Bacterial host adaptation through sequence and structural variations of a single type III effector gene.

Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.

Bacterial eradication by a low-energy pulsed electron beam generator.

Low-energy electron beams (LEEB) are a safe and practical sterilization solution for in-line industrial applications, such as sterilizing medical products. However, their low dose rate induces product degradation, and the limited maximal energy prohibits high-throughput applications. To address this, we developed a low-energy 'pulsed' electron beam generator (LEPEB) and evaluated its efficacy and mechanism of action. Bacillus pumilus vegetative cells and spores were irradiated with a 250 keV LEPEB system at a 100 Hz pulse repetition frequency and a pulse duration of only 10 ns. This produced highly efficient bacterial inactivation at a rate of >6 log10, the level required for sterilization in industrial applications, with only two pulses for vegetative bacteria (20 ms) and eight pulses for spores (80 ms). LEPEB induced no morphological or structural defects, but decreased cell wall hydrophobicity in vegetative cells, which may inhibit biofilm formation. Single- and double-strand DNA breaks and pyrimidine dimer formation were also observed, likely causing cell death. Together, the unique combination of high dose rate and nanosecond delivery of LEPEB enable effective and high-throughput bacterial eradication for direct integration into production lines in a wide range of industrial applications.

Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins.

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

In Depth Exploration of the Alternative Proteome of Drosophila melanogaster.

Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. In the present article, we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed 14 alternative proteins with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins.

S-Nitrosation of Arabidopsis thaliana Protein Tyrosine Phosphatase 1 Prevents Its Irreversible Oxidation by Hydrogen Peroxide.

Tyrosine-specific protein tyrosine phosphatases (Tyr-specific PTPases) are key signaling enzymes catalyzing the removal of the phosphate group from phosphorylated tyrosine residues on target proteins. This post-translational modification notably allows the regulation of mitogen-activated protein kinase (MAPK) cascades during defense reactions. Arabidopsis thaliana protein tyrosine phosphatase 1 (AtPTP1), the only Tyr-specific PTPase present in this plant, acts as a repressor of H2O2 production and regulates the activity of MPK3/MPK6 MAPKs by direct dephosphorylation. Here, we report that recombinant histidine (His)-AtPTP1 protein activity is directly inhibited by H2O2 and nitric oxide (NO) exogenous treatments. The effects of NO are exerted by S-nitrosation, i.e., the formation of a covalent bond between NO and a reduced cysteine residue. This post-translational modification targets the catalytic cysteine C265 and could protect the AtPTP1 protein from its irreversible oxidation by H2O2. This mechanism of protection could be a conserved mechanism in plant PTPases.

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.

The final step of 40S ribosomal subunit maturation is controlled by a dual key lock.

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

A Proteomic- and Bioinformatic-Based Identification of Specific Allergens from Edible Insects: Probes for Future Detection as Food Ingredients.

The increasing development of edible insect flours as alternative sources of proteins added to food and feed products for improving their nutritional value, necessitates an accurate evaluation of their possible adverse side-effects, especially for individuals suffering from food allergies. Using a proteomic- and bioinformatic-based approach, the diversity of proteins occurring in currently consumed edible insects such as silkworm (Bombyx mori), cricket (Acheta domesticus), African migratory locust (Locusta migratoria), yellow mealworm (Tenebrio molitor), red palm weevil (Rhynchophorus ferrugineus), and giant milworm beetle (Zophobas atratus), was investigated. Most of them consist of phylogenetically-related protein allergens widely distributed in the different groups of arthropods (mites, insects, crustaceans) and mollusks. However, a few proteins belonging to discrete protein families including the chemosensory protein, hexamerin, and the odorant-binding protein, emerged as proteins highly specific for edible insects. To a lesser extent, other proteins such as apolipophorin III, the larval cuticle protein, and the receptor for activated protein kinase, also exhibited a rather good specificity for edible insects. These proteins, that are apparently missing or much less represented in other groups of arthropods, mollusks and nematods, share well conserved amino acid sequences and very similar three-dimensional structures. Owing to their ability to trigger allergic responses in sensitized people, they should be used as probes for the specific detection of insect proteins as food ingredients in various food products and thus, to assess their food safety, especially for people allergic to edible insects.

Application of pulsed electric fields for the biocompatible extraction of proteins from the microalga Haematococcus pluvialis.

