L’azote (N) est un facteur majeur limitant la croissance des plantes. Sa disponibilité peut également avoir un impact sur la résistance des plantes aux pathogènes en régulant leur immunité. Afin de mieux comprendre les liens entre la nutrition azotée et les défenses de la plante, nous avons analysé l’impact de la disponibilité en N sur la résistance de Medicago truncatula à un pathogène racinaire, Aphanomyces euteiches, en prenant en compte la variabilité génétique de la plante. Cet oomycète est considéré comme un des facteurs limitant le plus la production des légumineuses. Deux conditions de nutrition azotée, non limitante ou carencée en N, et dix génotypes de M. truncatula ont été testés in vitro. Les résultats ont montré que la résistance est modulée par les conditions nutritionnelles, dépendament du génotype. Les analyses d’expression de gènes impliqués dans le métabolisme azoté et dans les réponses de défense ainsi que la quantification des teneurs en acides aminés et des composés métaboliques secondaires ont montré des réponses différentes selon les génotypes et la condition nutritive. Elles ont souligné en particulier le rôle potentiellement important de la glutamine dans ce pathosystème. De plus, nous avons mis en évidence l’importance de l’homéostasie du monoxyde d’azote (NO) dans la résistance de M. truncatula à A. euteiches et que la disponibilité en azote impactait l’homéostasie du NO en affectant les niveaux de S-nitrosothiols et l’activité de la S-nitrosoglutathion réductase dans les racines. Ces résultats soulignent l’importance du métabolisme azoté et de son interaction avec le génotype de la plante dans les réactions de défense chez M. truncatula. / Nitrogen (N) is a major limiting factor for plant growth. N availability can also impact plant resistance to pathogens by regulating plant immunity. To better understand the links between N nutrition and plant defense, we analyzed the impact of N availability of plant on Medicago truncatula resistance to the root pathogen, Aphanomyces euteiches, taking into account plant genetic variability. This oomycete is considered as the most limiting factor for legume production. Two conditions of N nutrition, non-limiting or deprived in N, and ten plant genotypes were tested in vitro. The results showed that the resistance is modulated by nutritional conditions, depending on plant genotype. Analysis of the expression of genes involved in N metabolism and defense and quantification of different amino-acids contents and secondary metabolic compounds showed different responses of the genotypes and highlighted a potential role of glutamine in this pathosystem. Furthermore, our work underlined the importance of nitric oxide (NO) homeostasis for M. truncatula resistance to A. euteiches and that N availability impacts NO homeostasis by affecting S-nitrosothiol levels and S-nitrosoglutathione reductase activity in roots. These studies highlight, therefore, the importance of N metabolism and its interaction with plant genotype in defense responses in M. truncatula.

