Diatoms associated with foraminifers of the genus Amphistegina were assessed using a combination of morphological and molecular techniques. These included: 1) microscopic identification of diatoms cultured from the host, 2) sequencing of portions of the small subunit of the ribosomal RNA gene (18S) and the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase [i.e., RubisCO] gene (rbcL) from DNA extracted directly from the Amphistegina hosts and also from diatoms cultured from these hosts, and 3) denaturing gradient gel electrophoresis (DGGE) profiles of rbcL and internal transcribed spacer 1 (ITS1) PCR amplicons from DNA extracted directly from hosts and from cultures. Consistent with previous culture studies, multiple species of pennate diatoms of the genera Nitzschia, Fragilaria (including Nanofrustulum), Amphora, and Navicula, were cultured from >900 host specimens collected from >20 sites in the western Atlantic and four sites in the Pacific. Diatoms of the genus Nitzschia grew in about half of all successful cultures. The genetic identities of selected cultures were consistent with those based on morphological taxonomy. Diatom sequences from DNA extracted directly from the cytoplasm of the Amphistegina hosts were species specific and distinct from sequences obtained from cultured diatoms and from sequences in GenBank of diatom taxa previously reported as endosymbionts. Multiple phylogenetic analyses revealed that the 18S and rbcL diatom sequences from specimens of A. gibbosa collected from the Atlantic sites and of Amphistegina spp. from Hawai’i were most similar to the 18S and rbcL sequences of an unnamed Fragilariaceae diatom in GenBank (Accession # JX413542.1 for 18S and JX413559.1 for rbcL) and other closely related diatoms in that family. Of diatom taxa previously reported as endosymbionts of larger foraminifers, Nanofrustulum shiloi was the most similar, but not identical, to the sequences from hosts collected from the Atlantic and Hawai’i. The 18S and rbcL diatom sequences from the Atlantic host species, A. gibbosa, were all nearly identical, but small intra-species differences (subclades) were observed from specimens collected from the deepest (75 m) site in the Florida Keys and also from the eastern-most site, Young Island near St. Vincent. The 18S and rbcL diatom sequences from the two host species from Hawai’i, A. lobifera and A. lessonii, were more variable but still within the family Fragilariaceae. The diatom sequences from A. radiata collected from two sites in Papua New Guinea (PNG) were most similar to diatoms of the family Plagiogrammaceae and therefore distinct from sequences obtained from other Amphistegina species in this study, as well as from all diatoms previously reported as endosymbionts. A small difference was observed between the diatom sequences from host specimens collected from a Pacific site as compared to a Bismarck Sea site. The ITS1 DGGE profiles of DNA extracted directly from A. gibbosa specimens at different depths, locations, and seasons in the western Atlantic were nearly identical. Differences were seen between rbcL DGGE profiles of DNA extracted directly from the different Amphistegina host species. The rbcL DGGE profiles directly from all hosts were clearly different from those extracted from diatoms cultured from the same host specimens, as well as from Nitzschia laevis, a commonly reported diatom endosymbiont in past culture-based studies. My findings are consistent with ultrastructural studies of endosymbionts of Amphistegina published in the early 1980s and congruent with recent molecular studies of endosymbionts in other diatom-bearing foraminifers, all of which indicate specificity. Nevertheless, the consistency with which several diatom taxa have been reported in culture studies from all oceans indicates the possibility of some relationship with Amphistegina spp., either as important food items, epiphytes, or minor opportunistic symbionts that can thrive in culture media.

The symbiotic relationships often represent not only the permanent cohabitation of two organisms, but they can evolve and change over time. Many papers published so far focused on the the specificity of lichens, characterising both highly specific species and those that can establish a symbiosis with many algal genotypes. In this second case we can imagine the possibility of a photobiont exchange during the life of lichen - the dynamics of lichen symbiosis. In this thesis I compared the occurence and diversity of photobionts within the thalli of lichens growing on the disturbed localities and on the the localities without the disturbance. These two types of localities were similar to species richness of photobionts, but the disturbed localities dominated by the number of photobiont genera. These localities also reached a higher value of phylogenetic diversity. Conversely, non-phylogenetic analysis revealed the higher level of photobiont diversity on non-disturbed localities, where the core of diversity was formed by different lineages of Trebouxia. I have also found the lichens, which cooperate with different algae on disturbed and non-disturbed localities. The photobiont distribution presented in this paper shows the possibility of photobiont exchange during the community succesion. The...

