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International audience; In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

Background Understanding the mechanisms involved in climacteric fruit ripening is key to improve fruit harvest quality and postharvest performance. Kiwifruit (Actinidia deliciosa cv. ‘Hayward’) ripening involves a series of metabolic changes regulated by ethylene. Although 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) or ozone (O3) exposure suppresses ethylene-related kiwifruit ripening, how these molecules interact during ripening is unknown. Results Harvested ‘Hayward’ kiwifruits were treated with 1-MCP and exposed to ethylene-free cold storage (0 °C, RH 95%) with ambient atmosphere (control) or atmosphere enriched with O3 (0.3 μL L− 1) for up to 6 months. Their subsequent ripening performance at 20 °C (90% RH) was characterized. Treatment with either 1-MCP or O3 inhibited endogenous ethylene biosynthesis and delayed fruit ripening at 20 °C. 1-MCP and O3 in combination severely inhibited kiwifruit ripening, significantly extending fruit storage potential. To characterize ethylene sensitivity of kiwifruit following 1-MCP and O3 treatments, fruit were exposed to exogenous ethylene (100 μL L− 1, 24 h) upon transfer to 20 °C following 4 and 6 months of cold storage. Exogenous ethylene treatment restored ethylene biosynthesis in fruit previously exposed in an O3-enriched atmosphere. Comparative proteomics analysis showed separate kiwifruit ripening responses, unraveled common 1-MCP- and O3-dependent metabolic pathways and identified specific proteins associated with these different ripening behaviors. Protein components that were differentially expressed following exogenous ethylene exposure after 1-MCP or O3 treatment were identified and their protein-protein interaction networks were determined. The expression of several kiwifruit ripening related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1), ethylene receptor (ETR1), lipoxygenase (LOX1), geranylgeranyl diphosphate synthase (GGP1), and expansin (EXP2), was strongly affected by O3, 1-MCP, their combination, and exogenously applied ethylene. Conclusions Our findings suggest that the combination of 1-MCP and O3 functions as a robust repressive modulator of kiwifruit ripening and provide new insight into the metabolic events underlying ethylene-induced and ethylene-independent ripening outcomes. Electronic supplementary material The online version of this article (10.1186/s12870-018-1584-y) contains supplementary material, which is available to authorized users.

BackgroundParasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biocontrol, and infecting aphids requires overcoming both aphid defenses and their defensive endosymbionts.ResultsWe present thede novogenome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids:Aphidius erviandLysiphlebus fabarum(Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp), highly syntenic, and the most AT-rich reported thus far for any arthropod (GC content: 25.8% and 23.8%). This nucleotide bias is accompanied by skewed codon usage, and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and age-specific energy demands. We identify expansions of F-box/Leucine-rich-repeat proteins, suggesting that diversification in this gene family may be associated with their broad host range or with countering defenses from aphids’ endosymbionts. The absence of some immune genes (Toll and Imd pathways) resembles similar losses in their aphid hosts, highlighting the potential impact of symbiosis on both aphids and their parasitoids.ConclusionsThese findings are of fundamental interest for insect evolution and beyond. This will provide a strong foundation for further functional studies including coevolution with respect to their hosts, the basis of successful infection, and biocontrol. Both genomes are available athttps://bipaa.genouest.org.

AGAP : équipe GE2pop; Improving nutritional seed quality is an important challenge in grain legume breeding. However, the genes controlling the differential accumulation of globulins, which are major contributors to seed nutritional value in legumes, remain largely unknown. We combined a search for protein quantity loci with genome-wide association studies on the abundance of 7S and 11S globulins in seeds of the model legume species Medicago truncatula. Identified genomic regions and genes carrying polymorphisms linked to globulin variations were then cross-compared with pea (Pisum sativum), leading to the identification of candidate genes for the regulation of globulin abundance in this crop. Key candidates identified include genes involved in transcription, chromatin remodeling, post-translational modifications, transport and targeting of proteins to storage vacuoles. Inference of a gene coexpression network of 12 candidate transcription factors and globulin genes revealed the transcription factor ABA-insensitive 5 (ABI5) as a highly connected hub. Characterization of loss-of-function abi5 mutants in pea uncovered a role for ABI5 in controlling the relative abundance of vicilin, a sulfur-poor 7S globulin, in pea seeds. This demonstrates the feasibility of using genome-wide association studies in M. truncatula to reveal genes that can be modulated to improve seed nutritional value.

