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Nitrogen-fixing root nodulation is confined to four plant orders, including > 14 000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Highlight Persistence of intracellular rhizobia in legumes involves plant and bacterial genes. Here we show that DNF2, bacA, SymCRK/RSD and nifA/nifH successively prevent bacterial death during the symbiotic process., Medicago truncatula belongs to the legume family and forms symbiotic associations with nitrogen fixing bacteria, the rhizobia. During these interactions, the plants develop root nodules in which bacteria invade the plant cells and fix nitrogen for the benefit of the plant. Despite massive infection, legume nodules do not develop visible defence reactions, suggesting a special immune status of these organs. Some factors influencing rhizobium maintenance within the plant cells have been previously identified, such as the M. truncatula NCR peptides whose toxic effects are reduced by the bacterial protein BacA. In addition, DNF2, SymCRK, and RSD are M. truncatula genes required to avoid rhizobial death within the symbiotic cells. DNF2 and SymCRK are essential to prevent defence-like reactions in nodules after bacteria internalization into the symbiotic cells. Herein, we used a combination of genetics, histology and molecular biology approaches to investigate the relationship between the factors preventing bacterial death in the nodule cells. We show that the RSD gene is also required to repress plant defences in nodules. Upon inoculation with the bacA mutant, defence responses are observed only in the dnf2 mutant and not in the symCRK and rsd mutants. In addition, our data suggest that lack of nitrogen fixation by the bacterial partner triggers bacterial death in nodule cells after bacteroid differentiation. Together our data indicate that, after internalization, at least four independent mechanisms prevent bacterial death in the plant cell. These mechanisms involve successively: DNF2, BacA, SymCRK/RSD and bacterial ability to fix nitrogen.

In many parts of the world local people are convinced that forests, trees and rainfall are related in more than one way: forests and trees not only depend on rainfall but help to generate it. Scientists confronted with this perspective have always denied such effects, or at best been ‘agnostic’, as it seemed impossible in their data to find evidence. New evidence on credible mechanisms for forest and tree effects on rainfall is, however, emerging. It can revolutionize current climate negotiations that all focus on greenhouse gas emissions.

International audience; Rhizobia and legumes establish symbiotic interactions leading to the production of root nodules, in which bacteria fix atmospheric nitrogen for the plant's benefit. This symbiosis is efficient because of the high rhizobia population within nodules. Here, we investigated how legumes accommodate such bacterial colonization. We used a reverse genetic approach to identify a Medicago truncatula gene, SymCRK, which encodes a cysteine-rich receptor-like kinase that is required for rhizobia maintenance within the plant cells, and performed detailed phenotypic analyses of the corresponding mutant. The Medicago truncatula symCRK mutant developed nonfunctional and necrotic nodules. A nonarginine asparate (nonRD) motif, typical of receptors involved in innate immunity, is present in the SymCRK kinase domain. Similar to the dnf2 mutant, bacteroid differentiation defect, defense-like reactions and early senescence were observed in the symCRK nodules. However, the dnf2 and symCRK nodules differ by their degree of colonization, which is higher in symCRK. Furthermore, in contrast to dnf2, symCRK is not a conditional mutant. These results suggest that in M. truncatula at least two genes are involved in the symbiotic control of immunity. Furthermore, phenotype differences between the two mutants suggest that two distinct molecular mechanisms control suppression of plant immunity during nodulation.

International audience; Rhizobia and legumes are able to interact in a symbiotic way leading to the development of root nodules. Within nodules, rhizobia fix nitrogen for the benefit of the plant. These interactions are efficient because spectacularly high densities of nitrogen fixing rhizobia are maintained in the plant cells. DNF2, a Medicago truncatula gene has been described as required for nitrogen fixation, bacteroid's persistence and to prevent defense-like reactions in the nodules. This manuscript shows that a Rhizobium mutant unable to differentiate is not sufficient to trigger defense-like reactions in this organ. Furthermore, we show that the requirement of DNF2 for effective symbiosis can be overcome by permissive growth conditions. The dnf2 knockout mutants grown in vitro on agarose or Phytagel as gelling agents are able to produce nodules fixing nitrogen with the same efficiency as the wild-type. However, when agarose medium is supplemented with the plant defense elicitor ulvan, the dnf2 mutant recovers the fix- phenotype. Together, our data show that plant growth conditions impact the gene requirement for symbiotic nitrogen fixation and suggest that they influence the symbiotic suppression of defense reactions in nodules.

La fixation symbiotique d'azote par l'ajonc d'Europe (Ulex europaeus) représente une source importante d'azote dans la forêt cultivée de pins maritimes des 'Landes de Gascogne'. Cette thèse a pour objectif la création d'un modèle conceptuel de dynamique de population de l'ajonc dans la région en vue de la prédiction du flux d'azote associé. Pour cela plusieurs étapes du cycle de vie de l'espèce ont été étudiées: la production et la prédation des graines, puis le recrutement de nouveaux individus depuis le stock de graines du sol. Nos résultats suggèrent que le maintien de cette espèce héliophile à l'ombre de la canopée des pins est facilité par la diminution de l'allocation de ressources à la reproduction, lui permettant de maintenir sa croissance, et par la diminution de la prédation des graines. Par ailleurs, le recrutement de nouveaux ajoncs semble influencé par des facteurs écologiques identifiables (e.g. humidité du sol). Mais ces facteurs sont fortement variables dans l'espace et dans le temps, et aucun micro habitat n'apparait comme plus favorable à la régénération de l'espèce. Ces résultats sont intégrés à un modèle conceptuel, représentant les différentes étapes du cycle de vie de l'espèce, et l'impact des opérations sylvicoles sur ces étapes. Les connaissances issues de cette thèse pourraient aussi bénéficier à d'autres problématiques liées à l'espèce (e.g. c'est une espèce invasive dans de nombreuses régions du monde) et à la compréhension de l'écologie de la régénération des espèces ligneuses. En effet, l'allocation des ressources à la reproduction pourrait être plastique et permettre une réponse adaptative à un environnement changeant; et la variabilité spatiotemporelle de la régénération des ligneux pourrait reposer sur l'existence de niches écologiques de régénération définies dans l'espace écologique., Symbiotic nitrogen fixation by European gorse (Ulex europaeus) is a major source of nitrogen in the 'Landes de Gascogne' cultivated forest of maritime pine. This thesis aims to build a conceptual model of the population dynamic of gorse in the region, with a view to predict the associated flux of nitrogen. To achieve this, several steps of the life cycle of the species were studied: production and predation of seeds, and the recruitment of new plants from the seedbank.The results suggest that the maintenance of this light demanding species in the shade of the canopy of pines is improved by the decrease of reproductive allocation, which enable to maintain its growth, and by the decrease of the predation of seeds. The recruitment of new gorse plants seems controlled by identifiable ecological factors (e.g. soil moisture), however these factors are highly variable in space and time, and no specific micro-habitat appears to be more favourable for the species regeneration.These results are integrated into a conceptual model based on the different steps of the life cycle of the species, and taking into consideration the influence of forestry practices on these steps. Furthermore, the production of knowledge could benefit other subjects related to the species (e.g gorse is a noxious weed in many region in the world) and the understanding of the ecology of woody plants' regeneration. Allocation of resources to reproduction may be plastic and enable an adaptative response to a changing environment; and spatiotemporal variability of the regeneration of woody plants may rely on the existence of ecological regeneration niches defined in the ecological space.