as the major QTLs. Moreover, bioinformatics analysis revealed the presence of 6 candidate genes, which showed differential expression under drought and control conditions. CONCLUSION: These QTLs/genes may be deployed for marker-assisted pyramiding to improve drought tolerance in the existing rice varieties.

Comparative transcriptome profiling reveals differential defense responses among Alternaria brassicicola resistant Sinapis alba and susceptible Brassica rapa.

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba, a close relative of Brassicas, and the highly susceptible Brassica rapa. The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa. Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola, which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.

Niche differentiation of belowground microorganisms and their functional signatures in Assam type tea (Camellia sinensis var. assamica).

We employed an Illumina-based high-throughput metagenomics sequencing approach to unveil the rhizosphere and root endosphere microbial community associated with an organically grown Camellia population located at the Experimental Garden for Plantation Crops, Assam (India). The de novo assembled tea root endosphere metagenome contained 24,231 contigs (total 7,771,089 base pairs with an average length of 321 bps), while tea rhizosphere soil metagenome contained 261,965 sequences (total 230,537,174 base pairs, average length 846). The most prominent rhizobacteria belonged to the genera, viz., Bacillus (10.35%), Candidatus Solibacter (6.36%), Burkholderia (5.19%), Pseudomonas (3.9%), Streptomyces (3.52%), and Bradyrhizobium (2.77%), while the root endosphere was dominated by bacterial genera, viz., Serratia (46.64%), Methylobacterium (8.02%), Yersinia (5.97%), Burkholderia (2.05%), etc. The presence of few agronomically important bacterial genera, Bradyrhizobium, Rhizobium (each 0.93%), Sinorhizobium (0.34%), Azorhizobium, and Flavobacterium (0.17% each), was also detected in the root endosphere. KEGG pathway mapping indicated the presence of microbial metabolic pathway genes related to tyrosine metabolism, tryptophan metabolism, glyoxylate, and dicarboxylate metabolism which play important roles in endosphere activities, including survival, growth promotion, and host adaptation. The root endosphere microbiome also contained few important plant growth promoting traits related to phytohormone production, abiotic stress alleviation, mineral solubilization, and plant disease suppression.

Understanding the thermal response of rice eukaryotic transcription factor eIF4A1 towards dynamic temperature stress: insights from expression profiling and molecular dynamics simulation.

Eukaryotic translation initiation factors (eIFs) are the group of regulatory proteins that are involved in the initiation of translation events. Among them, eIF4A1, a member of the DEAD-box RNA helicase family, participates in a wide spectrum of activities which include, RNA splicing, ribosome biogenesis, and RNA degradation. It is well known that ATP-binding and subsequent hydrolysis activities are crucial for the functionality of such helicases. Although the stress-responsive upregulation of eIF4A1 has been reported in plants during stress, it is difficult to anticipate the functionality of the corresponding protein product. Therefore, to understand the activity of eIF4A1 in rice in response to temperature stress, we first conducted an expression analysis of the gene and further investigated the structural stability of the eIF4A1-ATP/Mg2+ complex through molecular dynamics (MD) simulations at different temperature conditions (277 K, 300 K, and 315 K). Our results demonstrated a three to fourfold increased expression of rice eIF4A1 both in root and shoot at 42 °C compared to control. Furthermore, the MD simulation portrayed strong ATP/Mg2+ binding at a higher temperature in comparison to control and cold temperature. Overall, the increased expression pattern of eIF4A1 and strong ATP/Mg2+ binding at higher temperature indicated the heat stress-tolerant capacity of the gene in rice. The results from our study will help in understanding the activity of gene and guide the researchers for screening of novel stress inducible candidate genes for the engineering of temperature stress tolerant plants.Communicated by Ramaswamy H. Sarma.

Combined CADD and Virtual Screening to Identify Novel Nonpeptidic Falcipain-2 Inhibitors.