The availability of a large variety of specific blockers, which inhibit different K(+) currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca(2+)-activated K(+) channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using (125)I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the (125)I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family.

Reductions in sulfur dioxide emissions and the use of sulfur-free mineral fertilizers are decreasing soil sulfur levels and threaten the adequate fertilization of most crops. To provide knowledge regarding legume adaptation to sulfur restriction, we subjected Medicago truncatula, a model legume species, to sulfur deficiency at various developmental stages, and compared the yield, nutrient allocation and seed traits. This comparative analysis revealed that sulfur deficiency at the mid-vegetative stage decreased yield and altered the allocation of nitrogen and carbon to seeds, leading to reduced levels of major oligosaccharides in mature seeds, whose germination was dramatically affected. In contrast, during the reproductive period, sulfur deficiency had little influence on yield and nutrient allocation, but the seeds germinated slowly and were characterized by low levels of a biotinylated protein, a putative indicator of germination vigor that has not been previously related to sulfur nutrition. Significantly, plants deprived of sulfur at an intermediary stage (flowering) adapted well by remobilizing nutrients from source organs to seeds, ensuring adequate quantities of carbon and nitrogen in seeds. This efficient remobilization of photosynthates may be explained by vacuolar sulfate efflux to maintain leaf metabolism throughout reproductive growth, as suggested by transcript and metabolite profiling. The seeds from these plants, deprived of sulfur at the floral transition, contained normal levels of major oligosaccharides but their germination was delayed, consistent with low levels of sucrose and the glycolytic enzymes required to restart seed metabolism during imbibition. Overall, our findings provide an integrative view of the legume response to sulfur deficiency.

Alfalfa (Medicago sativa L.) yield is severely compromised by soil salinity, especially at the level of seedling establishment. This question was addressed by proteomics to decipher whether specific changes in protein accumulation correlate with germination performance of alfalfa seeds submitted to a salinity stress as obtained by imbibing seeds in the presence of NaCl. This study used alfalfa seeds submitted to an osmopriming invigoration treatment that proved very efficient in counteracting the negative effect of salinity stress on germination performance. Comparative proteomic analyses disclosed 94 proteins commonly characterizing the response of both the untreated control and osmoprimed seeds to the experimental salinity stress. Remarkably, many of them, representing 84 proteins, showed contrasting accumulation patterns when comparing the untreated control and osmoprimed seeds submitted to the same salt stress. Thus numerous changes observed in the proteome of the untreated control seeds imbibed in the presence of salt, and presumably accounting for the loss in seed vigour associated with salinity stress, can be substantially reversed in osmoprimed seeds undergoing this stress. These data therefore provide a biochemical understanding of the increase in seed vigour generally observed with primed seeds.

Venom composition of parasitoid wasps attracts increasing interest – notably molecules ensuring parasitism success on arthropod pests – but its variation within and among taxa is not yet understood. We have identified here the main venom proteins of two braconid wasps, Psyttalia lounsburyi (two strains from South Africa and Kenya) and P. concolor, olive fruit fly parasitoids that differ in host range. Among the shared abundant proteins, we found a GH1 β-glucosidase and a family of leucine-rich repeat (LRR) proteins. Olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. Assuming that Psyttalia host larvae sequester ingested glycosides, the injected venom GH1 β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. Venom LRR proteins are similar to truncated Toll-like receptors and may possibly scavenge the host immunity. The abundance of one of these LRR proteins in the venom of only one of the two P. lounsburyi strains evidences intraspecific variation in venom composition. Altogether, venom intra- and inter-specific variation in Psyttalia spp. were much lower than previously reported in the Leptopilina genus (Figitidae), suggesting it might depend upon the parasitoid taxa.