Les légumineuses peuvent interagir avec les bactéries du sol de la famille des Rhizobiaceae. Cette interaction aboutit à la formation d'un organe spécialisé appelé nodosité. Au sein des cellules symbiotiques des nodosités, les rhizobia sont capables de fixer l'azote atmosphérique et de la convertir en ammoniac, qui est une source d'azote assimilable par les plantes. Chez la Légumineuse Medicago truncatula, les cellules symbiotiques produisent une large famille de peptides riches en cystéines appelées (NCRs) spécifiquement exprimés dans les nodosités. Ces NCRs induisent la différenciation des bactéroïdes qui se traduit par un allongement cellulaire couplé à une forte endoréplication du génome (les bactéroïdes deviennent polyploïdes) contribuant ainsi à une augmentation importante de la taille des cellules, ainsi qu’une perméabilité membranaire accrue et une perte de toute capacité reproductrice. Les peptides NCRs ressemblent à des défensines, des peptides antimicrobiens, acteurs clés de l’immunité innée. L'analyse de l'expression de 334 gènes NCR dans 267 différentes conditions expérimentales en utilisant la base de données MtGEA (Medicago truncatula Gene Expression Atlas) a révélé que l'ensemble des gènes NCR testés (sauf quatre) n'est exprimé que dans les nodosités, ils ne sont pas exprimés dans d’autres organes de la plante, ni lors d’une infection par des agents pathogènes. De plus l’expression des NCRs n’est induite en réponse à aucune interaction biotique ou abiotique testée ou à des facteurs Nod. Les gènes NCR sont activés en vagues successives au cours de l’organogenèse nodulaire et ce profil temporel est en corrélation avec une localisation spatiale spécifique de leurs transcrit de la zone apicale à la partie proximale de nodosités. En outre, nous avons montré que les NCRs ne sont pas induites pendant la sénescence des nodules. Ces analyses expérimentales ensemble avec des calculs d’entropie de Shannon, une métric pour la spécificité d’expression, montrent que les gènes NCR sont parmi les gènes les plus fortement et le plus spécifiquement exprimés chez M. truncatula. Ainsi, l'expression des NCRs est soumise à une régulation extrêmement stricte et ils sont activés exclusivement pendant l’organogenèse et au cours du développement nodulaire dans les cellules symbiotiques polyploïdes. Cette analyse a suggéré l'implication de la régulation épigénétique des gènes NCR. La formation des cellules symbiotiques s'exerce par une endoreplication et est associée à une reprogrammation transcriptionnelle. En utilisant le tri par cytométrie en flux des noyaux, en fonction de leur contenu en ADN, nous avons montré que les vagues transcriptionnelles sont en correlation avec les niveaux croissants de ploïdie et resultent des modifications épigénétiques durant les cycles d’endoréplication. Nous avons étudié la méthylation de l'ADN génomique et l'accessibilité à la chromatine, ainsi que la présence des marqueurs répresseurs (H3K27me3) ou activateurs transcriptionnels (H3K9ac) sur des gènes spécifiques des nodosités. La méthylation différentielle de l'ADN n'a été trouvée que dans un petit sous-ensemble de gènes symbiotiques spécifiques aux nodosités. Néanmoins, plus que la moitié des gènes NCR était différentiellement méthyles. D'autre part, l'expression des gènes était corrélée avec la décondensation de la chromatine (ouverture), un enrichissement du marqueur H3K9ac et une diminution du marqueur H3K27me3. Nos résultats suggèrent que l’endoréplication, pendant la différenciation cellulaire dans les nodosités, fasse partie des mécanismes qui lèvent l’inactivation transcriptionnelle des gènes spécifiques des nodosités, ceci résultant de modifications des codes épigénétiques au niveau de la chromatine. / Legume plants are able to interact with soil bacteria from the Rhizobiaceae family. This interaction leads to the development of a specialized organ called root nodule. Inside the symbiotic nodule cells, rhizobia are capable to fix atmospheric nitrogen and convert it to ammonia, which is a usable nitrogen source for the plant. In the legume Medicago truncatula the symbiotic cells produce high amounts of Nodule-Specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes. The expression analysis of 334 NCR genes in 267 different experimental conditions using the Medicago truncatula Gene Expression Atlas (MtGEA) revealed that all the NCR genes except five are exclusively expressed in nodules. No NCR expression is induced in any other plant organ or in response to biotic, abiotic stress tested or to Nod factors. The NCR genes are activated in consecutive waves during nodule organogenesis, which correlated with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. Moreover, we showed that NCRs are not induced during nodule senescence. According to their Shannon entropy, a metric for tissue specificity, NCR genes are among the most specifically and highest expressed genes in M. truncatula. Thus, NCR gene expression is subject to an extreme tight regulation since they are only activated during nodule organogenesis in the polyploid symbiotic cells. This analysis suggested the involvement of epigenetic regulation of the NCR genes. The formation of the symbiotic cells is driven by endoreduplication and is associated with transcriptional reprogramming. Using sorted nodule nuclei according to their DNA content, we demonstrated that the transcriptional waves correlate with growing ploidy levels and investigated how the epigenome changes during endoreduplication cycles. We studied genome-wide DNA methylation and chromatin accessibility as well as the presence of repressive H3K27me3 and activating H3K9ac histone tail modifications on selected genes. Differential DNA methylation was found only in a small subset of symbiotic nodule-specific genes, including over half of the NCR genes, while in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes’ active or repressed state. On the other hand, expression of these genes correlated with ploidy-dependent opening of the chromatin and in a subset of tested genes with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in differentiation of symbiotic cells.

