Protonation controls ASIC1a activity via coordinated movements in multiple domains.

Acid-sensing ion channels (ASICs) are neuronal Na(+)-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity.

Role of retinoic acid signaling pathway in endoderm antero-posterior patterning

Summary : During vertebrate embryonic development, the endoderm gives rise to the digestive tract and associated organs such as thyroid, lung, liver and pancreas. Earlier studies have shown that extracellular signals coming from the lateral plate mesoderm pattern the endoderm along the antero-posterior axis specifying different organ primordia. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. In this study, we focused on the role of the retinoic acid (RA) signaling pathway in the regionalization of the future gut tube along the main body axis. We show that the RA-synthesizing enzyme Raldh2 is expressed in mesoderm close to the endoderm during gastrulation and during somitogenesis. During the same period, all retinoic acid receptors (RARs), which directly activate gene transcription, are expressed in endoderm suggesting that endoderm can be responsive to RA. Activation or inhibition of RA signaling was achieved by adding RA or RAR inhibitors tither on beads or in the medium to cultured chick embryos. Branchial arch (BA) endoderm markers were shifted posteriorly upon depletion of RA at gastrulation, but were not shifted after this stage. Conversely, exposure to exogenous RA repressed the most-anterior BA markers and shifted more posterior BA markers anteriorly. This suggests that graded levels of RA activity in the foregut define gene boundaries and expression levels. The posterior foregut and midget markers Pdxl and CdxA require RA for their expression, but elevated RA does not shift their expression domain along the antero-posterior axis. In addition, we investigated if RA signaling pathway interacts with other signaling pathways to pattern the endoderm. Although both RA and FGFs block anterior foregut marker expression, our experiments suggest that FGF signaling does not depend on RA in anterior endoderm. To validate our chick data in mammalians and evaluate whether RA acts directly on endoderm, we have further generated a conditional loss-of-function system in the mouse, which is still under examination.

Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes.

BACKGROUND: A concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency. METHODS: In 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers. RESULTS: Patients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate. CONCLUSION: The positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.

Growth and metabolic activity of the extramatrical mycelium of endomycorrhizal maize plants

The objective of this experiment was to quantify the extramatrical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus etunicatum (Becker & Gerdemann) grown on maize (Zea mays L. var. Piranão) provided with various levels of phosphate fertilizer and harvested at 30, 60 and 90 days after planting (DAP). Total extramatrical mycelium (TEM) was extracted from soil using a modified membrane filtration method, followed by quantification using a grid intersection technique. Active extramatrical mycelium (AEM) proportion was determined using an enzymatic method which measured dehydrogenase activity by following iodonitrotetrazolium reduction. At low levels of added P, there was relatively less TEM than at high levels of added P, but the AEM proportion at low soil P availability was significantly greater than at high soil P.

Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity.

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Phosphate forms in plant and their internal buffering in five soybean cultivars

Differences among plants in their ability to support nutritional stress periods may be caused by a differential vacuole capacity of ion storage and release and may also depend on the intensity of nutrient re-translocation under such conditions. In five soybean cultivars, submitted to eight days of P deprivation, the dry matter production and the contents of three phosphorus (P) forms - inorganic (Pi), organic (Po), and acid-soluble total (Pts) of different plant organs were determined. Pi release velocity (RSPi) was estimated as the tangent to the equations obtained for Pi f(t) at the point t = 2 days (the mean point in the period of greatest Pi decrease), considering that -deltaPi/deltat expresses the rate of Pi release. The internal Pi buffering capacity (IBCPi) was calculated as the inverse of the RSPi. Cultivars' differences in size of the non-metabolic Pi pool, RSPi, and the ability to transport Pi from less to more actively metabolizing regions were evaluated. The preferential Pi source and sink compartments under limited P absorption conditions were also evaluated. The cultivar Santa Rosa showed the highest Pi storage ability when the external supply was high, and a more intensive release under low P supply conditions than IAC8 and UFV1. The cultivar Uberaba was superior to Doko in its ability to store and use Pi. In all cultivars, upper leaves and roots were the main sink of Pi stored in the middle and lower leaves. Roots and upper leaves showed larger RSPi and lower IBCPi values than middle and lower leaves.