Fruit development and ripening depends on highly coordinated phyto-hormonal activities. Although the role of synthetic cytokinin N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) in promoting fruit growth has been established, knowledge regarding the underlying mechanism is still lacking. Here, we characterize the effect of CPPU application 20 d after full bloom at pre- and post-harvest biology of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. ‘Hayward’). Data revealed that CPPU stimulates kiwifruit growth through the enlargement of small cells. During fruit development, the abundance of 16 proteins that are mainly related to defence was increased by CPPU while CPPU altered the expression of 19 polar metabolites in outer pericarp. Sugar homeostasis, cell wall modifications, TCA cycle and myo-inositol pathway were mostly affected by CPPU in kiwifruit during development. Upon postharvest ripening at 20 °C following 2 months of cold storage (0 °C), CPPU suppressed ethylene production and retained central placenta softening, indicating that CPPU induced tissue-dependent disturbances in climacteric ripening. Nineteen central placenta proteins and up to 15 metabolites of outer pericarp and central placenta tissues were affected by CPPU in ripened kiwifruits. These observations amplified our understanding in the regulation of fruit development and ripening by exogenously supplied cytokinins. Biological significance This study demonstrates that CPPU application, apart from fruit development, influenced also the kiwifruit climacteric ripening behaviour. An insight on the action of CPPU during kiwifruit development is provided, showing that it is partially based on a general stimulation of TCA cycle and myo-inositol pathway along with alternation in sugar and cell wall metabolism. Data also revealed that CPPU regulates ethylene biosynthesis and influences central placenta softening, indicating that this tissue may play a prominent role in kiwifruit ripening. Also, this work provides a first characterization of the ripening-affected central placenta proteins that offers insights into kiwifruit ripening. The current study provides a baseline of information for understanding the metabolic processes that are regulated by exogenous cytokinin during fruit development and ripening.

Background Endoparasitoid wasps are important natural enemies of the widely distributed aphid pests and are mainly used as biological control agents. However, despite the increased interest on aphid interaction networks, only sparse information is available on the factors used by parasitoids to modulate the aphid physiology. Our aim was here to identify the major protein components of the venom injected at oviposition by Aphidius ervi to ensure successful development in its aphid host, Acyrthosiphon pisum. Results A combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified. Conclusions Our data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-342) contains supplementary material, which is available to authorized users.

Endoparasitoid wasps inject venom proteins with their eggs to protect them from the host immune response and ensure successful parasitism. Here we report identification of Cu,Zn superoxide dismutase (SOD) transcripts for both intracellular SOD1 and extracellular SOD3 in the venom apparatus of two Leptopilina species, parasitoids of Drosophila. Leptopilina SODs show sequence and structure similarity to human SODs, but phylogenetic analyses indicate that the extracellular SODs are more related to cytoplasmic vertebrate SODs than to extracellular SODs, a feature shared by predicted insect extracellular SODs. We demonstrate that L. boulardi SOD3 is indeed secreted and active as monomeric glycosylated forms in venom. Our results also evidence quantitative variation in SOD3 venom contents between closely related parasitoid species, as sod3 is 100-fold less expressed in Leptopilina heterotoma venom apparatus and no protein and SOD activity are detected in its venom. Leptopilina recombinant SOD3s as well as a mammalian SOD in vitro inhibit the Drosophila phenoloxidase activity in a dose-dependent manner, demonstrating that SODs may interfere with the Drosophila melanization process and, therefore, with production of cytotoxic compounds. Although the recombinant L. boulardi SOD3 quantity needed to observe this effect precludes a systemic effect of the wasp venom SOD3, it is still consistent with a local action at oviposition. This work provides the first demonstration that insect extracellular SODs are indeed secreted and active in an insect fluid and can be used as virulence factors to counteract the host immune response, a strategy largely used by bacterial and fungal pathogens but also protozoan parasites during infection.

