Rajesh Kumar Pathak

Modeling of the MAPK machinery activation in response to various abiotic and biotic stresses in plants by a system biology approach

Mitogen-Activated Protein Kinases (MAPKs) cascade plays an important role in regulating plant growth and development, generating cellular responses to the extracellular stimuli. MAPKs cascade mainly consist of three sub-families i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK), several cascades of which are activated by various abiotic and biotic stresses. In this work we have modeled the holistic molecular mechanisms essential to MAPKs activation in response to several abiotic and biotic stresses through a system biology approach and performed its simulation studies. As extent of abiotic and biotic stresses goes on increasing, the process of cell division, cell growth and cell differentiation slow down in time dependent manner. The models developed depict the combinatorial and multicomponent signaling triggered in response to several abiotic and biotic factors. These models can be used to predict behavior of cells in event of various stresses depending on their time and exposure through activation of complex signaling cascades.

Systems Biology for Smart Crops and Agricultural Innovation: Filling the Gaps between Genotype and Phenotype for Complex Traits Linked with Robust Agricultural Productivity and Sustainability.

In recent years, rapid developments in several omics platforms and next generation sequencing technology have generated a huge amount of biological data about plants. Systems biology aims to develop and use well-organized and efficient algorithms, data structure, visualization, and communication tools for the integration of these biological data with the goal of computational modeling and simulation. It studies crop plant systems by systematically perturbing them, checking the gene, protein, and informational pathway responses; integrating these data; and finally, formulating mathematical models that describe the structure of system and its response to individual perturbations. Consequently, systems biology approaches, such as integrative and predictive ones, hold immense potential in understanding of molecular mechanism of agriculturally important complex traits linked to agricultural productivity. This has led to identification of some key genes and proteins involved in networks of pathways involved in input use efficiency, biotic and abiotic stress resistance, photosynthesis efficiency, root, stem and leaf architecture, and nutrient mobilization. The developments in the above fields have made it possible to design smart crops with superior agronomic traits through genetic manipulation of key candidate genes.

In Silico Identification of Mimicking Molecules as Defense Inducers Triggering Jasmonic Acid Mediated Immunity against Alternaria Blight Disease in Brassica Species

Alternaria brassicae and Alternaria brassicicola are two major phytopathogenic fungi which cause Alternaria blight, a recalcitrant disease on Brassica crops throughout the world, which is highly destructive and responsible for significant yield losses. Since no resistant source is available against Alternaria blight, therefore, efforts have been made in the present study to identify defense inducer molecules which can induce jasmonic acid (JA) mediated defense against the disease. It is believed that JA triggered defense response will prevent necrotrophic mode of colonization of Alternaria brassicae fungus. The JA receptor, COI1 is one of the potential targets for triggering JA mediated immunity through interaction with JA signal. In the present study, few mimicking compounds more efficient than naturally occurring JA in terms of interaction with COI1 were identified through virtual screening and molecular dynamics simulation studies. A high quality structural model of COI1 was developed using the protein sequence of Brassica rapa. This was followed by virtual screening of 767 analogs of JA from ZINC database for interaction with COI1. Two analogs viz. ZINC27640214 and ZINC43772052 showed more binding affinity with COI1 as compared to naturally occurring JA. Molecular dynamics simulation of COI1 and COI1-JA complex, as well as best screened interacting structural analogs of JA with COI1 was done for 50 ns to validate the stability of system. It was found that ZINC27640214 possesses efficient, stable, and good cell permeability properties. Based on the obtained results and its physicochemical properties, it is capable of mimicking JA signaling and may be used as defense inducers for triggering JA mediated resistance against Alternaria blight, only after further validation through field trials.

Virtual screening of natural inhibitors to the predicted HBx protein structure of Hepatitis B Virus using molecular docking for identification of potential lead molecules for liver cancer.

The HBx protein in Hepatitis B Virus (HBV) is a potential target for anti-liver cancer molecules. Therefore, it is of interest to screen known natural compounds against the HBx protein using molecular docking. However, the structure of HBx is not yet known. Therefore, the predicted structure of HBx using threading in LOMET was used for docking against plant derived natural compounds (curcumin, oleanolic acid, resveratrol, bilobetin, luteoline, ellagic acid, betulinic acid and rutin) by Molegro Virtual Docker. The screening identified rutin with binding energy of -161.65 Kcal/mol. Thus, twenty derivatives of rutin were further designed and screened against HBx. These in silico experiments identified compounds rutin01 (-163.16 Kcal/mol) and rutin08 (- 165.76 Kcal/mol) for further consideration and downstream validation.

Computer aided screening of natural compounds targeting the E6 protein of HPV using molecular docking.

The cancer profile in the Indian state of Uttarakhand reveals that the breast cancer is the most prevalent type of cancers in females followed by cervical and ovarian type. Literature survey shows that the E6 protein of Human Papilloma Virus-16 (HPV-16) is responsible for causing several forms of cancer in human. Therefore, it is of interest to screen HPV-16 E6 target protein with known natural compounds using computer aided molecular modeling and docking tools. The complete structure of E6 is unknown. Hence, the E6 structure model was constructed using different online servers followed by molecular docking of Colchine, Curcumin, Daphnoretin, Ellipticine and Epigallocatechin-3-gallate; five known natural compounds with best E6 protein model predicted by Phyre2 server. The screening exercise shows that Daphnoretin (with binding free energy of -8.3 kcal/mol), a natural compound derived from Wikstroemia indica has the top binding properties. Thus, it is of interest to consider the compound for further validation.