Activity of phosphorus of synthetic Fe-K-P compounds in superphosphate fertilizers as affected by pH and ionic strength

The concentration of orthophosphate ions released from Fe-K-P compounds (Fe3KH8(PO4)6 .6H2O and Fe3KH14(PO4)8 .4H2O) present in superphosphates increases with pH, which initially suggests that the agronomic effectiveness of P fertilizers containing high amounts of these compounds would also increase with soil pH but studies considering activity, instead of concentration, are necessary. With this purpose, both compounds were synthesized under laboratory conditions, characterized by elemental chemical analysis, optical microscopy, X ray diffractometry, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), and used in a solubility study. Solutions of 0.01, 0.05 and 0.1 mol L-1 NaCl with pH adjusted to 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 were prepared for the solubility study of H8-syn, H14-syn and a phosphate rock (PR) from Brazil. The orthophosphate activity as H2PO4- and HPO4(2-) was calculated in each situation as related to pH and ionic strength using software MINTEQ. The remaining precipitates after equilibrium were chemically analyzed and subjected to X ray, SEM and EDS. Results of chemical analysis and instrumental techniques confirmed the preparation method. The activity of orthophosphate ions of both compounds tended to decrease under increasing pH and/or ionic strength of the solution, which in turn suggests that an increase in the solution pH does not necessarily promote an increase in the P bioavailability for plant uptake. This can be important when evaluating agronomic data of P fertilizers with high contents of these two Fe-K-P compounds.

Genetic Variants of Serum Uric Acid and Gout: An Analysis of > 170,000 Individuals

Background/Purpose: Gout is a common and excruciatingly painful inflammatory arthritis caused by hyperuricemia. In addition to various lifestyle risk factors, a substantial genetic predisposition to gout has long been recognized. The Global Urate Genetics Consortium (GUGC) has aimed to comprehensively investigate the genetics of serum uric acid and gout using data from _ 140,000 individuals of European-ancestry, 8,340 individuals of Indian ancestry, 5,820 African-Americans, and 15,286 Japanese. Methods: We performed discovery GWAS meta-analyses of serum urate levels (n_110,347 individuals) followed by replication analyses (n_32,813 different individuals). Our gout analysis involved 3,151 cases and 68,350 controls, including 1,036 incident gout cases that met the American College of Rheumatology Criteria. We also examined the association of gout with fractional excretion of uric acid (n_6,799). A weighted genetic urate score was constructed based on the number of risk alleles across urate-associated loci, and their association with the risk of gout was evaluated. Furthermore, we examined implicated transcript expression in cis (expression quantitative trait loci databases) for potential insights into the gene underlying the association signal. Finally, in order to further identify urate-associated genomic regions, we performed functional network analyses that incorporated prior knowledge on molecular interactions in which the gene products of implicated genes operate. Results: We identified and replicated 28 genome-wide significant loci in association with serum urate (P 5_10_8), including all previously-reported loci as well as 18 novel genetic loci. Unlike the majority of previouslyidentified loci, none of the novel loci appeared to be obvious candidates for urate transport. Rather, they were mapped to genes that encode for purine production, transcription, or growth factors with broad downstream responses. Besides SLC2A9 and ABCG2, no additional regions contained SNPs that differed significantly (P _ 5_10_8) between sexes. Urateincreasing alleles were associated with an increased risk of gout for all loci. The urate genetic risk score (ranging from 10 to 45) was significantly associated with an increased odds of prevalent gout (OR per unit increase, 1.11; 95% CI, 1.09-1.14) and incident gout (OR, 1.10; 95% CI, 1.08-1.13). Associations for many of the loci were of similar magnitude in individuals of non-European ancestry. Detailed characterization of the loci revealed associations with transcript expression and the fractional excretion of urate. Network analyses implicated the inhibins-activins signaling pathways and glucose metabolism in systemic urate control. Conclusion: The novel genetic candidates identified in this urate/gout consortium study, the largest to date, highlight the importance of metabolic control of urate production and urate excretion. The modulation by signaling processes that influence metabolic pathways such as glycolysis and the pentose phosphate pathway appear to be central mechanisms underpinned by the novel GWAS candidates. These findings may have implications for further research into urate-lowering drugs to treat and prevent gout.

Alterations in microRNA expression contribute to fatty acid-induced pancreatic beta-cell dysfunction.