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H(2)O(2)) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mm H(2)O(2) on several oxidative features such as protein carbonylation, endogenous H(2)O(2) and lipid peroxidation levels. We report that H(2)O(2) treatment of the pea seeds increased their endogenous H(2)O(2) content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H(2)O(2) in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H(2)O(2) , whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H(2)O(2) in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

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 [35S]-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.

International audience; We have purified the AaTX1 peptide from the Androctonus australis (Aa) scorpion venom, previously cloned and sequenced by Legros and collaborators in a venom gland cDNA library from Aa scorpion. AaTX1 belongs to the alpha-Ktx15 scorpion toxins family (alpha KTx15-4). Characterized members of this family share high sequence similarity and were found to block preferentially I-A-type voltage-dependent K+ currents in rat cerebellum granular cells in an irreversible way. In the current work, we studied the effects of native AaTX1 (nAaTX1) using whole-cell patch-clamp recordings of I-A current in substantia nigra pars compacta dopaminergic neurons. At 250 nM, AaTX1 induces 90% decrease in I-A current amplitude. Its activity was found to be comparable to that of rAmmTX3 (alpha KTx15-3), which differs by only one conserved (R/K) amino acid in the 19th position suggesting that the difference between R19 and K19 in AaFX1 and AmmTX3, respectively, may not be critical for the toxins' effects. Molecular docking of both toxins with Kv4.3 channel is in agreement with experimental data and suggests the implication of the functional dyade K27-Y36 in toxin-channel interactions. Since AaTX1 is not highly abundant in Aa venom, it was synthesized as well as AmmTX3. Synthetic peptides, native AaTX1 and rAmmTX3 peptides showed qualitatively the same pharmacological activity. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on Kv4.3 channel. (C) 2014 Elsevier Ltd. All rights reserved.

International audience; The arms race between immune suppressive parasites that produce virulence factors and hosts that evolve resistance to these factors is suggested to be a key driver for the diversification of both partners. However, little is known regarding the diversity of virulence factors in closely related parasites or the mechanisms underlying the variation of virulence. One of the best-described model to address this issue is the interaction between Leptopilina parasitic wasps and their Drosophila hosts, in which variation of virulence is well documented. Thanks to a combined transcriptomic and proteomic approach, we have identified the main secreted proteins in the venom of Leptopilina heterotoma (Gotheron strain, 66 proteins) and of two well-characterized strains of Leptopilina boulardi, ISm and ISy (65 and 49 proteins, respectively). Results revealed significant quantitative differences in venom components between the L. boulardi strains, in agreement with their different virulence properties. Strikingly, the two related Leptopilina species did not share any abundant venom protein. The main identified proteins in L boulardi were RhoGAPs and serpins while an aspartylglucosaminidase (AGA) was found abundant in L heterotoma. The extensive quantitative variation observed between these species may be related with their use of different virulence strategies and/or to differences in their host range (specialist versus generalist). Altogether, our data suggests that parasitoid venom can quickly evolve, mainly through rapid changes in regulation of gene expression. It also evidences venom evolutionary processes largely described in other venomous animals i.e. the convergent recruitment of venom proteins between phylogenetically unrelated organisms, and the role of duplications in the emergence of multigenic families of virulence factors.

International audience; Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.

