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L'article original est publié par The American Society of Plant Biologists; International audience; LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogenfixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box–dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant–microbe interactions and development through interacting with receptor-like kinases is discussed.

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes., Author Summary Parasitic nematodes are microscopic worms that cause major diseases of plants, animals and humans. During compatible interactions, root-knot nematodes (RKN) induce the formation of galls in which redifferentiation of root cells into multinucleate and hypertrophied giant cells is essential for nematode growth and reproduction. The importance of glutathione (GSH), a major antioxidant molecule involved in plant development, in plant microbe interaction and in abiotic stress response, was analyzed during the plant-RKN interaction. Our analyses demonstrated that the gall development and functioning are characterized by an adapted GSH metabolism and that depletion of GSH content impairs nematode reproduction and modified sex ratio. This phenotype is linked to specific modifications of carbon metabolism which do not occur in uninfected roots indicating a peculiar metabolism of this neoformed organ. This first metabolomic analysis during the plant-RKN interaction highlights the regulatory role played by GSH in this pathogenic interaction and completes our vision of the role of GSH during plant-pathogen interactions. RKN sex ratio modification has previously been observed under unfavorable nematode feeding conditions suggesting that the GSH-redox system could be a general sensor of gall fitness in natural conditions.

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction

International audience; To facilitate the phenotypic characterization of Medicago truncatula, our aim was to provide a framework of analysis of flowering in response to environmental factors. The flowering of the line A17 was analysed in different conditions of temperature, duration of vernalization and photoperiod. Flowering was characterized using three descriptors at the axis level: the position of the first reproductive node (1RN), the date of beginning of flowering (DBF) and the florochron (RFa1) corresponding to the reciprocal of the rate of progression of flowering along each axis. As for vegetative development, it was found that flowering could be analysed as a function of thermal time using a base temperature (Tb) of 5 °C. Vernalization displayed a sound impact on the flowering. For all the studied axes, increasing the duration of vernalization lowered the 1RN and hastened the DBF. By contrast, for most of the studied axes, RFa1 was only slightly affected by vernalization. For the branch B0, RFa1 was a genotypic constant when thermal time was used. Considering B0 as a reference axis, an ecophysiological model was developed to simulate the impact of environmental factors on the three components of flowering. Concrete practical applications of the model-based framework presented herein are proposed for helping the genetic and genomic studies of M. truncatula.

Nitrogen-fixing Sinorhizobium sp. bacteria interact with Medicago truncatula plants, forming root nodules in this nitrogen fixation symbiosis. When rhizobia colonize rhizosphere’s legumes, symbiotic specificity is expressed at two levels, among-species for nodulation ability and within-species for nodulation competitivity. We investigated the genetic diversity of 223 Sinorhizobium sp. strains isolated from nodules sampled from four Medicago truncatula fixed lines, each issued from distinct geographic areas, and cultivated on the same French soil. The DNA molecular polymorphism of isolates was analyzed by CE-SSCP and RFLP on 5 loci chosen either for species characterization or for their involvement in symbiosis and effectors translocation pathways. Although more than 90% of the sample bacteria belonged to Sinorhizobium medicae (others belonging to S. meliloti), statistical tests showed a significant differentiation among M. truncatula lines for bacterial species proportion. Conversely, on the basis of the four other loci analysed, we could not detect any significant among-lines population differentiation, for each bacterial species. Nevertheless, several rare strains isolated were specific from a single plant line. Thus, we compared the nodulation ability and the fixation efficiency of these isolates on the different M. truncatula lines used for trapping. Moreover, to enlighten the role played by bacterial genetic diversity on nodulation competitivity, intraspecific competition assays were performed with genetically different strains on 80 plants. Hence, SNP molecular genotyping of 200 nodules allowed the choice of the most competitive strain for each of the two Sinorhizobium species. Therefore, those two strains were tested for their ability to compete in the formation of nodules on the four Medicago truncatula lines, with inoculation of strains at different ratios (90/10, 50/50 and 10/90 proportions) on 30 plants each. Results led us to formulate hypotheses explaining the S. medicae versus S. meliloti proportions found initially in this bacterial trapping. This work outlines the influence of the genetic diversity of the host plant on the distribution of symbiotic genotypes in rhizospheric bacterial populations, and allows a better understanding of bacterial selection in natural and cultivated ecosystems.

International audience; The genetic structure of bacterial and fungal communities was characterized in the rhizosphere of Medicago truncatula Gaertn. cv. Jemalong line J5 at five developmental stages (three vegetative and two reproductive stages), and in three compartments (bulk soil, rhizosphere soil and root tissues). The genetic structure of microbial communities was determined by cultivation-independent methods using directly extracted DNA that was characterized by automated ribosomal intergenic spacer analysis (ARISA). Principal component analyses (PCA) indicate that, for all developmental stages, the genetic structure of microbial communities differed significantly by compartment, with a major shift in the community in root tissues corresponding to the most intimate compartment with the plant. Differences were also recorded during plant development, the most significant being observed during the transition between vegetative and reproductive stages. Throughout this period, plants were shown to establish the highest level of symbiotic association (mycorrhization, nodulation) with arbuscular mycorrhizal fungi and Rhizobia. During the reproductive stages, the dynamics of the genetic structure differed between bacterial and fungal communities. At the last reproductive stage, the genetic structure of bacterial communities became close to that recorded during the first vegetative stages, suggesting a resilience phenomenon, whereas the genetic structure of fungal communities remained different from the vegetative stages and also from the early reproductive stages, suggesting a persistence of the rhizosphere effect