OBJECTIVE: Visceral obesity and elevated plasma free fatty acids are predisposing factors for type 2 diabetes. Chronic exposure to these lipids is detrimental for pancreatic beta-cells, resulting in reduced insulin content, defective insulin secretion, and apoptosis. We investigated the involvement in this phenomenon of microRNAs (miRNAs), a class of noncoding RNAs regulating gene expression by sequence-specific inhibition of mRNA translation. RESEARCH DESIGN AND METHODS: We analyzed miRNA expression in insulin-secreting cell lines or pancreatic islets exposed to palmitate for 3 days and in islets from diabetic db/db mice. We studied the signaling pathways triggering the changes in miRNA expression and determined the impact of the miRNAs affected by palmitate on insulin secretion and apoptosis. RESULTS: Prolonged exposure of the beta-cell line MIN6B1 and pancreatic islets to palmitate causes a time- and dose-dependent increase of miR34a and miR146. Elevated levels of these miRNAs are also observed in islets of diabetic db/db mice. miR34a rise is linked to activation of p53 and results in sensitization to apoptosis and impaired nutrient-induced secretion. The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in beta-cell exocytosis. Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis. Treatment with oligonucleotides that block miR34a or miR146 activity partially protects palmitate-treated cells from apoptosis but is insufficient to restore normal secretion. CONCLUSIONS: Our findings suggest that at least part of the detrimental effects of palmitate on beta-cells is caused by alterations in the level of specific miRNAs.

Mitochondrial Fatty Acid Oxidation in Obesity

Significance: Current lifestyles with high-energy diets and little exercise are triggering an alarming growth in obesity. Excess of adiposity is leading to severe increases in associated pathologies, such as insulin resistance, type 2 diabetes, atherosclerosis, cancer, arthritis, asthma, and hypertension. This, together with the lack of efficient obesity drugs, is the driving force behind much research. Recent Advances: Traditional anti-obesity strategies focused on reducing food intake and increasing physical activity. However, recent results suggest that enhancing cellular energy expenditure may be an attractive alternative therapy. Critical Issues: This review evaluates recent discoveries regarding mitochondrial fatty acid oxidation (FAO) and its potential as a therapy for obesity. We focus on the still controversial beneficial effects of increased FAO in liver and muscle, recent studies on how to potentiate adipose tissue energy expenditure, and the different hypotheses involving FAO and the reactive oxygen species production in the hypothalamic control of food intake. Future Directions: The present review aims to provide an overview of novel anti-obesity strategies that target mitochondrial FAO and that will definitively be of high interest in the future research to fight against obesity-related disorders. Antioxid. Redox Signal. 00, 000000.

Characterization of the PHO1 gene family and the responses to phosphate deficiency of Physcomitrella patens.

PHO1 was previously identified in Arabidopsis (Arabidopsis thaliana) as a protein involved in loading inorganic phosphate (Pi) into the xylem of roots and its expression was associated with the vascular cylinder. Seven genes homologous to AtPHO1 (PpPHO1;1-PpPHO1;7) have been identified in the moss Physcomitrella patens. The corresponding proteins harbor an SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the conserved C-terminal hydrophobic portion, both common features of the plant PHO1 family. Northern-blot analysis showed distinct expression patterns for the PpPHO1 genes, both at the tissue level and in response to phosphate deficiency. Transgenic P. patens expressing the beta-glucuronidase reporter gene under three different PpPHO1 promoters revealed distinct expression profiles in various tissues. Expression of PpPHO1;1 and PpPHO1;7 was specifically induced by Pi starvation. P. patens homologs to the Arabidopsis PHT1, DGD2, SQD1, and APS1 genes also responded to Pi deficiency by increased mRNA levels. Morphological changes associated with Pi deficiency included elongation of caulonemata with inhibition of the formation of side branches, resulting in colonies with greater diameter, but reduced mass compared to Pi-sufficient plants. Under Pi-deficient conditions, P. patens also increased the synthesis of ribonucleases and of an acid phosphatase, and increased the ratio of sulfolipids over phospholipids. These results indicate that P. patens and higher plants share some common strategies to adapt to Pi deficiency, although morphological changes are distinct, and that the PHO1 proteins are well conserved in bryophyte despite the lack of a developed vascular system.

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis.

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi-deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi-sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi-deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.

Effect of a temperature increase in the non-noxious range on proton-evoked ASIC and TRPV1 activity.