International audience; Post-harvest ozone application has recently been shown to inhibit the onset of senescence symptoms on fleshy fruit and vegetables; however, the exact mechanism of action is yet unknown. To characterize the impact of ozone on the post-harvest performance of kiwifruit (Actinidia deliciosa cv. 'Hayward'), fruits were cold stored (0 °C, 95% relative humidity) in a commercial ethylene-free room for 1, 3, or 5 months in the absence (control) or presence of ozone (0.3 μl l(-1)) and subsequently were allowed to ripen at a higher temperature (20 °C), herein defined as the shelf-life period, for up to 12 days. Ozone blocked ethylene production, delayed ripening, and stimulated antioxidant and anti-radical activities of fruits. Proteomic analysis using 1D-SDS-PAGE and mass spectrometry identified 102 kiwifruit proteins during ripening, which are mainly involved in energy, protein metabolism, defence, and cell structure. Ripening induced protein carbonylation in kiwifruit but this effect was depressed by ozone. A set of candidate kiwifruit proteins that are sensitive to carbonylation was also discovered. Overall, the present data indicate that ozone improved kiwifruit post-harvest behaviour, thus providing a first step towards understanding the active role of this molecule in fruit ripening.

Marylin Torrentino-Madamet1, Lionel Almeras2, Christelle Travaillé1, Véronique Sinou1, Matthieu Pophillat3, Maya Belghazi4, Patrick Fourquet3, Yves Jammes5, Daniel Parzy11UMR-MD3, Université de la Méditerranée, Antenne IRBA de Marseille (IMTSSA, Le Pharo), 2Unité de Recherche en Biologie et Epidémiologie Parasitaires, Antenne IRBA de Marseille (IMTSSA, Le Pharo), 3Centre d'Immunologie de Marseille Luminy, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de la Méditerranée, 4Centre d'Analyse Protéomique de Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, 5UMR-MD2, Physiologie et Physiopathologie en Conditions d'Oxygénations Extrêmes, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, Marseille, FranceObjectives: Although human respiratory metabolism is characterized by the mitochondrial electron transport chain, some organisms present a “branched respiratory chain.” This branched pathway includes both a classical and an alternative respiratory chain. The latter involves an alternative oxidase. Though the Plasmodium falciparum alternative oxidase is not yet identified, a specific inhibitor of this enzyme, salicylhydroxamic acid (SHAM), showed a drug effect on P. falciparum respiratory function using oxygen consumption measurements. The present study aimed to highlight the metabolic pathways that are affected in P. falciparum following SHAM exposure.Design: A proteomic approach was used to analyze the P. falciparum proteome and determine the metabolic pathways altered following SHAM treatment. To evaluate the SHAM effect on parasite growth, the phenotypic alterations of P. falciparum after SHAM or/and hyperoxia exposure were observed.Results: After SHAM exposure, 26 proteins were significantly deregulated using a fluorescent two dimensional-differential gel electrophoresis. Among these deregulated proteins, some were particularly involved in energetic metabolism. And the combinatory effect of SHAM/hyperoxia seems deleterious for the growth of P. falciparum.Conclusion: Our results indicated that SHAM appears to activate glycolysis and decrease stress defense systems. These data provide a better understanding of parasite biology.Keywords: Plasmodium falciparum, salicylhydroxamic acid, hyperoxia, glycolysis, proteomic

International audience; Alfalfa, the most widely grown leguminous crop in the world, is generally exposed to severe salinity stress in Tunisia, notably affecting its germination performance. Toward a better understanding of alfalfa seed vigor, we have used proteomics to characterize protein changes occurring during germination and osmopriming, a pretreatment that accelerates germination and improves seedling uniformity particularly under stress conditions. The data revealed that germination was accompanied by dynamic changes of 79 proteins, which are mainly involved in protein metabolism, cell structure, metabolism, and defense. Comparative proteomic analysis also revealed 63 proteins specific to osmopriming, 65 proteins preferentially varying during germination, and 14 proteins common to both conditions. Thus, the present study unveiled the unexpected finding that osmopriming cannot simply be considered as an advance of germination-related processes but involves other mechanisms improving germination such as the mounting of defense mechanisms enabling osmoprimed seeds to surmount environmental stresses potentially occurring during germination. The present results therefore provide novel avenues toward understanding the mechanisms of invigoration of low vigor seeds by priming treatments that are widely used both in commercial applications and in developing countries (on farm seed priming) to better control crop yields.

