Gohar Taj

MAPK machinery in plants: Recognition and response to different stresses through multiple signal transduction pathways

The mitogen-activated protein kinase (MAPK) cascades play diverse roles in intra- and extra-cellular signaling in plants. MAP kinases are the component of kinase modules which transfer information from sensors to responses in eukaryotes including plants. They play a pivotal role in transduction of diverse extracellular stimuli such as biotic and abiotic stresses as well as a range of developmental responses including differentiation, proliferation and death. Several cascades are induced by different biotic and abiotic stress stimuli such as pathogen infections, heavy metal, wounding, high and low temperatures, high salinity, UV radiation, ozone, reactive oxygen species, drought and high or low osmolarity. MAPK signaling has been implicated in biotic stresses and has also been associated with hormonal responses. The cascade is regulated by various mechanisms, including not only transcriptional and translational regulation but through post-transcriptional regulation such as protein-protein interactions. Recent detailed analysis of certain specific MAP kinase pathways have revealed the specificity of the kinases in the cascade, signal transduction patterns, identity of pathway targets and the complexity of the cascade. The latest insights and finding are discussed in this paper in relation to the role of MAPK pathway modules in plant stress signaling.

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.

Genome-Wide Comparative in silico Analysis of Calcium Transporters of Rice and Sorghum

The mechanism of calcium uptake, translocation and accumulation in Poaceae has not yet been fully understood. To address this issue, we conducted genome-wide comparative in silico analysis of the calcium (Ca2+) transporter gene family of two crop species, rice and sorghum. Gene annotation, identification of upstream cis-acting elements, phylogenetic tree construction and syntenic mapping of the gene family were performed using several bioinformatics tools. A total of 31 Ca2+ transporters, distributed on 9 out of 12 chromosomes, were predicted from rice genome, while 28 Ca2+ transporters predicted from sorghum are distributed on all the chromosomes except chromosome 10 (Chr 10). Interestingly, most of the genes on Chr 1 and Chr 3 show an inverse syntenic relationship between rice and sorghum. Multiple sequence alignment and motif analysis of these transporter proteins revealed high conservation between the two species. Phylogenetic tree could very well identify the subclasses of channels, ATPases and exchangers among the gene family. The in silico cis-regulatory element analysis suggested diverse functions associated with light, stress and hormone responsiveness as well as endosperm- and meristem-specific gene expression. Further experiments are warranted to validate the in silico analysis of the predicted transporter gene family and elucidate the functions of Ca2+ transporters in various biological processes.

Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.).

Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca(2+)/H(+) antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca(2+) ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca(2+) ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet.

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.

Identification and characterization of calcium transporter gene family in finger millet in relation to grain calcium content

Calcium (Ca) is an essential mineral for proper growth and development of plants as well as animals. In plants including cereals, calcium is deposited in seed during its development which is mediated by specialized Ca transporters. Common cereal seeds contain very low amounts of Ca while the finger millet (Eleusine coracana) contains exceptionally high amounts of Ca in seed. In order to understand the role of Ca transporters in grain Ca accumulation, developing seed transcriptome of two finger millet genotypes (GP-1, low Ca and GP-45 high Ca) differing in seed Ca content was sequenced using Illumina paired-end sequencing technology and members of Ca transporter gene family were identified. Out of 109,218 and 120,130 contigs, 86 and 81 contigs encoding Ca transporters were identified in GP-1 and GP-45, respectively. After removal of redundant sequences, a total of 19 sequences were confirmed as Ca transporter genes, which includes 11 Ca(2+) ATPases, 07 Ca(2+)/cation exchangers and 01 Ca(2+) channel. The differential expressions of all genes were analyzed from transcriptome data and it was observed that 9 and 3 genes were highly expressed in GP-45 and GP-1 genotypes respectively. Validation of transcriptome expression data of selected Ca transporter genes was performed on different stages of developing spikes of both genotypes grown under different concentrations of exogenous Ca. In both genotypes, significant correlation was observed between the expression of these genes, especially EcCaX3, and on the amount of Ca accumulated in seed. The positive correlation of seed mass with the amount of Ca concentration was also observed. The efficient Ca transport property and responsiveness of EcCAX3 towards exogenous Ca could be utilized in future biofortification program.

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.