BACKGROUND: Plasmodium falciparum is the most dangerous and widespread diseasecausing species of malaria. Falcipain-2 (FP2) of Plasmodium falciparum, is a potential target for antimalarial chemotherapy since it is involved in an essential cellular function such as hemoglobin degradation during the parasite's life cycle. However, despite their central role in the life cycle of the parasite, no commercial drug targeting Falcipain-2 has been developed to date. Prior efforts to develop peptide-based drugs against Plasmodium have been futile due to their susceptibility to being degraded by host enzymes. OBJECTIVE: Here, we report computer-aided drug design of new nonpeptidic inhibitors against FP2, which are likely to be safe from degradation by host enzymes. METHODS: We have virtually screened for the probable FP2 inhibitors from the PubChem database by submitting the well-equilibrated 3-D structure of FP2. Furthermore, virtual screenings and dockings were carried out using PyRx and Discovery Studio. RESULTS: We found 15 top-ranking molecules with carbaldehyde pharmacophore having a good fit with the target protein. Based on the C-Docker values, the top 4 hits (PubChem 44138738, Pub- Chem 20983198, PubChem 20983081 and PubChem 28951461) for FP2 were identified. These four hits have been observed to bound to the active cleft of the protein. Moreover, their complexes were also found to be stable from the RMSD and Radius of Gyration analysis. CONCLUSION: The selected compounds 2-(benzylamino)-8-methylquinoline-3-carbaldehyde (Pub- Chem44138738), 6-bromo-2-(3,4-dihydro-1H-isoquinolin-2-yl)quinoline-3-carbaldehyde (Pub- Chem 20983198), 2-(3,4-dihydro-1H-isoquinolin-2-yl)-6-ethylquinoline-3-carbaldehyde(PubChem 20983081)and 2-[benzyl(methyl)amino]quinoline-3-carbaldehyde (PubChem 28951461) may be the starting point for further modification as a new type of nonpeptidic drug for malaria disease.

Mechanism of interaction of an endofungal bacterium Serratia marcescens D1 with its host and non-host fungi.

Association of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ and an outer membrane associated murein lipoprotein also showed significant up-regulation during the interaction process, suggesting the involvement of T6SS in the invasion process.

Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study.

Enzymes from natural sources protect the environment via complex biological mechanisms, which aid in reductive immobilization of toxic metals including chromium. Nevertheless, progress was being made in elucidating high-resolution crystal structures of reductases and their binding with flavin mononucleotide (FMN) to understand the underlying mechanism of chromate reduction. Therefore, herein, we employed molecular dynamics (MD) simulations, principal component analysis (PCA), and binding free energy calculations to understand the dynamics behavior of these enzymes with FMN. Six representative chromate reductases in monomeric and dimeric forms were selected to study the mode, dynamics, and energetic component that drive the FMN binding process. As evidenced by MD simulation, FMN prefers to bind the cervix formed between the catalytic domain surrounded by strong conserved hydrogen bonding, electrostatic, and hydrophobic contacts. The slight movement and reorientation of FMN resulted in breakage of some crucial H-bonds and other nonbonded contacts, which were well compensated with newly formed H-bonds, electrostatic, and hydrophobic interactions. The critical residues aiding in tight anchoring of FMN within dimer were found to be strongly conserved in the bacterial system. The molecular mechanics combined with the Poisson-Boltzmann surface area binding free energy of the monomer portrayed that the van der Waals and electrostatic energy contribute significantly to the total free energy, where, the polar solvation energy opposes the binding of FMN. The proposed proximity relationships between enzyme and FMN binding site presented in this study will open up better avenues to engineer enzymes with optimized chromate reductase activity for sustainable bioremediation of heavy metals.

Molecular characterization and sequence analyses of Banana bunchy top virus infecting banana cultivar Jahaji (Dwarf Cavendish) in Assam, India.

Several isolates of Banana bunchy top virus (BBTV) have been reported worldwide. They are members of either the Pacific Indian Ocean (PIO) or the South East Asian (SEA) group. However, there is only one completely sequenced isolate published from the northeastern part of India till date. Therefore, we obtained the complete sequences of all the six genomic components of a BBTV isolate from the northeastern Indian state of Assam. The isolate was named as BBTV-As-JOR, and its genome showed the presence of the reported conserved motifs. Nevertheless, like other Indian BBTV isolate, the major common regions in DNA-R and DNA-U3 of BBTV-As-JOR had deletions of 26 and 36 nucleotides, respectively. Phylogenetic analysis based on 312 sequences of BBTV DNA-R classified BBTV-As-JOR as a member of the PIO group; similar phylogenetic patterns were also found with the other genomic segments. Analysis with Recombination Detection Program revealed two intra-segment recombination events involving DNA-C of geographically distinct BBTV isolates. On the other hand, DNA-U3 and DNA-N were found to be involved in few inter-segment recombination events in BBTV-As-JOR. This is the first report of a BBTV isolate from Assam and also of another PIO isolate from the region (the other isolate, BBTV-Umiam, was much closer to the SEA group). The detected possible recombinants could emerge as a major future threat for the banana cultivations in the country considering the asexual nature of propagation of banana crop.

Revealing shared differential co-expression profiles in rice infected by virus from reoviridae and sequiviridae group.