Background Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. Methodology/Principal Findings Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. Conclusions/Significance The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation.

Sulfate is required for the synthesis of sulfur-containing amino acids and numerous other compounds essential for the plant life cycle. The delivery of sulfate to seeds and its translocation between seed tissues is likely to require specific transporters. In Arabidopsis (Arabidopsis thaliana), the group 3 plasmalemma-predicted sulfate transporters (SULTR3) comprise five genes, all expressed in developing seeds, especially in the tissues surrounding the embryo. Here, we show that sulfur supply to seeds is unaffected by T-DNA insertions in the SULTR3 genes. However, remarkably, an increased accumulation of sulfate was found in mature seeds of four mutants out of five. In these mutant seeds, the ratio of sulfur in sulfate form versus total sulfur was significantly increased, accompanied by a reduction in free cysteine content, which varied depending on the gene inactivated. These results demonstrate a reduced capacity of the mutant seeds to metabolize sulfate and suggest that these transporters may be involved in sulfate translocation between seed compartments. This was further supported by sulfate measurements of the envelopes separated from the embryo of the sultr3;2 mutant seeds, which showed differences in sulfate partitioning compared with the wild type. A dissection of the seed proteome of the sultr3 mutants revealed protein changes characteristic of a sulfur-stress response, supporting a role for these transporters in providing sulfate to the embryo. The mutants were affected in 12S globulin accumulation, demonstrating the importance of intraseed sulfate transport for the synthesis and maturation of embryo proteins. Metabolic adjustments were also revealed, some of which could release sulfur from glucosinolates.

International audience; Diseases caused by arthropod-borne viruses are a significant threat to the health of human and animal populations throughout the world. Better knowledge of the molecules synthesized in the salivary gland and saliva of hematophagous arthropods could be of use for improving the control of pathogen transmission. Recently, a sialome analysis of three Aedes aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) carried out in our laboratory allowed us to identify 44 saliva proteins. Of these secreted proteins, none was exclusively expressed in one colony, suggesting that expression of salivary proteins is highly conserved across populations. In another study, we reported that some of these salivary proteins could be used as the genus-specific markers for travelers' exposure to mosquito vectors. Here, comparison of salivary gland protein profiles between these same three Ae. aegypti colonies was performed using the one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis method. As observed at the saliva level, no significant differences were detected between these three colonies. The salivary gland protein repertoire from the Ae. aegypti mosquito was analyzed using a proteomic approach. One hundred and twenty proteins were identified in these salivary glands representing the largest description of the Ae. aegypti salivary gland protein catalog. We succeeded in identifying 15 secreted proteins, some of which have already been reported as being involved in blood feeding. A comparison of the proteins identified between the salivary glands and the sialome is discussed.

Background Sulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole. Results In the present study, we demonstrated that the Sultr4;1 gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the Sultr4;2 gene. A characterization of dry mature seeds from a Sultr4;1 T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the Sultr4;1 seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes. Conclusions This study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of sultr4;1 mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds.

Mature angiosperm seeds consist of an embryo surrounded by the endosperm and the testa. The endosperm cap that covers the radicle plays a regulatory role during germination and is a major target of abscisic acid-induced inhibition of germination. Cress (Lepidium sativum) is a close relative of the model plant Arabidopsis thaliana (Arabidopsis). Cress seeds offer the unique possibility of performing tissue-specific proteomics due to their larger size while benefiting the genomic tools available for Arabidopsis. This work provides the first description of endosperm cap proteomics during seed germination. An analysis of the proteome of the cress endosperm cap at key stages during germination and after radicle protrusion in the presence and absence of abscisic acid led to the identification of 144 proteins, which were clustered by the changes in their abundances and categorized by function. Proteins with a function in energy production, protein stability and stress response were overrepresented among the identified endosperm cap proteins. This strongly suggests that the cress endosperm cap is not a storage tissue as the cereal endosperm but a metabolically very active tissue regulating the rate of radicle protrusion.