Differential induction of two different cystatin genes during pathogenesis of Karnal bunt (Tilletia indica) in wheat under the influence of jasmonic acid

In the present study, expression patterns of two different wheat cystatins (WCs) were studied under the influence of jasmonate signaling in triggering resistance against Karnal bunt (KB). Cystatins are cysteine proteinase inhibitors (CPI) constituting a multigene family which regulate the activity of endo- and/or exogenous cysteine proteinases (CP). Two wheat varieties HD-29 (resistant, R) and WH-542 (susceptible, S) were pre-conditioned with jasmonate and then artificially inoculated with sporidial suspension of Tilletia indica to study its influence in inducing defense by regulating cystatin genes. On the transcriptional level, WC4 and WC5 gave different temporal expression patterns. Expression of WC4 was higher in boot emergence stage which is most susceptible to KB and then slowly declined in both varieties. Expression of WC5 showed an entirely reverse pattern of expression, which kept on rising as the grains matured. Cystatin activity determination by inhibitor assay gave higher activity in resistant variety and under JA treatment. Estimation of specific activity of total cystatin at different days after inoculation (DAI) showed that JA positively induced cystatin expression in both varieties but R variety always registered a greater cystatin expression than the susceptible one (P<0.05). In plants inoculated with pathogen, initially there was a rise in cystatin activity which gradually decreased 7 DAI when compared with the un-inoculated plants. Based on these findings it is clearly demonstrated that jasmonate acts as a potential activator of induced resistance by up-regulating cystatin expression and provides the conditioning effect prior to infection through the maintenance of critical balance of CP/CPI interaction. However, different cystatin genes show different temporal expression patterns and may play different roles at various developmental stages of the grain.

PREDICTION OF DOWNSTREAM INTERACTION OF TRANSCRIPTION FACTORS WITH MAPK3 IN ARABIDOPSIS THALIANA USING PROTEIN SEQUENCE INFORMATION

Protein*Protein interactions (PPIs) are vital to most biological processes thus the identification of PPIs is of primary importance. In the present work, we endeavor to identify the downstream interaction partners of Mitogen Activated Protein Kinase3 (MAPK3) in Arabidopsis Thaliana using the information of protein sequences through Support Vector Machine (SVM) approach. The approach here used is supervised learning based on physiochemical properties of protein sequences through which we predict whether the MAPK3 proteins interact with downstream transcription factor proteins viz., Myb, bZIP, WRKY, Myb*related proteins, AP2/EREBP, and NAC with which its interaction is almost unknown. The Myb*related transcription factor family is showing maximum interaction percentage i.e. 71.14% with MAPK3 while minimum interaction percentage is 21.15% which is shown by NAC transcription factor family. The interaction percentage shown by the gene loci of rest transcription factor family i.e . Myb, bZIP, AP2/EREBP, WRKY are 67.78%, 68.05%, 21.91% and 58.33% respectively. The results of our study clearly revealed the complexity of MAPK3 interaction with several variants of different transcription factors and the same can be verified by different methodology of wet lab experimentation for elucidating the role in various biological processes.

MAPK Signaling Cascades and Transcriptional Reprogramming in Plant–Pathogen Interactions

In plants, innate immunity is triggered through pattern recognition receptors (PRRs) in response to microbe-associated molecular patterns (MAMPs) to provide the first line of inducible defense. Plant receptor protein kinases (RPKs) represent the main plasma membrane PRRs perceiving diverse MAMPs. RPKs trigger mitogen-activated protein kinase (MAPK) module which is one of the earliest signaling events after plant sensing of the invading pathogen as they link the perception of external stimuli to cellular responses. MAPK signaling networks serve specific and overlapping roles in controlling the activities and synthesis of a plethora of transcription factors (TFs), enzymes, hormones, peptides, and antimicrobial chemicals, contributing to resistance against bacteria, oomycetes, and fungi. Transcriptional reprogramming has been carried out by one of the most studied WRKY family of transcription factors. Recently, genetic evidence directly proved its significance as positive and negative regulators of disease resistance. WRKY genes were shown to be functionally connected forming a transcriptional network composed of positive and negative feedback loops and feed-forward modules. Within a web of partially redundant elements, some WRKY factors hold central positions mediating fast and efficient activation of defense programs.