Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.     

Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and evolutionary perspect

The extracellular matrix (ECM) or cell wall is a dynamic system and serves as the first line mediator in cell signaling to perceive and transmit extra- and intercellular signals in many pathways. Although ECM is a conserved compartment ubiquitously present throughout evolution, a compositional variation does exist among different organisms. ECM proteins account for 10% of the ECM mass, however, comprise several hundreds of different molecules with diverse functions. To understand the function of ECM proteins, we have developed the cell wall proteome of a crop legume, chickpea (Cicer arietinum). This comprehensive overview of the proteome would provide a basis for future comparative proteomic efforts for this important crop. Proteomic analyses revealed new ECM proteins of unknown functions vis-à-vis the presence of many known cell wall proteins. In addition, we report here evidence for the presence of unexpected proteins with known biochemical activities, which have never been associated with ECM.     

Optimization of redox reactions employing whole cell biocatalysis

The ability of Saccharomyces cerevisiae to catalyse the reduction reaction of carboxylic acids into alcohols is described. Earlier reports have led to the characterization of the reduction of carbonyl groups into alcohols mediated by the enzyme alcohol dehydrogenase. We investigated the ability of this organism to catalyse the said conversion using the carboxylic acids, acetic acid and butyric acid. In the absence of any previous characterization, whole cell catalysis proved effective. The uptake of these acids from the medium was estimated using a plate assay method involving litmus-agar. The plate assay was found to be a convenient and extremely adaptable method for quantitation of acids in organic as well as aqueous medium. The comparison of existing paradigms in pure protein catalysis with whole cells catalysis proved anomalous. We report that it is solvent toxicity rather than hydrophobic index that correlates with the activity observed in non-aqueous conditions for whole cell biocatalysis. Reduction of acetic acid as well as butyric acid occurred, with efficiency of reaction with butyric acid being marginally higher. The reduction therefore occurs for both the short chain carboxylic acids used in this study. We therefore illustrate the reduction route of acids into alcohols and propose a model two-step pathway for the reaction. Process optimization may be further attempted to enhance the presently moderate reaction efficiencies. Steps made in the direction by studying the pH dependency and use of sacrificial substrate have yielded encouraging results.     

Expression of estrogen receptor {alpha} in the anteroventral periventricular nucleus of hypogonadal mice

Gonadotropin-releasing hormone-1 (GnRH-1) neurons play critical roles in the development and maintenance of reproductive function in all vertebrates. Due to a truncation in the GnRH-1 gene, hypogonadal (hpg) mice are unable to synthesize GnRH-1 and are infertile. These animals develop in the complete absence of exposure to gonadal steroid hormones, making them an interesting model for understanding brain sexual differentiation and dimorphism. We studied expression of the estrogen receptors (ERs) alpha and beta in the medial anteroventral periventricular nucleus (mAVPV), an important reproductive neuroendocrine brain region, in wild-type and hpg mice of both sexes. Adult wild-type and hpg mice of the same genetic background were used to quantify numbers of ERalpha and ERbeta immunoreactive cells in the mAVPV using a stereologic approach. Quantitative analyses showed that ERalpha cell numbers were significantly higher in hpg than wild-type mice, irrespective of sex. Qualitatively, ERalpha-immunoreactive cells were concentrated more densely along the ventricular zone of the AVPV of wild-type female mice compared with wild-type male mice or hpg male and female mice. No ERbeta-immunoreactive cells were detected in the mAVPV of any mice, a result that was surprising because ERbeta expression is abundant in the mAVPV of rats. These results on ERalpha provide additional evidence that the female brain is not the "default" organizational pattern, because neither ERalpha cell number nor its distribution in hpg mice, which develops with a deficiency of reproductive hormones, resembles that of the wild-type female mouse. Differences in ERalpha expression may be due in part to the absence of gonadal steroid hormones, although they more likely to also involve other factors, potentially GnRH itself.     

Climate change and multitrophic interactions in soil: the primacy of plants and functional domains