International audience; In this study, we have characterized the immunological and pharmacological properties of the three major alpha-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of alpha-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as alpha-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.

International audience; Flaviviruses are positive-stranded RNA viruses that are a public health problem because of their widespread distribution and their ability to cause a variety of diseases in humans. West Nile virus is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. Clinical manifestations of West Nile virus infection are diverse, and their pathogenic mechanisms depend on complex virus-cell interactions. In the present work, we used proteomics technology to analyze early Vero cell response to West Nile infection. The differential proteomes were resolved 24 h postinfection using two-dimensional DIGE followed by mass spectrometry identification. Quantitative analysis (at least 2-fold quantitative alteration, p < 0.05) revealed 127 differentially expressed proteins with 68 up-regulated proteins and 59 down-regulated proteins of which 93 were successfully identified. The implication for mammalian cellular responses to this neurotropic flavivirus infection was analyzed and made possible more comprehensive characterization of the virus-host interactions involved in pathogenesis. The present study thus provides large scale protein-related information that should be useful for understanding how the host metabolism is modified by West Nile infection and for identifying new potential targets for antiviral therapy.

International audience; Many species of parasitoid wasps inject polydnavirus particles in order to manipulate host defenses and development. Because the DNA packaged in these particles encodes almost no viral structural proteins, their relation to viruses has been debated. Characterization of complementary DNAs derived from braconid wasp ovaries identified genes encoding subunits of a viral RNA polymerase and structural components of polydnavirus particles related most closely to those of nudiviruses—a sister group of baculoviruses. The conservation of this viral machinery in different braconid wasp lineages sharing polydnaviruses suggests that parasitoid wasps incorporated a nudivirus-related genome into their own genetic material. We found that the nudiviral genes themselves are no longer packaged but are actively transcribed and produce particles used to deliver genes essential for successful parasitism in lepidopteran hosts.

International audience; In this paper were described the purification, the sequencing, and the immunological and biological characterization of a new Kaliotoxin analog, Aam-KTX, from the venom of the scorpion Androctonus amoreuxi. The toxin effects on three cloned Kv channels (Kv1.1, Kv1.2, and Kv1.3) were investigated in Xenopus oocytes using electrophysiology experiments. The Aam-KTX preference for Kv1.3 channel versus Kv1.2 was expected (EC(50) values, 1.1+/-0.02 and 10.4+/-1.5 nM, respectively) but its total inefficacy on Kv1.1 was very surprising. 3D molecular modeling of Aam-KTX brought putative answers to this difference in selectivity.

A variety of mechanisms have been proposed to account for the extension of life span in seeds (seed longevity). In this work, we used Arabidopsis (Arabidopsis thaliana) seeds as a model and carried out differential proteomics to investigate this trait, which is of both ecological and agricultural importance. In our system based on a controlled deterioration treatment (CDT), we compared seed samples treated for different periods of time up to 7 d. Germination tests showed a progressive decrease of germination vigor depending on the duration of CDT. Proteomic analyses revealed that this loss in seed vigor can be accounted for by protein changes in the dry seeds and by an inability of the low-vigor seeds to display a normal proteome during germination. Furthermore, CDT strongly increased the extent of protein oxidation (carbonylation), which might induce a loss of functional properties of seed proteins and enzymes and/or enhance their susceptibility toward proteolysis. These results revealed essential mechanisms for seed vigor, such as translational capacity, mobilization of seed storage reserves, and detoxification efficiency. Finally, this work shows that similar molecular events accompany artificial and natural seed aging.