Acid-sensing ion channels (ASICs) are neuronal H(+)-gated cation channels, and the transient receptor potential vanilloid 1 channel (TRPV1) is a multimodal cation channel activated by low pH, noxious heat, capsaicin, and voltage. ASICs and TRPV1 are present in sensory neurons. It has been shown that raising the temperature increases TRPV1 and decreases ASIC H(+)-gated current amplitudes. To understand the underlying mechanisms, we have analyzed ASIC and TRPV1 function in a recombinant expression system and in dorsal root ganglion (DRG) neurons at room and physiological temperature. We show that temperature in the range studied does not affect the pH dependence of ASIC and TRPV1 activation. A temperature increase induces, however, a small alkaline shift of the pH dependence of steady-state inactivation of ASIC1a, ASIC1b, and ASIC2a. The decrease in ASIC peak current amplitudes at higher temperatures is likely in part due to the observed accelerated open channel inactivation kinetics and for some ASIC types to the changed pH dependence of steady-state inactivation. The increase in H(+)-activated TRPV1 current at the higher temperature is at least in part due to a hyperpolarizing shift in its voltage dependence. The contribution of TRPV1 relative to ASICs to H(+)-gated currents in DRG neurons increases with higher temperature and acidity. Still, ASICs remain the principal pH sensors of DRG neurons at 35°C in the pH range ≥6.

Study of the regulation of antifungal activity in plant-associated pseudomas fluorescens

