Comparative functional proteomics of MAP Kinase signalling in Magnaporthe oryzae

Rice blast disease is caused by the heterothallic ascomycete fungus Magnaporthe oryzae and is one of the most severe diseases of cultivated rice throughout the world. The PMK1 (Pathogenicity Mitogen-activated protein Kinase) gene in M. oryzae has been identified to play diverse roles during pathogenesis-related development. PMK1 regulates appressorium formation and infectious hyphal growth. PMK1 is functionally related to Saccharomyces cerevisiae FUS3 and KSS1 MAPK genes which regulate the transcription factor Ste12. The STE12 homologue in M. oryzae, MST12, has also been identified and is known to be essential for appressorium mediated penetration and infectious growth. These observations imply that Pmk1 regulates a diverse set of targets important in both the initiation of appressorium development and the subsequent stages of invasive growth. In addition, the Mst12 transcription factor may function downstream of Pmk1 to regulate genes involved in appressorial penetration and infectious hyphal growth. We have used a comparative proteomic study between Guy11 (isogenic wild type), ∆mst12 and ∆pmk1 mutants to understand which genes are induced during appressorium formation and may be regulated by Mst12 and Pmk1. We carried out two-dimensional gel electrophoresis with proteins extracted from conidia that had germinated on a hydrophobic surface after 6h, 12h, 16h and 24h of incubation. M. oryzae underwent major changes in protein abundance and expression during the first 6 hours of spore germination in Guy11 which suggested that much of protein synthesis associated with appressorium morphogenesis and virulence occurs precociously during conidium germination on rice leaf surface. More than 394 differentially expressed proteins during conidium germination and appressorium formation have been identified by mass spectrometry. We confirmed many proteins already known as determinants of pathogenicity in M. oryzae such as enzymes involved in melanin biosynthesis or fatty acid β-oxidation. However, we also identified proteins absent or lowly induced in the Δpmk1 and Δmst12 mutants involved in cell wall reorganisation, secondary metabolism, lipid metabolism, photomorphogenesis or found as proteins of unknown functions. We generated 28 targeted gene deletion mutants to test the putative function of proteins predicted by proteomics analysis to be associated with appressorium development. We obtained 7 mutants showing a significant reduction in virulence and confirmed importance of regulated proteolysis during appressorium development but also elucidated new processes involved in infection such as the phosphoinositide pathway and three proteins of unknown function

Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms –a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice

Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [S-35]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment

Modulation of Chromatin Remodelling Induced by the Freshwater Cyanotoxin Cylindrospermopsin in Human Intestinal Caco-2 Cells

International audienceCylindrospermopsin (CYN) is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 mM for 24hrs) using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes), and DNA recombination and repair (with up-regulation of aptx and pms2 genes). Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2) involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5) and dimethylated histone H3 (Lys4), two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN

Ultraviolet photodissociation for non-target screening-based identification of organic micro-pollutants in water samples

Non-target screening (NTS) based on the combination of liquid chromatography coupled to high-resolution mass spectrometry has become the key method to identify organic micro-pollutants (OMPs) in water samples. However, a large number of compounds remains unidentified with current NTS approaches due to poor quality fragmentation spectra generated by suboptimal fragmentation methods. Here, the potential of the alternative fragmentation technique ultraviolet photodissociation (UVPD) to improve identification of OMPs in water samples was investigated. A diverse set of water-relevant OMPs was selected based on k-means clustering and unsupervised artificial neural networks. The selected OMPs were analyzed using an Orbitrap Fusion Lumos equipped with UVPD. Therewith, information-rich MS2 fragmentation spectra of compounds that fragment poorly with higher-energy collisional dissociation (HCD) could be attained. Development of an R-based data analysis workflow and user interface facilitated the characterization and comparison of HCD and UVPD fragmentation patterns. UVPD and HCD generated both unique and common fragments, demonstrating that some fragmentation pathways are specific to the respective fragmentation method, while others seem more generic. Application of UVPD fragmentation to the analysis of surface water enabled OMP identification using existing HCD spectral libraries. However, high-throughput applications still require optimization of informatics workflows and spectral libraries tailored to UVPD.ISSN:1420-304

Finite element dq-model for MTPA flux control of Synchronous Reluctance Motor (SynRM)

This article focuses on the modelling of SynRM including magnetic saturation and cross-saturation to obtain accurate dynamic simulations from models obtained by Finite Element Analysis (FEA). It is shown here that in the presence of nonlinear magnetic materials, the use of flux instead of current is of interest for both simulation and control design. From this observation, dynamic flux simulation model is obtained. The accuracy of the modelling is illustrated though the design of a Maximum Torque Per Ampere (MTPA) based flux control. Simulation results, close to the real SynRM obtained from FEA are conducted to validate the development and its practical usefulness

Proteomic investigation of the effect of salicylic acid on arabidopsis seed germination and establishment of early defense mechanisms 1[W][OA]

International audienc

Proteomic Investigation of the Effect of Salicylic Acid on Arabidopsis Seed Germination and Establishment of Early Defense Mechanisms

The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [(35)S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor

Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer

We are submitting a manuscript describing the application of proton transfer charge reduction to increase the sequence coverage for proteins > 30 kDa using Orbitrap FTMS