L'importance des symbioses dans l'évolution du vivant est désormais admise et les associations symbiotiques sont observées dans une grande diversité d'habitats. Notre étude porte sur une symbiose au sein d'un écosystème réduit, les sources hydrothermales de l'océan profond. Bathymodiolus azoricus est un bivalve hydrothermal vivant le long de la ride Médio-Atlantique, qui héberge dans des cellules branchiales spécialisées, deux types de γ-protéobactéries différentes : des méthanotrophes (MOX) et des sulfo-oxydantes (SOX). Ces dernières sont capables d'oxyder les composés réduits présents dans le fluide hydrothermal fournissant ainsi énergie et/ou source de carbone à leur hôte. Cette double endosymbiose est plastique ainsi, l'abondance relative du type de symbionte hébergé (SOX vs. MOX) varie en fonction des concentrations en composés réduits présent dans le milieu (H2S, CH4). L'objectif de ce travail de thèse est d'identifier les acteurs moléculaires impliqués dans l'acquisition, le maintien et la régulation des bactéries symbiotiques. Pour ce faire, une analyse OMICs globale (protéomique -nano LC-MS/MS- et transcriptomique -micro-array-) a été mise en ¿uvre sur des individus symbiotiques issus de population naturelle (site hydrothermal Lucky Strike, -1700m) et sur des individus ayant expérimentalement perdu ou maintenu leurs symbiotes. Suite à cette approche globale et exploratoire, une approche plus spécifique a été menée sur des familles de protéines impliquées dans des processus immunitaire et/ou d'interactions hôte/symbiotes. Cette thèse apporte un éclaircissement sur les mécanismes régissant les relations et la communication hôte/symbiote. / Hydrothermal vents are located on the mid-ocean ridges, and are characterized by challenging physico-chemical conditions. Despite these conditions dense hydrothermal communities develop down around hydrothermal fluid emissions. The presence of marine invertebrates relies on their capacity to cope with these challenging factors, and, for those forming most of the biomass, on their ability to live in symbiosis with chemoautotrophic bacteria. Bathymodiolus azoricus is one of these symbiotic species that harbors two types of γ-proteobacteria, a sulfide-oxidizing bacterium (SOX) (using the oxidation of H2S as the source of energy and CO2 as source of carbon) and a methane-oxidizing bacterium (MOX) (that uses the oxidation of CH4 as both a source of energy and carbon). These bacteria are located in specific epithelial cells in the gill tissue of the mussel. The proportion and number of these symbiont types (SOX vs. MOX) in B.azoricus can change in response to environmental conditions, and especially on the relative concentration of reduced compounds. The aim of our study is to understand the molecular mechanisms of acquisition, regulation and maintenance of the symbiotic charge in B .azoricus gills. We therefore, performed a global OMICs analysis (proteomics –nano LC-MS/MS and transcriptomics- micro-array) on mussels from natural population (Lucky Strike, -1700m) and on mussels that experimentally loose or maintain their symbiotic rate. This exploratory approach was followed by a more specific approach on family of proteins involved in immunity process and/or in host/symbiont interactions. This PhD provides hypotheses on the mechanisms governing the relationship and communication between host and symbionts.