Anthracnose, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. Cloning and characterization of the host resistance (R) genes against the pathogen will improve our knowledge of molecular mechanisms underlying host resistance and facilitate the development of resistant alfalfa cultivars. However, the intractable genetic system of cultivated alfalfa, owing to its tetrasomic inheritance and outcrossing nature, limits the ability to carry out genetic analysis in alfalfa. Nonetheless, the model legume Medicago truncatula, a close relative of alfalfa, provides a surrogate for cloning the counterparts of many agronomically important genes in alfalfa. In this study, we used genetic map-based approach to clone RCT1, a host resistance gene against C. trifolii race 1, in M. truncatula. The RCT1 locus was delimited within a physical interval spanning ~200 kilo-bases located on the top of M. truncatula linkage group 4. Complementation tests of three candidate genes on the susceptible alfalfa clones revealed that RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad spectrum anthracnose resistance. Thus, RCT1 offers a novel resource to develop anthracnose-resistant alfalfa cultivars. Furthermore, the cloning of RCT1 also makes a significant contribution to our understanding of host resistance against the fungal genus Colletotrichum.

Trabalho de investigação desenvolvido no Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, na Faculdade de Engenharia da Universidade do Porto e no Instituto de Biologia Molecular e Celular da Universidade do Porto / Tese de mestrado integrado. Bioengenharia - Ramo de Biotecnologia Molecular. Faculdade de Engenharia. Universidade do Porto. 2012

Alfalfa, Medicago sativa L., cultivars, breeding lines and germplasm releases (populations) and Rhizobium meliloti L. strains that exhibit nitrogen fixation efficiency and tolerance to salinity and drought stresses should enhance seedling establishment, increase yields, reduce nitrogen dependency on petroleum-based nitrogen fertilizers, and allow wider use of irrigated lands in semiarid and a rid regions. In vitro experiments were conducted to determine relative salt (sodium chloride -- NaCl) tolerance: l) during germination and early seedling growth of 229 alfalfa populations from North Africa, the Middle East and the United States, 2) survival and growth of 92 rhizobial strains obtained from different laboratories in the United States or isolated from host plants growing in saline and arid regions of the Intermountain west, 3) compatibility, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strains, and 4) survival, nodulation and nitrogen fixation efficiency of the most salt-tolerant alfalfa populations and rhizobial strain tested under drought conditions. Results from screening of alfalfa at salinity levels of 0 to 3.2 S m-1 (0.1 S m-1 = 640 mg L-1 = 10 mM NaCl at 25° C) indicated 29 populations exhibited salt tolerance at 2.8 S m-1. At salinity levels of 0 to 7.2 S m-1, 14 rhizobial strains exhibited salt tolerance at 6.4 s m-1. Compatibility, nodulation and nitrogen fixation efficiency in the 29 alfalfa populations and the 14 rhizobial strains under controlled conditions showed that four of the rhizobial strains were highly compatible with all 29 populations. These 29 populations and four strains were then exposed to 0 to 1.6 S m-1 NaCl, with the result that nitrogen was fixed with highly compatible symbionts at 1.6 S m-1 NaCl. From these results, the six most highly compatible alfalfa populations and the best overall rhizobial strain were combined and tested under simulated drought stress. These populations were able to fix more nitrogen under drought stress (-0.76 MPa) (0.088 nmol seedling-1 s-1) than they did under salt stress (1.6 S m-1) (0.066 nmol seedling-1 s-1). The in vitro screening technique for acetylene reduction appears to be a useful tool for detecting physiological changes due to salinity and water stresses and for measuring seedling nitrogen fixation efficiency. Breeding for drought and salinity tolerance in conjunction with high nitrogen fix ing potential may be more realistic than breeding strictly for nitrogen fixation without regard for environmental adaptation.