This study aims to employ a pulsed electric field (PEF) treatment for the biocompatible (non-destructive) extraction of proteins from living cells of the green microalga Haematococcus pluvialis. Using a field strength of 1 kV cm-1, we achieved the extraction of 10.2 µg protein per mL of culture, which corresponded to 46% of the total amount of proteins that could be extracted by complete destructive extraction (i.e. the grinding of biomass with glass beads). We found that the extraction yield was not improved by stronger field strengths and was not dependent on the pulse frequency. A biocompatibility index (BI) was defined as the relative abundance of cells that remained alive after the PEF treatment. This index relied on measurements of several physiological parameters after a PEF treatment. It was found that at 1 kV cm-1 that cultures recovered after 72 h. Therefore, these PEF conditions constituted a good compromise between protein extraction efficiency and culture survival. To characterize the PEF treatment further at a molecular level, mass spectrometry-based proteomics analyses of PEF-prepared extracts was used. This led to the identification of 52 electro-extracted proteins. Of these, only 16 proteins were identified when proteins were extracted with PEF at 0.5 cm-1. They belong to core metabolism, stress response and cell movement. Unassigned proteins were also extracted. Their physiological implications and possible utilization in food as alimentary complements are discussed.

The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection.

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.

Proteomic Analysis of Two Weight Classes of Mule Duck "foie gras" at the End of an Overfeeding Period.

The weight of the liver is one of the important selection criteria in the quality of "foie gras". This factor is highly variable despite the fact that individuals are reared, overfed and slaughtered in the same way. In this study, we performed an analysis of the proteome profile of two weight classes of light (between 550 and 599 g) and heavy (more than 700 g) livers. For the analysis of the proteic extracts, a liquid chromatographic analysis coupled with mass spectrometry was carried out. In low-weight livers, aerobic energy metabolism, protein metabolism and lipid metabolism oriented toward export and beta-oxidation were overexpressed. On the contrary, high weight livers were characterized by anaerobic energy metabolism and a more active protein catabolism associated with cell apoptosis and reorganization of the cell structure.

Metabolomic and proteomic changes induced by growth inhibitory concentrations of copper in the biofilm-forming marine bacterium Pseudoalteromonas lipolytica.

Copper is an essential element for living cells but this metal is present in some marine environments at such high concentrations that it can be toxic for numerous organisms. In polluted areas, marine organisms may develop specific adaptive responses to prevent cell damage. To investigate the influence of copper on the metabolism of a single organism, a dual approach combining metabolomics and proteomics was undertaken on the biofilm-forming bacterial strain Pseudoalteromonas lipolytica TC8. In order to highlight differential adaptation according to the phenotype, the response of P. lipolytica TC8 to copper stress was studied in planktonic and biofilm culture modes under growth inhibitory copper concentrations. As expected, copper exposure led to the induction of defense and detoxification mechanisms. Specific metabolite and protein profiles were thus observed in each condition (planktonic vs. biofilm and control vs. copper-treated cultures). Copper exposure seems to induce drastic changes in the lipid composition of the bacterial cell membrane and to modulate the abundance of proteins functionally known to be involved in copper cell homeostasis in both planktonic and biofilm culture modes. Much more proteins differentially expressed after copper treatment were observed in biofilms than in planktonic cells, which could indicate a more heterogeneous response of biofilm cells to this metallic stress.

Insights into the Allergenic Potential of the Edible Yellow Mealworm (Tenebrio molitor).

The edible yellow mealworm (Tenebrio molitor), contains an extremely diverse panel of soluble proteins, including proteins with structural functions such as muscle proteins, as well as proteins involved in metabolic functions such as enzymes. Most of these proteins display a more or less pronounced allergenic character toward previously sensitized people, especially people allergic to shrimps and other shellfish. A mass spectrometry approach following the separation of a mealworm protein, extracted by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, allowed us to identify up to 106 distinct protein fractions including molecules with structural and functional functions, susceptible to developing an allergenic potential due to the possibility of immunoglobulin E-binding cross-reactions with their counterparts occurring in shellfish. In this respect, most of the sera from people allergic to shrimps reacted with the mealworm protein extract in Western blot experiments. Moreover, the potential mealworm allergens triggered the in vitro degranulation of rat leukemic basophils transfected with the human high-affinity IgE receptor (FcεRI), upon sensitization by the IgE-containing sera from people allergic to shrimps and other shellfish foods. Owing to the large repertoire of IgE-binding cross-reacting allergens the yellow mealworm shares with other phylogenetically-related groups of arthropods, it would seem prudent to inform the consumers, especially those allergic to shellfish, by appropriate labeling on edible mealworm packages about the potential risk of developing an allergic reaction.