Les bivalves côtiers Codakia orbicularis et C. orbiculata, de la famille des Lucinidae, abritent des Gammaprotéobactéries endosymbiotiques sulfo-oxydantes dans leurs branchies. Ces deux bivalves vivent dans les herbiers à Thalassia testudinum et hébergent la même bactérie symbiotique selon les analyses effectuées à partir des séquences d’ADNr 16S. Lors de période de stabulation, la population bactérienne symbiotique décroit alors qu’il n’y a pas, dans le même temps, de relargage des symbiotes observé. Des analyses en cytochimie ont montré une forte activité d’enzymes lysosomales lors de ces épisodes de privation de nourriture et de soufre. Il a ainsi été montré que les symbiotes peuvent servir directement de source de nourriture aux bivalves pour survivre lors de ces périodes de crise. Le transfert de carbone des symbiotes vers l’hôte peut être flexible et pourrait consister en un simple transfert de matière organique ou "milking", dans des conditions normales de nutrition et de digestion des symbiotes et devenir du "farming", dans des conditions de stabulation. Jusqu’à ce jour, le symbiote reste non cultivable. De ce fait, l’utilisation de techniques indépendantes de la culture comme les approches –omics ont été mises en place pour étudier la physiologie de cette bactérie symbiotique. Le génome du symbiote a été analysé par Next Generation Sequencing (NGS) permettant ainsi d’obtenir les bases du protéome et ainsi de pouvoir analyser la physiologie du symbiote. Dans ce travail, le protéome des bactéries a été analysé sous différentes conditions. L’oxydation des sulfures est une des voies métaboliques clés du symbiote de Codakia. Cette voie fait très probablement appel au système Sox périplasmique, ainsi qu’à une sulfite réductase cytoplasmique (DsrAB), une APS réductase (AprAB) et une ATP sulfurylase (SopT). De plus, deux autres enzymes additionnelles d’oxydation des sulfures ont pu être mises en évidence dans l’espace périplasmique du symbiote telle que la quinone réductase (Sqr) et la sulfide déhydrogénase (FccAB). Les gènes des enzymes du cycle de Calvin Benson Bassham (CBB) ne semblent pas être tous présents dans le génome du symbiote. Les protéines de la RuBisCO sont abondamment exprimées. Il semblerait que la régénération du ribulose-1,5-bisphosphate soit effectuée de façon non conventionnelle via une phosphofructokinase PPi-dépendante. Une autre caractéristique du CBB est qu’il y a deux formes différentes de RuBisCO codées dans le génome du symbiote. Les deux formes sont exprimées en même temps, mais la forme I de la RuBisCO est 50 fois plus exprimée que la forme II. En plus de la vie autotrophique, plusieurs gènes nécessaires à une vie hétérotrophique sont présents dans le génome. Dans le protéome, les enzymes de la glycolyse et du cycle TCA sont faiblement exprimées. Les protéines du métabolisme du glycogène ont également été identifiées dans le protéome. De plus, plusieurs types de transporteurs comme ABC, TRAP et PTS sont présents dans le génome et certaines formes d’expression de ces transporteurs ont pu être suspectées, y compris lors de la vie intracellulaire du symbiote. De façon inattendue, un groupe de gènes nif est présent dans le génome permettant la fixation de l’azote atmosphérique par le symbiote. Les protéines codées par les gènes clés, comme la nitrogénase NifH/K/D, ont été abondamment trouvées dans le protéome. De plus, l’analyse du protéome montre une régulation forte de ces protéines dans des conditions de stabulation du bivalve hôte. La rubrerythrine est fortement exprimée et servirait à protéger la nitrogénase de l’oxygène au sein des bacteriocytes. L’endosymbiote bactérien code également pour un système de sécrétion de type 6 (T6SS) pour le transport de molécules bactériennes effectrices à travers les membranes du cytoplasme de la cellule hôte et jouerait un rôle possible de communication directe avec l’hôte. / The shallow water bivalves Codakia orbicularis and Codakia orbiculata, both belonging to the family Lucinidae, harbor endosymbiotic sulfur-oxidizing gamma-Proteobacteria in their gills. The bivalves live in seagrass beds of Thalassia testudinum and harbor the same bacterial symbionts according to 16S rDNA sequence analysis. During starvation, the symbiont population decreases while no release of symbionts were observed. We observed lysosomal enzyme activity during sulfide and food starvation with cytochemical staining methods. We suggest that the host uses symbionts as a nutrient source to survive a hunger crisis. The carbon transfer from the symbionts to the host could be flexible and could consist in transfer of organic matter, "milking", under normal feeding conditions and digestion of the symbionts, "farming", under starved conditions. Until now the symbiont alone is not cultivable. Therefore, cultivation-independent techniques, like -omics approaches were used to analyze the physiology of the symbiont. Next generation sequencing (NGS) was employed to sequence the genomes of symbionts from both hosts, display the backbone for proteomics. The soluble- and membrane-associated symbiont proteomes were analyzed during different conditions. The oxidation of sulfide is one key metabolic pathway of the Codakia symbiont, most probably using the periplasmic Sox-system, a cytoplasmatic sulfite reductase (DsrAB), an APS reductase (AprAB) and an ATP sulfurylase (SopT). Furthermore, indications for two additional putative sulfide oxidation systems in the periplasmic space, the sulfide quinone reductase (Sqr) and the sulfide dehydrogenase (FccAB), could be found. The Calvin Benson Bassham cycle (CBB) of the symbiont is not completely encoded in the genome. The key genes, RuBisCO, are abundantly expressed. It is assumed that the regeneration of the ribulose-1,5-bisphosphate is performed unconventionally via a PPi-dependent phosphofructokinase. Another feature of the CBB is that two different forms of RuBisCO are encoded in the genome. Both are expressed at the same time, but RuBisCO form I is about 50x times more expressed. Additional to the autotrophic lifestyle, all genes for the heterotrophic lifestyle are encoded in the genome. In the proteome, the enzymes related to glycolysis and TCA-cycle were low expressed. Interestingly, proteins for glycogen metabolism were identified in the proteome. Additionally, several types of transporters like ABC, TRAP and PTS are encoded in the genome. In the proteome several indications were found for an expression of these transporters, even in the endosymbiotic lifestyle. Unexpectedly, in the genome a nif gene cluster is encoded for gaseous nitrogen fixation as ammonium source. The key genes, the nitrogenase NifH/K/D, were abundantly identified in proteome. Further, the proteome analyses indicate a strictly down-regulation of these proteins under starvation conditions. Rubrerythrin, a strongly expressed protein and is predicted to protect the nitrogenase against oxygen stress. The bacterial endosymbionts encode a specialized secretion system type 6 (T6SS) for the transport of bacterial effector molecules through the membranes to the host cytoplasm and display one possibility for a direct "communication" with the host. In summary, genomics and proteomics analyses of the Codakia symbiont improved the knowledge about the metabolism of the symbiont in lucinid bivalves.. The genomics and proteomics data generated in this study can be used as a basis for further in-depth analyses of the physiology of the symbionts and interaction with the host.

Human domination of global nutrient cycles is profoundly altering our planet. Yet on coral reefs, the effects of changing nutrient regimes have likely been over-simplified. This dissertation investigates the complexity of animal-nutrient interactions at the organismal level and explores how the outcomes of these interactions cascade through levels of biological organization. To do so, I examined the effects of nitrogen (N) and phosphorus (P) on corals and macroalgae, and how these effects in turn influenced reef communities and entire ecosystems. I show that P consistently increases coral growth rates while N has variable, often negative, effects on coral growth. The majority of this variability was explained by the contrasting responses of corals to ammonium, which had negligible effects on coral growth, versus nitrate, which consistently had negative effects on corals. Experimental manipulations of nutrient regimes revealed that these effects could be attributed, in part, to increased damage to the photosynthetic components of the corals’ endosymbionts. Nitrogen and P-enrichment also impacted macroalgae, increasing the nutrient content of algal tissue and in turn, consumption patterns of herbivorous fishes. Initial phase parrotfishes and juvenile surgeonfishes increased their feeding rates on algae rich in N and P respectively. However, adults from both species were irresponsive to algal nutrient content. At the community level, the effects of N and P on corals, algae and herbivory were linked to the development of distinct benthic communities. Algae cover was lower and coral growth rates higher around reef structures that were consistently enriched with N and P excreted by sheltering fishes. At the ecosystem level, I found that the responses of corals to N and P enrichment were similar to those of other nutrient-sharing mutualists. Across terrestrial and marine environments, I show that N and P enrichment consistently decouples mutualism performance, benefiting one partner at the expense of the other. Thus, collectively this dissertation demonstrates that the impacts of global nutrient loading resonate from single organisms through whole ecosystems.