A comparative study of proteome and transcriptome changes during Medicago truncatula ( cultivar Jemalong) seed development has been carried out. Transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. These data, combined with the analysis of transcript and protein distribution in the isolated seed coat, endosperm, and embryo, demonstrated the major contribution made to the embryo by the surrounding tissues. First, a remarkable compartmentalization of enzymes involved in methionine biosynthesis between the seed tissues was revealed that may regulate the availability of sulfur-containing amino acids for embryo protein synthesis during seed filling. This intertissue compartmentalization, which was also apparent for enzymes of sulfur assimilation, is relevant to strategies for modifying the nutritional value of legume seeds. Second, decreasing levels during seed filling of seed coat and endosperm metabolic enzymes, including essential steps in Met metabolism, are indicative of a metabolic shift from a highly active to a quiescent state as the embryo assimilates nutrients. Third, a concomitant persistence of several proteases in seed coat and endosperm highlighted the importance of proteolysis in these tissues as a supplementary source of amino acids for protein synthesis in the embryo. Finally, the data revealed the sites of expression within the seed of a large number of transporters implied in nutrient import and intraseed translocations. Several of these, including a sulfate transporter, were preferentially expressed in seeds compared with other plant organs. These findings provide new directions for genetic improvement of grain legumes.

International audience; The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifH::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.

Increased cellular levels of reactive oxygen species are known to occur during seed development and germination, but the consequences in terms of protein degradation are poorly characterized. In this work, protein carbonylation, which is an irreversible oxidation process leading to a loss of function of the modified proteins, has been analyzed by a proteomic approach during the first stages of Arabidopsis (Arabidopsis thaliana) seed germination. In the dry mature seeds, the legumin-type globulins (12S cruciferins) were the major targets. However, the acidic α-cruciferin subunits were carbonylated to a much higher extent than the basic (β) ones, consistent with a model in which the β-subunits are buried within the cruciferin molecules and the α-subunits are more exposed to the outside. During imbibition, various carbonylated proteins accumulated. This oxidation damage was not evenly distributed among seed proteins and targeted specific proteins as glycolytic enzymes, mitochondrial ATP synthase, chloroplastic ribulose bisphosphate carboxylase large chain, aldose reductase, methionine synthase, translation factors, and several molecular chaperones. Although accumulation of carbonylated proteins is usually considered in the context of aging in a variety of model systems, this was clearly not the case for the Arabidopsis seeds since they germinated at a high rate and yielded vigorous plantlets. The results indicate that the observed specific changes in protein carbonylation patterns are probably required for counteracting and/or utilizing the production of reactive oxygen species caused by recovery of metabolic activity in the germinating seeds.

Nitrile hydratases (NHases) are industrially significant iron- and cobalt-containing enzymes used in the large-scale synthesis of acrylamide. Previous reports have shown that the active site peptides of NHases are post-translationally modified by oxidation of cysteine residues, and that these modifications are essential for catalysis. We report mass spectrometric evidence of the oxidation states of the active site cysteines in the iron coordination spheres of two iron-containing nitrile hydratases, namely R312 NHase from Rhodococcus rhodochrous strain R312 and NI1 NHase from Comamonas testosteroni. At least one of these cysteines is oxidised to a sulfinic acid (SO(2)H) and there is also evidence suggesting an additional oxidation to a sulfenic acid (SOH). This is the first evidence for the presence of these oxidation states for full-length NHases and for Fe-NHases from different microorganisms. The presence of these covalent modifications was confirmed by performing mass spectrometry on the active site peptide of R312 NHase, under native, reduced and carboxymethylated conditions. We also show the nitrosylation of the iron by mass spectrometry, as well as the release of NO by photoirradiation.

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.

Background The emergence of Plasmodium falciparum resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in P. falciparum have not yet been clearly defined, particularly at the protein level. Methods A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ). Results After doxycycline treatment, 32 and 40 P. falciparum proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis. Conclusions In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.

Background Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers. Methods In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission. Results Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach. Conclusion Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.