This study evaluated the early establishment phase of various monocultures and mixed species plantings of Agropyron desertorum (AGDE), Medicago sativa (MESA), and Atriplex canescens (ATCA). The study was conducted under both greenhouse and field conditions. In the green-house, the experimental design was a split-plot, randomized-block design in time, using transplanted seedlings. The treatments were exposed to three water levels to determine species response to varying moisture availability. Plastic pots of 15.4 cm (diameter) by 17.9 cm (depth) were filled with 2,800 g of sandy-loam soil and brought to field capacity. The pots were weighted twice weekly and the water lost to evapotranspiration was replaced. The plants were measured for plant height on six occasions at two week intervals. On the last measurement date, plants were harvested for above-and-below ground biomass. The field used the species treatment as in the greenhouse study and these were hand sown in 1 m2 plots. Using a line-source sprinkler system, species responses at four water levels were examined. Soil water content in the various field plots was determined at various depths via access tubes and a neutron soil moisture probe. The experimental design was a split-plot design in time. Seedling emergence was recorded daily for the first 35 days after seeding. Plant height was measured six times during the growing season. All the species were clipped, oven-dried, and weighed at the end of the experimental period in 1982. In the greenhouse, AGDE was the dominant species, yet produced more in the MESA combination. MESA exhibited a marked decrease in production when grown with AGDE. ATCA showed a remarkable ability to survive under high water stress. Variable soil water content created in the field resulted in significant differences in germination, emergence, and establishment among planting treatments. MESA germinated and emerged faster than AGDE and ATCA, allowing it to utilize water and nutrient resources earlier than the competing species. The greatest shoot production occurred when MESA grew alone. In the AGDE-MESA combination, shading apparently reduced AGDE growth and shoot production at the two most favorable water levels. ATCA exhibited poor germination and emergence in treatments with low soil water availability.

Phoma medicaginis is a necrotrophic fungal pathogen, commonly found infecting Medicago truncatula and M. sativa in temperate regions of Australia. To identify, characterize and differentiate eight P. medicaginis isolates from Western Australia, morphological phenotypes and five gene regions (actin, â- tubulin, calmodulin, internal transcribed spacer, translation elongation factor 1-á) were examined. Sequence comparisons showed that specimens isolated from M. truncatula in Western Australia formed a group that was consistently different from, but closely allied to, a P. medicaginis var. medicaginis type specimen. Characterization of three P. medicaginis genotypes showed that all exhibited a narrow host range, causing disease only in M. sativa and M. truncatula among eight commonly cultivated legume species sampled. Infection of 85 M. truncatula accessions showed a continuous distribution in disease phenotypes, with the majority of accessions susceptible. Differences in disease phenotypes suggest that M. truncatula harbours specific and diverse sources of resistance to individual P. medicaginis genotypes. To characterize the genetic basis of resistance to P. medicaginis two F2 populations derived from crosses between the resistant accession SA27063 and the susceptible accessions SA3054 and A17 were phenotyped for disease symptoms. Highly significant recessive QTLs for resistance to P. medicaginis OMT5 were identified in each mapping population. In SA27063 x A17 a QTL named resistance to the necrotroph Phoma medicaginis one (rnpm1) was identified on the short arm of LG4. In SA27063 x SA3054 a QTL (rnpm2) was identified on the long arm of LG8. Further fine mapping of the areas surrounding the QTLs is underway to identify the genes underlying rnpm1 and rnpm2. Examination of the recombination frequencies between genetic markers on the long arms of chromosomes 4 and 8 in the SA27063 x A17 cross revealed an apparent genetic linkage between these chromosomes. Subsequent analysis of other crosses showed this unexpected linkage relationship is characteristic for genetic maps derived from A17. Furthermore F1 individuals derived from crosses involving A17 showed 50% pollen viability or less. This semisterility and the unexpected linkage relationships provide good evidence for a reciprocal translocation in A17 between chromosomes four and eight. The implications of the distinctive chromosomal rearrangement in A17 on genetic mapping, genome sequencing and comparative mapping are discussed. The Mt16kOLI1plus microarray was used to identify transcriptional changes in M. truncatula expressed in defence against P. medicaginis. Three-hundred-and-thirty-four differentially expressed transcripts showed a change of two-fold or more in either the resistant or susceptible interaction, and most of the Phoma-regulated genes could be assigned to functional categories which have been reported to be involved in plant defence responses. RT-qPCR and HPLCUV confirmed involvement of the octadecanoid and phenylpropanoid pathways in response to P. medicaginis infection. Faster induction of lipoxygenase genes and constitutively higher levels of certain phenolic metabolites were observed in resistant plants.