Differential co-expression is a cutting-edge approach to analyze gene expression data and identify both shared and divergent expression patterns. The availability of high-throughput gene expression datasets and efficient computational approaches have unfolded the opportunity to a systems level understanding of functional genomics of different stresses with respect to plants. We performed the meta-analysis of the available microarray data for reoviridae and sequiviridae infection in rice with the aim to identify the shared gene co-expression profile. The microarray data were downloaded from ArrayExpress and analyzed through a modified Weighted Gene Co-expression Network Analysis (WGCNA) protocol. WGCNA clustered the genes based on the expression intensities across the samples followed by identification of modules, eigengenes, principal components, topology overlap, module membership and module preservation. The module preservation analysis identified 4 modules; salmon (638 genes), midnightblue (584 genes), lightcyan (686 genes) and red (562 genes), which are highly preserved in both the cases. The networks in case of reoviridae infection showed neatly packed clusters whereas, in sequiviridae, the clusters were loosely connected which is due to the differences in the correlation values. We also identified 83 common transcription factors targeting the hub genes from all the identified modules. This study provides a coherent view of the comparative aspect of the expression of common genes involved in different virus infections which may aid in the identification of novel targets and development of new intervention strategy against the virus.

Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

Identification and validation of plant miRNA from NGS data-an experimental approach.

miRNAs are class of endogenously initiated noncoding RNAs, which are most critical in gene expression and regulation at posttranscriptional level. They do so either by cleavage of the target mRNA or by translational repression. miRNAs are being given enough attention in recent years because of its role in myriad developmental processes including tumorogenesis and host-pathogen interaction. Advent of Next Generation Sequencing (NGS) technology and computational approach made it possible to pinpoint the precise role of miRNA and their target. Identification of miRNAs and their target has several approaches depending on efficiency, cost and time. The present review summarizes the developments in the field of plant miRNA w.r.t. to experimental approaches that are being followed to identify and validate the miRNAs and their targets.

Bacillus megaterium adapts to acid stress condition through a network of genes: Insight from a genome-wide transcriptome analysis.

RNA-seq analysis of B. megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stress-responsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B. megaterium G18 to adapt and survive in acid stress condition.

Novel insights into structure-function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus.

The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue-specific expression studies to explain its structure-function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future.

Molecular recognition of avirulence protein (avrxa5) by eukaryotic transcription factor xa5 of rice (Oryza sativa L.): insights from molecular dynamics simulations.

The avirulence gene avrxa5 of bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) recognized by the resistant rice lines having corresponding resistance (xa5) gene in a gene-for-gene manner. We used a combinatorial approach involving protein-protein docking, molecular dynamics (MD) simulations and binding free energy calculations to gain novel insights into the gene-for-gene mechanism that governs the direct interaction of R-Avr protein. From the best three binding poses predicted by molecular docking, MD simulations were performed to explore the dynamic binding mechanism of xa5 and avrxa5. Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) techniques were employed to calculate the binding free energy and to uncover the thriving force behind the molecular recognition of avrxa5 by eukaryotic transcription factor xa5. Binding free energy analysis revealed van der Waals term as the most constructive component that favors the xa5 and avrxa5 interaction. In addition, hydrogen bonds (H-bonds) and essential electrostatic interactions analysis highlighted amino acid residues Lys54/Asp870, Lys56/Ala868, Lys56/Ala866, Lys56/Glu871, Ile59/His862, Gly61/Phe858, His62/Arg841, His62/Leu856, Ser101/Ala872 and Ser105/Asp870 plays pivotal role for the energetically stability of the R-Avr complex. Insights gained from the present study are expected to unveil the molecular mechanisms that define the transcriptional activator mediated transcriptome modification in host plants.

E-microsatellite markers for Centella asiatica (Gotu Kola) genome: validation and cross-transferability in Apiaceae family for plant omics research and development.

Abstract Centella asiatica (Gotu Kola) is a plant that grows in tropical swampy regions of the world and has important medicinal and culinary use. It is often considered as part of Ayurvedic medicine, traditional African medicine, and traditional Chinese medicine. The unavailability of genomics resources is significantly impeding its genetic improvement. To date, no attempt has been made to develop Expressed Sequence Tags (ESTs) derived Simple Sequence Repeat (SSR) markers (eSSRs) from the Centella genome. Hence, the present study aimed to develop eSSRs and their further experimental validation and cross-transferability of these markers in different genera of the Apiaceae family to which Centella belongs. An in-house pipeline was developed for the entire analyses by combining bioinformatics tools and perl scripts. A total of 4443 C. asiatica EST sequences from dbEST were processed, which generated 2617 nonredundant high quality EST sequences consisting 441 contigs and 2176 singletons. Out of 1776.5 kb of examined sequences, 417 (15.9%) ESTs containing 686 SSRs were detected with a density of one SSR per 2.59 kb. The gene ontology study revealed 282 functional domains involved in various processes, components, and functions, out of which 64 ESTs were found to have both SSRs and functional domains. Out of 603 designed EST-SSR primers, 18 pairs of primers were selected for validation based on the optimum parameter value. Reproducible amplification was obtained for six primer pairs in C. asiatica that were further tested for cross-transferability in nine other important genera/species of the Apiaceae family. Cross-transferability of the EST-SSR markers among the species were examined and Centella javanica showed highest transferability (83.3%). The study revealed six highly polymorphic EST-SSR primers with an average PIC value of 0.95. In conclusion, these EST-SSR markers hold a big promise for the genomics analysis of Centella asiatica, to facilitate comparative map-based analyses across other related species within the Apiaceae family, and future marker-assisted breeding programs. To the best of our knowledge, this is the first report of development of EST-SSRs in Centella asiatica by in silico approaches, which offers a veritable potential in further use in plant omics research and development.