RésuméEn agriculture d'énormes pertes sont causées par des champignons telluriques pathogènes tels que Thielaviopsis, Fusarium, Gaeumannomyces et Rhizoctonia ou encore l'oomycète Pythium. Certaines bactéries dites bénéfiques, comme Pseudomonas fluorescens, ont la capacité de protéger les plantes de ces pathogènes par la colonisation de leur racines, par la production de métabolites secondaires possédants des propriétés antifongiques et par l'induction des mécanismes de défenses de la plante colonisée. P. fluorescens CHAO, une bactérie biocontrôle isolée d'un champ de tabac à Payerne, a la faculté de produire un large spectre de métabolites antifongiques, en particulier le 2,4- diacétylphloroglucinol (DAPG), la pyolutéorine (PLT), le cyanure d'hydrogène (HCN), la pyrrolnitrine (PRN) ainsi que des chélateurs de fer.La plante, par sécrétion racinaire, produit des rhizodéposites, source de carbone et d'azote, qui profitent aux populations bactériennes vivant dans la rhizosphere. De plus, certains stresses biotiques et abiotiques modifient cette sécrétion racinaire, en terme quantitatif et qualitatif. De leur côté, les bactéries bénéfiques, améliorent, de façon direct et/ou indirect, la croissance de la plante hôte. De nombreux facteurs biotiques et abiotiques sont connus pour réguler la production de métabolites secondaires chez les bactéries. Des études récentes ont démontré l'importance de la communication entre la plante et les bactéries bénéfiques afin que s'établisse une interaction profitant à chacun des deux partis. Il est ainsi vraisemblable que les populations bactériennes associées aux racines soient capables d'intégrer ces signaux et d'adapter spécifiquement leur comportement en conséquence.La première partie de ce travail de thèse a été la mise au point d'outils basés sur la cytométrie permettant de mesurer l'activité antifongique de cellules bactériennes individuelles dans un environnent naturel, les racines des plantes. Nous avons démontré, grâce à un double marquage aux protéines autofluorescentes GFP et mCherry, que les niveaux d'expression des gènes impliqués dans la biosynthèse des substances antifongiques DAPG, PLT, PRN et HCN ne sont pas les mêmes dans des milieux de cultures liquides que sur les racines de céréales. Par exemple, l'expression de pltA (impliqué dans la biosynthèse du PLT) est quasiment abolie sur les racines de blé mais atteint un niveau relativement haut in vitro. De plus cette étude a mis en avant l'influence du génotype céréalien sur l'expression du gène phlA qui est impliqué dans la biosynthèse du DAPG.Une seconde étude a révélé la communication existant entre une céréale (orge) infectée par le pathogène tellurique Pythium ultimum et P. fluorescens CHAO. Un système de partage des racines nous a permis de séparer physiquement le pathogène et la bactérie bénéfique sur la plante. Cette méthode a donné la possibilité d'évaluer l'effet systémique, causé par l'attaque du pathogène, de la plante sur la bactérie biocontrôle. En effet, l'infection par le phytopathogène modifie la concentration de certains composés phénoliques dans les exsudats racinaires stimulant ainsi l'expression de phi A chez P.fluorescens CHAO.Une troisième partie de ce travail focalise sur l'effet des amibes qui sont des micro-prédateurs présents dans la rhizosphere. Leur présence diminue l'expression des gènes impliqués dans la biosynthèse du DAPG, PLT, PRN et HCN chez P.fluorescens CHAO, ceci en culture liquide et sur des racines d'orge. De plus, des molécules provenant du surnageant d'amibes, influencent l'expression des gènes requis pour la biosynthèse de ces antifongiques. Ces résultats illustrent que les amibes et les bactéries de la rhizosphere ont développé des stratégies pour se reconnaître et adapter leur comportement.La dernière section de ce travail est consacrée à l'acide indole-acétique (LA.A), une phytohormone connue pour son effet stimulateur sur phlA. Une étude moléculaire détaillée nous a démontré que cet effet de l'IAA est notamment modulé par une pompe à efflux (FusPl) et de son régulateur transcriptionnel (MarRl). De plus, les gènes fusPl et marRl sont régulés par d'autres composés phénoliques tels que le salicylate (un signal végétal) et l'acide fusarique (une phytotoxine du pathogène Fusarium).En résumé, ce travail de thèse illustre la complexité des interactions entre les eucaryotes et procaryotes de la rhizosphère. La reconnaissance mutuelle et l'instauration d'un dialogue moléculaire entre une plante hôte et ses bactéries bénéfiques associées? sont indispensables à la survie des deux protagonistes et semblent être hautement spécifiques.SummaryIn agriculture important crop losses result from the attack of soil-borne phytopathogenic fungi, including Thielaviopsis, Fusarium, Gaeumannomyces and Rhizoctonia, as well as from the oomycete Pythium. Certain beneficial microorganisms of the rhizosphere, in particular Pseudomonas fluorescens, have the ability to protect plants against phytopathogens by the intense colonisation of roots, by the production of antifungal exoproducts, and by induction of plant host defences. P. fluorescens strain CHAO, isolated from a tobacco field near Payerne, produces a large array of antifungal exoproducts, including 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), hydrogen cyanide (HCN), pyrrolnitrin (PRN) and iron chelators. Plants produce rhizodeposites via root secretion and these represent a relevant source of carbon and nitrogen for rhizosphere microorganisms. Various biotic and abiotic stresses influence the quantity and the quality of released exudates. One the other hand, beneficial bacteria directly or indirectly promote plant growth. Biotic and abiotic factors regulate exoproduct production in biocontrol microorganisms. Recent studies have highlighted the importance of communication in establishing a fine-tuned mutualist interaction between plants and their associated beneficial bacteria. Bacteria may be able to integrate rhizosphere signals and adapt subsequently their behaviour.In a first part of the thesis, we developed a new method to monitor directly antifungal activity of individual bacterial cells in a natural environment, i.e. on roots of crop plants. We were able to demonstrate, via a dual-labelling system involving green and red fluorescent proteins (GFP, mCherry) and FACS-based flow cytometry, that expression levels of biosynthetic genes for the antifungal compounds DAPG, PLT, PRN, and HCN are highly different in liquid culture and on roots of cereals. For instance, expression of pltA (involved in PLT biosynthesis) was nearly abolished on wheat roots whereas it attained a relatively high level under in vitro conditions. In addition, we established the importance of the cereal genotype in the expression of phi A (involved in DAPG biosynthesis) in P. fluorescens CHAO.A second part of this work highlighted the systemic communication that exists between biocontrol pseudomonads and plants following attack by a root pathogen. A split-root system, allowing physical separation between the soil-borne oomycete pathogen Phytium ultimum and P. fluorescens CHAO on barley roots, was set up. Root infection by the pathogen triggered a modification of the concentration of certain phenolic root exudates in the healthy root part, resulting in an induction ofphlA expression in P. fluorescens CHAO.Amoebas are micro-predators of the rhizosphere that feed notably on bacteria. In the third part of the thesis, co-habitation of Acanthamoeba castellanii with P. fluorescens CHAO in culture media and on barley roots was found to significantly reduce bacterial expression of genes involved in the biosynthesis of DAPG, PLT, HCN and PRN. Interestingly, molecular cues present in supernatant of A. castelanii induced the expression of these antifungal genes. These findings illustrate the strategies of mutual recognition developed by amoeba and rhizosphere bacteria triggering responses that allow specific adaptations of their behaviour.The last section of the work focuses on indole-3-acetic acid (IAA), a phytohormone that stimulates the expression of phi A. A detailed molecular study revealed that the IAA-mediated effect on phi A is notably modulated by an efflux pump (FusPl) and its transcriptional regulator (MarRl). Remarkably, transcription of fusPl and marRl was strongly upregulated in presence of other phenolic compounds such as salicylate (a plant signal) and fusaric acid (a phytotoxin of the pathogenic fungus Fusarium).To sum up, this work illustrates the great complexity of interactions between eukaryotes and prokaryotes taking place in the rhizosphere niche. The mutual recognition and the establishment of a molecular cross-talk between the host plant and its associated beneficial bacteria are essential for the survival of the two partners and these interactions appear to be highly specific.