Project description:Global food production is reliant on the application of finite phosphorus (P) fertilisers. Numerous negative consequences associated with intensive P fertilisation have resulted in a high demand to find alternative sustainable methods that will enhance crop P uptake. Bacteroidetes, primarily from the genus Flavobacterium, have recently been shown to be abundant members of the plant microbiome, but their general ecological role and potential to mobilise P in the rhizosphere remains very poorly characterised. Here, we sought to determine the P mobilisation potential of Flavobacterium strains isolated from the rhizosphere of oilseed rape (Brassica napus L.). In contrast to other abundant rhizosphere bacteria, such as Pseudomonas, all Flavobacterium strains exhibited constitutive phosphatase activity independent of external phosphate (Pi) concentrations. Interestingly, a combination of exoproteomic analysis and molecular microbiology techniques revealed that Flavobacterium have a complex and largely unique repertoire of proteins to mobilise and acquire Pi. This includes the expression of novel, as yet unidentified, phosphatases, and numerous proteins of unknown function. We also discovered that Flavobacterium expresses certain SusCD-like transporters, whose role is typically associated with specialised carbon acquisition, in response to Pi-starvation. Furthermore, the genes encoding these unusual Pi-responsive proteins were enriched in plant-associated Flavobacterium strains suggesting that this machinery represents niche-adaptive strategies for overcoming P scarcity in this genus. We propose that abundant rhizosphere-dwelling Flavobacterium spp. have evolved unique mechanisms for coping with Pi-stress which may provide novel solutions for future sustainable agricultural practices.
Project description:We present here evidence that histones H3.1 and H4 can be imported into the nucleus as monomers in human cells. Using a tether-and-release system to study the cytosolic phase and import dynamics of newly synthesised histones, we find that H3.1 and H4 can be maintained as stable monomers in the cytosol in a tethered state. Cytosolically tethered histones are bound tightly to Importin- proteins (predominantly IPO4), but not to the histone specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, dissociation with IPO4 and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones under steady-state conditions reveals an excess of H3 specifically associated with sNASP, suggesting that NASP can maintain a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol, with sNASP acting as a potential nuclear receptor for monomeric histone H3.
Project description:Recent evidence suggests that the ubiquitin-proteasome system (UPS) is involved in several aspects of plant immunity and a range of plant pathogens subvert the UPS to enhance their virulence. Here we show that proteasome activity is strongly induced during basal defense in Arabidopsis. Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas syringae pv. maculicola ES4326. Both proteasome subunits are required for Pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic-acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type-III secretion dependent manner. A screen for type-III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1 and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass-spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome leading to its inhibition. Thus, the proteasome is an essential component of PTI and SAR, which is targeted by multiple bacterial effectors.
Project description:PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is involved in both chromosome segregation during mitotic exit, and potentially establishment of cell polarity during zygote development in the mosquito midgut, suggesting that chemical inhibitors of PP1 may be explored for blocking parasite transmission
Project description:We designed a bottom-up proteomic approach combining two complementary strategies as GeLC-MS/MS (protein samples fractionated by SDS-PAGE are analyzed by nanoLC-MS/MS after in-gel digestion) and shotgun (protein samples are directly analyzed by nanoLC-MS/MS after in-solution digestion) to give an exhaustive view of the proteome of the chicken amniotic fluid at the 11th day of the development, before the substantial transfer of egg white (which completely changes the global protein profile).
Project description:We characterized the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.
Project description:Trimethylation of histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved Bromo-Adjacent Homology (BAH) module of BAHCC1 (BAHCC1BAH) ‘recognizes’ H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and ChIP-seq-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-lysine-binding ‘cage’ formed by BAHCC1BAH, mediating co-localization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is overexpressed in human acute leukemias and interacts with transcriptional co-repressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1BAH:H3K27me3 interaction, causes de-repression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germ-line mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study unveils a novel H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH ‘reader’.
Project description:Plasmepsin X (PM X) is an aspartate protease expressed in the malaria parasie Plasmodium falciparum. Following synthesis PM X undergoes proteolytic processing. This project aims to understand the significance of PM X processing. To identify the cleavage sites, the processed PM X bands were excised from gels and subjected to LC-MSMS.
Project description:Only a few small regulatory RNAs (sRNAs) have been characterized in B. subtilis, the paradigm of Gram-positive bacteria, and one of the major challenges is target identification. Here we use global in vivo RNA psoralen cross-linking to identify RNA-RNA partners in Bacillus subtilis. Two sRNAs, RoxS and FsrA, play key roles in balancing the metabolic state of the cell in response to carbon sources and iron limitation, respectively. In this study, we identify new mRNA targets for both RoxS and FsrA, and a small RNA (S345/RosA) that is able to interact with both sRNAs. We report that RosA controls the maturation and degradation of RoxS and acts as a sponge to limit the efficacy of RoxS on its targets. Expression of RosA is catabolically repressed by the transcription factor CcpA. We provide evidence that the RosA/RoxS interaction plays a key role in regulating metabolism in response to a switches in carbon source.
Project description:This project mainly aims to characterize the complex toxic components present in the venom of Indian cobra (Naja naja) from the Western Ghats of India. Naja naja (NN) is native to the Indian subcontinent and is also found in Pakistan, Sri Lanka, Bangladesh and Southern Nepal. It is a highly venomous snake species of genus Naja of the Elapidae family. They are seen in wide habitats like plains, dense or open forests, rocky terrains, wetlands, agricultural lands, and outskirts of villages and even in highly populated urban areas. This species has been included in the ‘Big 4’ category of venomous snake species that accounts for majority of morbidity and mortality cases in India. Therefore, exploring the venom proteome of Naja naja is decisive to develop and design new antivenom and therapeutics against its envenomation. The venom proteome of Naja naja was characterized through various orthogonal separation strategies and identification strategies. In order to achieve this the crude venom components were resolved on a 12% SDS page. Also, the venom was decomplexed through reversed-phase HPLC followed by SDS analysis. Further each of the bands were subjected to in-gel digestion using trypsin, chymotrypsin and V8 proteases. All the digested peptides were then subjected to Q-TOF LC-MS/MS analysis.