Analysis of durum wheat proteome changes under marine and fungal biostimulant treatments using large-scale quantitative proteomics: A useful dataset of durum wheat proteins.

Durum wheat requires high nitrogen inputs to obtain the high protein concentration necessary to satisfy pasta and semolina quality criteria. Optimizing plant nitrogen use efficiency is therefore of major importance for wheat grain quality. Here, we studied the impact on grain yield, protein concentration, and for the first time on protein composition of a marine (DPI4913) and a fungal (AF086) biostimulants applied to plant leaves. A large-scale quantitative proteomics analysis of wheat flour samples led to a dataset of 1471 identified proteins. Quantitative analysis of 1391 proteins revealed 26 and 38 proteins with a significantly varying abundance after DPI4913 and AF086 treatment, respectively, with 14 proteins in common. Major effects affected proteins involved in grain technological properties like grain hardness, in storage functions with the gluten protein gamma-gliadin, in regulation processes with transcription regulator proteins, and in stress responses with biotic and abiotic stress defense proteins. The involvement of biostimulants in the abiotic stress response was further suggested by an increase in water-use efficiency for both DPI4913 (15.4%) and AF086 (9.9%) treatments. Overall, our work performed in controlled conditions showed that DPI4913 and AF086 treatments promoted grain yield while maintaining protein concentration, and positively affected protein composition for grain quality. Data are available via ProteomeXchange with identifier PXD012469.

Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8.

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways.

The infection of the model legume Medicago truncatula with Ralstonia solanacearum GMI1000 gives rise to bacterial wilt disease via colonisation of roots. The root and leaf responses to early infection (1 and 3 days post infection) were characterised to investigate the molecular mechanisms of plant resistance or susceptibility. A proteomics approach based on pools of susceptible and resistant recombinant inbred lines was used to specifically target the mechanisms for tolerance. Differential abundances were evidenced for proteins involved in defence (e.g., PR5, PR10, or Kunitz protease inhibitors) and signalling pathways (such as cyclophilin). R. solanacearum inoculation modifies expression levels of those genes, either in both genotypes (AOS1, LOX4, and proteinase inhibitors) or specifically in the resistant line (PR proteins). Exogenous application of salicylic acid (SA) enhanced tolerance to the bacteria, whereas methyl jasmonate (MeJA) enhanced short-term tolerance then promoted disease in the susceptible ecotype, suggesting that they may mediate defence responses. Conversely, proteomics-identified genes were also shown to be SA or MeJA responsive. This is the first description of differential response to R. solanacearum in M. truncatula. Our results suggest that root basal defence is activated at 1 dpi, together with the JA pathway. Specific resistance is then evidenced at three dpi, with the up-regulation of SA-dependent PR proteins.

Functional characterization of the chaperon-like protein Cdc48 in cryptogein-induced immune response in tobacco.

Cdc48, a molecular chaperone conserved in different kingdoms, is a member of the AAA+ family contributing to numerous processes in mammals including proteins quality control and degradation, vesicular trafficking, autophagy and immunity. The functions of Cdc48 plant orthologues are less understood. We previously reported that Cdc48 is regulated by S-nitrosylation in tobacco cells undergoing an immune response triggered by cryptogein, an elicitin produced by the oomycete Phytophthora cryptogea. Here, we inv estigated the function of NtCdc48 in cryptogein signalling and induced hypersensitive-like cell death. NtCdc48 was found to accumulate in elicited cells at both the protein and transcript levels. Interestingly, only a small proportion of the overall NtCdc48 population appeared to be S-nitrosylated. Using gel filtration in native conditions, we confirmed that NtCdc48 was present in its hexameric active form. An immunoprecipitation-based strategy following my mass spectrometry analysis led to the identification of about a hundred NtCdc48 partners and underlined its contribution in cellular processes including targeting of ubiquitylated proteins for proteasome-dependent degradation, subcellular trafficking and redox regulation. Finally, the analysis of cryptogein-induced events in NtCdc48-overexpressing cells highlighted a correlation between NtCdc48 expression and hypersensitive cell death. Altogether, this study identified NtCdc48 as a component of cryptogein signalling and plant immunity.

Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.

LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.