La plupart des espèces végétales terrestres vivent en symbiose avec les champignons mycorhiziens à arbuscules (MA). Il s’agit d’une symbiose très ancienne datant de plus de 400 millions d’années. Les champignons MA sont des champignons du sol qui appartiennent aux Gloméromycètes. Ils sont présents dans la plupart des écosystèmes terrestres. Ainsi, ils peuvent être considérés comme une composante intégrale des racines des plantes. Ils forment dans les cellules racinaires corticales des structures fonctionnelles essentielles appelées arbuscules où ils apportent à la plante des minéraux nutritifs en échange de sucres. L’auxine est une phytohormone impliquée dans de nombreux processus de développement des plantes, y compris la dominance apicale, les tropismes, la structuration vasculaire et la formation de racines latérales. Le principal objectif de notre travail était d’étudier de manière approfondie le rôle de l’auxine dans le processus de développement des mycorhizes. On sait déjà que la symbiose MA stimule la formation de racines latérales dans les plantes hôtes, ce qui pourrait être due à une modification du métabolisme de l’auxine, de son transport ou de sa perception. Les microARNs (miARNs) sont des molécules d’ARN non codantes de ~ 21 nucléotides capables de réprimer l’expression de gènes en ciblant et clivant spécifiquement leur ARNm correspondant. Plusieurs miARNs interagissent avec la signalisation de l’auxine et parmi eux miR393 qui cible les récepteurs à l’auxine. Nous avons étudié le rôle de miR393 dans la colonisation mycorhizienne. Nous mettons en évidence que chez Solanum lycopersicum (Solanacées), Medicago truncatula (Fabaceae) et Oryza sativa (Poaceae), l’expression des précurseurs de miR393 diminue lors de la mycorhization. En outre nous montrons que DR5-GUS, un gène rapporteur de réponse à l’auxine, est préférentiellement exprimé dans les cellules de la racine contenant les arbuscules. En sur-exprimant miR393 dans les racines et donc en régulant négativement l’expression des gènes de récepteurs à l’auxine, nous montrons également que les arbuscules ne se développent pas normalement. En tant que composantes des complexes récepteurs d’auxine, les protéines Aux/IAA jouent un rôle majeur dans la voie de signalisation de l’auxine en réprimant l’activité des facteurs de transcription de type ARF. Nous avons vérifié dans des racines de tomate mycorhizées l’expression de 25 gènes AUX/IAA. Nous nous sommes concentrés sur IAA27 dont l’expression est induite lors des premiers stades de la symbiose MA. Nous observons qu’une répression par ARNi de l’expression de IAA27 dans des plants de tomate conduit à une forte diminution de la colonisation MA et du nombre des arbuscules. Puis nous montrons par des approches différentes que la régulation positive de la mycorhization par IAA27 est liée à la biosynthèse des strigolactones. Globalement, ces résultats appuient fortement l’hypothèse selon laquelle la signalisation de l’auxine joue un rôle important aussi bien dans le stade précoce de la mycorhization que dans la formation des arbuscules. / Most land plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. This is a very ancient symbiosis dating back to 450 million years. AM fungi are soil fungi that belong to the Glomeromycota. They are present in most terrestrial ecosystems. Thus they can be considered as an integral root component of plants. They form essential functional structures called arbuscules in root cortical cells at which mineral nutrients are released to the plant in exchange of sugars. The phytohormone auxin is involved in many developmental processes in plants, including apical dominance, tropisms, vascular patterning and lateral root formation. The main objective of our work was to investigate further the role of auxin in the mycorrhizal developmental process. We already know that AM symbiosis stimulates the lateral root formation in host plants, which could be due to modification of auxin metabolism, transport or perception. The microRNAs (miRNAs) are ~21-nucleotides noncoding RNAs that target corresponding mRNA transcripts for cleavage and transcriptional repression. Several miRNAs interact with auxin signaling and among them miR393 that targets auxin receptors. We investigated the role of miR393 in AM root colonization. In Solanum lycopersicum (Solanaceae), Medicago truncatula (Fabaceae) and Oryza sativa (Poaceae), expression of the precursors of the miR393 was down-regulated during mycorrhization. In addition DR5-GUS, a reporter for auxin response, was found to be preferentially expressed in root cells containing arbuscules. By over-expressing miR393 in roots and therefore down-regulating auxin receptor genes, arbuscules could not develop normally. As components of auxin receptor complexes, Aux/IAA proteins play a major role in auxin signaling pathway by repressing the activity of ARF type transcription factors. We checked the expression of 25 AUX/IAA genes in AM roots. Among them, we focused on IAA27 that was significantly up-regulated during the early stages of AM symbiosis. IAA27 down-regulation in plants led to a strong decrease of AM colonization and arbuscule abundance. We showed by different approaches that the positive regulation of mycorrhization by IAA27 was linked to strigolactone biosynthesis. Overall these results strongly support the hypothesis that auxin signaling plays an important role both in the early stage of mycorrhization and in the arbuscule formation.

No description available.