Bibliography: leaves 164-193. This study aims to quantify the belowground input of organic carbon by barrel medic using techniques that account for root death and decomposition as well as root secretion and exudation. It also investigates the effect of defoliation on carbon allocation within the plant so as to determine the potential for optimising carbon input to the soil through grazing management.

Includes bibliographical references (p. B1-B24)

La capacidad de las leguminosas de establecer una asociación simbiotica con bacterias de suelo es de fundamental importancia para la incorporación de nitrógeno a los ecosistemas, particularmente en sistemas agronómicos. En el presente trabajo de tesis se estudió la dinámica de la asociación de transcriptos (mRNAs) y miRNAs a complejos traduccionales durante las etapas tempranas de la simbiosis. Para ello, inicialmente, se puso a punto de la técnica de purificación por afinidad de ribosomas y polisomas (TRAP) en Medicago truncatula. Se cuantificó la variación de los niveles de asociación a polisomas de quince mRNAs en respuesta a la inoculación con su simbionte Sinorhizobium meliloti. Se identificó un grupo de genes, cuyos mRNAs no varían significativamente a nivel de abundancia celular, pero que se regulan positivamente a nivel de su asociación a polisomas en respuesta al rizobio. Este grupo incluyó genes que codifican receptores de tipo quinasas requeridos para la infección bacteriana o la organogénesis del nódulo y factores de transcripción de la familia GRAS y NF-Y. Posteriormente, se evaluaron los niveles de asociación a polisomas de los mRNAs seleccionados en los tejidos específicos de la raíz involucrados en la formación de nódulos: epidermis, córtex y floema. Este análisis permitió identificar mRNAs que se asocian diferencialmente a polisomas en los distintos tipos celulares durante las etapas tempranas de la simbiosis. Por otro lado, se evaluó la presencia de pequeños RNAs (sRNAs) en los complejos traduccionales purificados mediante TRAP. Los sRNAs seleccionados, incluyendo miRNAs de 21 y 22 nts y tasiRNAs, se encontraron asociados a complejos traduccionales. En particular, los niveles de miR169 presentes en polisomas disminuyeron significativamente en respuesta a la inoculación. Esta dismunución se vió acompañada de un incremento en la asociación a polisomas de su gen blanco, NF-YA1, y de los niveles de la proteína en las raíces inoculadas. Estos resultados indican que tanto los mRNAs como los miRNAs estarían sometidos a un reclutamiento diferencial a polisomas y expone la importancia de la traducción selectiva durante la simbiosis. La extensión de la técnica TRAP a M. truncatula abre la posibilidad de profundizar este nivel de regulación tanto a nivel de raíz completa como de tipos celulares específicos en asociaciones simbióticas como la nodulación y la micorrización arbuscular. El presente trabajo de tesis contribuye a sustentar la relevancia de los niveles de regulación post-transcripcional en los cambios de la expresión génica que ocurren durante el establecimiento de una asociación simbiótica de importancia ecológica y agronómica.