Molecular characterization of EcCIPK24 gene of finger millet (Eleusine coracana) for investigating its regulatory role in calcium transport

Finger millet grains contain exceptionally high levels of calcium which is much higher compared to other cereals and millets. Since calcium is an important macronutrient in human diet, it is necessary to explore the molecular basis of calcium accumulation in the seeds of finger millet. CIPK is a calcium sensor gene, having role in activating Ca2+ exchanger protein by interaction with CBL proteins. To know the role of EcCIPK24 gene in seed Ca2+ accumulation, sequence is retrieved from the transcriptome data of two finger millet genotypes GP1 (low Ca2+) and GP45 (high Ca2+), and the expression was determined through qRT-PCR. The higher expression was found in root, shoot, leaf and developing spike tissue of GP45 compared to GP1; structural analysis showed difference of nine SNPs and one extra beta sheet domain as well as differences in vacuolar localization was predicted; besides, the variation in amino acid composition among both the genotypes was also investigated. Molecular modeling and docking studies revealed that both EcCBL4 and EcCBL10 showed strong binding affinity with EcCIPK24 (GP1) compared to EcCIPK24 (GP45). It indicates a genotypic structural variation, which not only affects the affinity but also calcium transport efficiency after interaction of CIPK-CBL with calcium exchanger (EcCAX1b) to pull calcium in the vacuole. Based on the expression and in silico study, it can be suggested that by activating EcCAX1b protein, EcCIPK24 plays an important role in high seed Ca2+ accumulation.

Identification and molecular characterization of Dof transcription factor gene family preferentially expressed in developing spikes of Eleusine coracana L.

Abstract We report 48 putative DNA binding with one finger (Dof) TF genes from genome and transcriptome data of finger millet (Eleusine coracana L.; FM), involved in plant developmental process. To characterize seed-specific Dof genes, transcript profiles of 32 EcDof identified from transcriptome data of developing spikes of FM genotypes were further analyzed in different tissues (root, stem, and leaf) and developmental stages of spikes (S1, S2, S3, and S4) in two FM genotypes [GE1437 (low protein genotype; LPG) and GE3885 (high protein genotype; HPG)]. More than 50% of identified EcDof genes showed expression during seed development processes. Among these, seven genes (EcDof 3, EcDof 5, EcDof 15, EcDof 18, EcDof 22, EcDof 23, and EcDof 31) expressed maximally at specific stages of seed development. Fourteen EcDof genes showed that differential transcript accumulation in vegetative tissue as well as in developing spikes suggests involvement during seed filling and also throughout the plant development. In addition, three EcDof genes (EcDof 9, EcDof 25, and EcDof 28) expressed preferentially at root and stem tissue. The 3D structural prediction of EcDof proteins showed variability in structural attributes. Molecular docking results showed strong binding affinity for seed-specific EcDof–EcO2 with α-prolamine promoters. The identified and characterized EcDof genes will help to dissect the roles of FM seed-specific Dof genes.

The role of tripartite interaction of calcium sensors and transporters in the accumulation of calcium in finger millet grain

Finger millet (Eleusine coracana) is one of important crops, and its grains contain an exceptionally high content of calcium. In order to investigate the molecular mechanism by which it orchestrate the accumulation of Ca2+ during grain filling, some candidate genes encoding calcium transporters [calcium exchangers (CAX1, CAX3)] and sensors [calcineurin-B like (CBL4 and 10)], a CBL-interacting protein kinase (CIPK24), and calmodulin (CaM) were identified using transcriptomics and differential expression analysis in two genotypes of finger millet differing in grain calcium content. These transporters and sensors are highly expressed in leaves and developing spikes of the genotype with a high grain Ca2+ indicating their potential role in Ca2+ accumulation. Calcium transporters, mainly CAXs, pump Ca2+ inside the cell through plasmalemma and tonoplast, and their activities are regulated by CaM dependent and independent Ca2+ sensor proteins of CaM and CBL-CIPK networks. Abundance of CaM in a high grain Ca2+ genotype is suggestive that CaM might also contribute for grain calcium accumulation by interaction with Ca2+ATPase. The upregulation of CAX1 in vegetative tissues and developing spikes and CAX3 only in developing spikes provides the most plausible clue for calcium transport and accumulation regulated by tripartite interaction in finger millet.

Systems biology of seeds: decoding the secret of biochemical seed factories for nutritional security

Seeds serve as biochemical factories of nutrition, processing, bio-energy and storage related important bio-molecules and act as a delivery system to transmit the genetic information to the next generation. The research pertaining towards delineating the complex system of regulation of genes and pathways related to seed biology and nutrient partitioning is still under infancy. To understand these, it is important to know the genes and pathway(s) involved in the homeostasis of bio-molecules. In recent past with the advent and advancement of modern tools of genomics and genetic engineering, multi-layered ‘omics’ approaches and high-throughput platforms are being used to discern the genes and proteins involved in various metabolic, and signaling pathways and their regulations for understanding the molecular genetics of biosynthesis and homeostasis of bio-molecules. This can be possible by exploring systems biology approaches via the integration of omics data for understanding the intricacy of seed development and nutrient partitioning. These information can be exploited for the improvement of biologically important chemicals for large-scale production of nutrients and nutraceuticals through pathway engineering and biotechnology. This review article thus describes different omics tools and other branches that are merged to build the most attractive area of research towards establishing the seeds as biochemical factories for human health and nutrition.