This thesis project is a proposal of a scenario of an industrial collaboration that could become the basis for the development of an Eco-Industrial Park inside the King Abdullah University of Science and Technology using the water desalination plant as the foundation of this network. It is defined why the university is a suitable place for a development of this type; what industrial networks can be formed and what environmental, economic and social benefits could be achieved if the Kingdom of Saudi Arabia implemented this project around the country. An Eco-Industrial Park is a collaboration between industries where they share resources and create an exchange network of wastes and by-products with the objective of obtaining positive environmental, economic and social impacts. In the proposed scenario, the initial collaboration is between a PV solar plant and the water desalination located inside the university. With the use of solar energy there are savings of CO2 emissions, which turn into positive impacts on the environment. The idea is that this initial collaboration could be extended in the future to provide the entire energy consumption of the KAUST community. The project also provides an insight into how an industrial symbiosis could be formed taking as a base this industrial collaboration. However, further studies and analysis are required in order to provide more solid information regarding the development of an Eco-Industrial Park.

Diplomová práce se zabývá transformací a novým návrhem funkčního využití území areálu výstaviště v Brně. Areál je vymezen od jihu velkým městským okruhem města Brna a od severu ulicí Hlinky. Cílem návrhu bylo oživit stávající hodnotné území tak, aby se z něho stal fungující městský organismus volně přístupný veřejnosti, který by tvořil symbiózu mezi historickou částí výstaviště a nově navrženou částí.

La légumineuse Medicago établie une interaction symbiotique avec des bactéries du sol fixatrices d’azote, les rhizobia. Cette interaction provoque la formation d’un nouvel organe racinaire, la nodosité, au sein de laquelle les bactéries infectent de manière massive et chronique les cellules de la plante. Malgré cette invasion, aucune réaction de défense n’est observée ce qui suggère l’existence de mécanismes symbiotiques locaux de contrôle de l’immunité. Les gènes de Medicago DNF2 et SymCRK codant une phospholipase C-like et un récepteur-like kinase riche en cystéines, semblaient intervenir dans ces mécanismes peu connus. Mon travail de thèse a consisté à mieux caractériser les mécanismes de tolérance aux rhizobia notamment ceux faisant intervenir ces deux gènes. Nos résultats indiquent que dnf2 et symCRK forment des nodosités non-fixatrices, nécrotiques, présentant une activation des défenses et une perte de viabilité des bactéroïdes (forme intracellulaire des bactéries). Par ailleurs, l’utilisation de mutants bactériens nous a permis de montrer que, chez la plante sauvage, la perte de viabilité des bactéroïdes et l’absence de fixation d’azote ne sont pas suffisantes pour stimuler les défenses. Nos résultats indiquent également que dnf2 et symCRK agissent successivement lors du processus symbiotique et que la nécessité de dnf2 pour l’établissement de la symbiose peut être contournée dans certaines conditions de culture. Enfin, nous avons réalisé une analyse du protéome de symCRK et des expériences de physiologie végétale qui ont mis en évidence la nécessité, pour le maintien d’une symbiose efficace, de réprimer la voie éthylène après internalisation des rhizobia dans les cellules végétales. Ensemble, nos données améliorent la compréhension du phénomène de tolérance observée dans les nodosités de Légumineuses. / The legume plant Medicago establishes symbiotic interaction with nitrogen fixing bacteria, called rhizobia. This interaction leads to the formation of root organs, the nodules. A massive and chronic infection of nodule cells is observed without induction of any plant defense suggesting that a symbiotic mechanism controls immunity in the nodules. The two Medicago genes, DNF2 and SymCRK encoding a phospholipase C-like protein and a cysteine-rich receptor-like kinase respectively were identified as potentially involved in the prevention of defenses during the late steps of the symbiosis. However, this phenomenon was poorly characterized. Herein we improved the characterization of the Legume tolerance to intracellular rhizobia with an emphasis on the role of DNF2 and SymCRK. Our results indicate that dnf2 and symCRK produce necrotic nodules that do not fix nitrogen, that develop defenses and in which bacteroids, the intracellular form of rhizobia, rapidly loose viability. Using bacterial mutants, we show that reduced bacteroid viability and/or nitrogen fixation defect are not per se enough to trigger defenses in wild type plants. Our results also indicate that DNF2 and SymCRK act successively during the symbiotic process and that artificial culture conditions can bypass DNF2 requirement for symbiosis. Finally, symCRK proteome analysis and physiological studies together indicate that the ethylene pathway has to be repressed after rhizobia internalization within the plant cells to maintain efficient symbiosis. Together our data improve the knowledge on the basis of legume tolerance to rhizobia.