Rediscovering medicinal plants' potential with OMICS: microsatellite survey in expressed sequence tags of eleven traditional plants with potent antidiabetic properties.

Herbal medicines and traditionally used medicinal plants present an untapped potential for novel molecular target discovery using systems science and OMICS biotechnology driven strategies. Since up to 40% of the world's poor people have no access to government health services, traditional and folk medicines are often the only therapeutics available to them. In this vein, North East (NE) India is recognized for its rich bioresources. As part of the Indo-Burma hotspot, it is regarded as an epicenter of biodiversity for several plants having myriad traditional uses, including medicinal use. However, the improvement of these valuable bioresources through molecular breeding strategies, for example, using genic microsatellites or Simple Sequence Repeats (SSRs) or Expressed Sequence Tags (ESTs)-derived SSRs has not been fully utilized in large scale to date. In this study, we identified a total of 47,700 microsatellites from 109,609 ESTs of 11 medicinal plants (pineapple, papaya, noyontara, bitter orange, bermuda brass, ratalu, barbados nut, mango, mulberry, lotus, and guduchi) having proven antidiabetic properties. A total of 58,159 primer pairs were designed for the non-redundant 8060 SSR-positive ESTs and putative functions were assigned to 4483 unique contigs. Among the identified microsatellites, excluding mononucleotide repeats, di-/trinucleotides are predominant, among which repeat motifs of AG/CT and AAG/CTT were most abundant. Similarity search of SSR containing ESTs and antidiabetic gene sequences revealed 11 microsatellites linked to antidiabetic genes in five plants. GO term enrichment analysis revealed a total of 80 enriched GO terms widely distributed in 53 biological processes, 17 molecular functions, and 10 cellular components associated with the 11 markers. The present study therefore provides concrete insights into the frequency and distribution of SSRs in important medicinal resources. The microsatellite markers reported here markedly add to the genetic stock for cross transferability in these plants and the literature on biomarkers and novel drug discovery for common chronic diseases such as diabetes.

Structure-based computational study of two disease resistance gene homologues (Hm1 and Hm2) in maize (Zea mays L.) with implications in plant-pathogen interactions.

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

Insights into the structure-function relationship of disease resistance protein HCTR in maize (Zea mays L.): a computational structural biology approach.

The disease resistance gene Hm1 of maize encodes a NADPH-dependent reductase enzyme, HC-toxin reductase (HCTR) that detoxifies the HC toxin secreted by the race specific fungus Cochliobolus carbonum race 1. HCTR enzyme shares 29.6% sequence identity with dihydroflavonol reductase (DFR) of grape, a key enzyme involved in flavonoid biosynthesis. Here we report the comparative modelling, molecular dynamics simulation and docking studies to explain the structure-function relationship and the mode of cofactor (NADPH) binding in HCTR enzyme at the molecular level. The nucleotide binding domain of modelled HCTR adopts a classic Rossmann fold and possesses a consensus glycine rich GxGxxG motif. Molecular simulation studies suggested that HCTR model retained stability throughout the simulation in aqueous solution. HCTR model showed considerable structural identities with the cofactor binding site of DFR, but significant difference in the catalytic site might be the reason of functional divergence between these families of proteins. Similarly electrostatic surface potential analysis of both HCTR and DFR revealed profound variations in the charge distribution over the substrate binding site, which can be correlated with the sequence variability and may suggest distinct substrate-binding patterns and differences in the catalytic mechanism. Docking results indicated Phe19, Gly21, Arg40, Thr90, Gly208, Arg218, Glu221 and Thr222 are important residues for cofactor (NADPH) binding through strong hydrogen bonding and electrostatic interactions. Alanine scanning and analysis of docking energies of mutant proteins suggested that Phe19, and Arg40 are two critical residues for the cofactor binding. The result from the present study is expected to pave the way for exploration of similar genes in other economically important crop varieties.