Soil multitrophic interactions transfer energy from plants as the predominant primary producer to communities of organisms that occupy different positions in the food chain and are linked by multiple ecological networks, which is the soil food web. Soil food web sequesters carbon, cycles nutrients, maintains soil health to suppress pathogens, helps plants tolerate abiotic and biotic stress, and maintains ecosystem resilience and sustainability. Understanding the influence of climate change on soil multitrophic interactions is necessary to maintain these essential ecosystem services. But summarising this influence is a daunting task due to a paucity of knowledge and a lack of clarity on the ecological networks that constitute these interactions. The scant literature is fragmented along disciplinary lines, often reporting inconsistent findings that are context and scale‐dependent. We argue for the differentiation of soil multitrophic interactions along functional and spatial domains to capture cross‐disciplinary knowledge and mechanistically link all ecological networks to reproduce full functionalities of the soil food web. Distinct from litter mediated interactions in detritosphere or elsewhere in the soil, the proposed ‘pathogen suppression’ and ‘stress tolerance’ interactions operate in the rhizosphere. A review of the literature suggests that climate change will influence the relative importance, frequency and composition of functional groups, their trophic interactions and processes controlling these interactions. Specific climate change factors generally have a beneficial influence on pathogen suppression and stress tolerance, but findings on the overall soil food web are inconsistent due to a high level of uncertainty. In addition to an overall improvement in the understanding of soil multitrophic interactions using empirical and modelling approaches, we recommend linking biodiversity to function, understanding influence of combinations of climatic factors on multitrophic interactions and the evolutionary ecology of multitrophic interactions in a changing climate as areas that deserve most attention.     

CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2''s association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.     

Protective immunity and granulomatous inflammation is mediated in vivo by T cells reactive to epitopes common to avirulent and listeriolysin-negative mutants of Listeria monocytogenes.

The ability of several listeriolysin O-negative mutants of the EGD and NCTC 7973 strains of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. T cell lines from different inbred mouse strains and derived T cell clones elicited by L. monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing revealed no differences between listeriolysin-positive and -negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined CD4+ T cell-mediated granuloma formation in the livers of mice previously immunized with viable bacteria of the virulent strain. Granulomatous inflammation could be elicited by iv application of heat-killed bacteria of listeriolysin-positive and of -negative bacteria. Protective immunity to listerial infections and granulomatous inflammation therefore appears to be mediated by T cells recognizing epitopes on listerial antigens that are shared by both pathogenic and nonpathogenic Listeria strains.     

An Escherichia coli Strain,PGB01, Isolated from Feral Pigeon Faeces,Thermally Fit to Survive in Pigeon,Shows High Level Resistance to Trimethoprim

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.     

Identification of potential targets in Staphylococcus aureus N315 using computer aided protein data analysis

Staphylococcus aureus is a gram positive bacterium, responsible for both community-acquired and hospital-acquired infection, resulting in a mortality rate of 39%. 43.2% resistance to methicilin and emerging resistance to Fluroquinolone and Oxazolidinone, have evoked the necessity of the establishment of alternative and effective therapeutic approach to treat this bacteria. In this computational study, various database and online software are used to determine some specific targets of Staphylococcus aureus N315 other than those used by Penicillin, Quinolone and Oxazolidinone. For this purpose, among 302 essential proteins, 101 nonhomologous proteins were accrued and 64 proteins which are unique in several metabolic pathways of S. aureus were isolated by using metabolic pathway analysis tools. Furthermore, 7 essentially unique enzymes involved in exclusive metabolic pathways were revealed by this research, which can be potential drug target. Along with these important enzymes, 15 non-homologous proteins located on membrane were identified, which can play a vital role as potential therapeutic targets for the future researchers.     

Abiotic elicitors mediated elicitation of innate immunity in tomato: an ex vivo comparison

Improvement of the host resistance by using hazard free chemical elicitors is emerging as an alternative approach in the field of plant disease management. In our present work, we have screened the efficacy and possible mechanism of abiogenic elicitors like Dipotassium hydrogen orthophosphate (K₂HPO₄), Oxalic acid (OA), Isonicotinic acid (INA), Salicylic acid (SA), Acetylsalicylate (AS), Arachidonic acid (AA) and Calcium chloride (CaCl₂) to stimulate innate immune responses in Lycopersicum esculentum Mill. Excised tomato leaves, treated with elicitors at three different concentrations, were found to stimulate defense and antioxidative enzymes, total phenol and flavonoid content after 24 h of incubation. CaCl₂ (0.5 %) followed by INA (2.5 mM) were found most effective in activation of all such defense molecules in tomato leaves. Furthermore, nitric oxide (NO), a key gaseous mediator in plant defense signaling, was also measured after subsequent elicitor application. Higher doses of elicitors showed an elevated level of reactive oxygen species (ROS) generation, enhanced lipid peroxidation rate and proline content, which indicates the extent of abiotic stress generation on the leaves. However, ROS production, lipid peroxidation rate and proline concentration remain significantly reduced as a result of CaCl₂ (0.5 %) and INA (2.5 mM) application. A sharp increase of total chlorophyll content was also recorded due to treatment of CaCl₂ (0.5 %). These results demonstrate the effects of different abiogenic elicitors to regulate the production of defense molecules. Results also suggest that among all such chemicals, CaCl₂ (0.5 %) and INA (2.5 mM) can be used as a potential elicitor in organic farming of tomato.