Stimulation du développement des racines latérales par des lipo-chitooligosaccharides (LCOs) symbiotiques chez Medicago truncatula

La légumineuse modèle Medicago truncatula peut s'associer avec deux types de microorganismes du sol pour former des endosymbioses racinaires : la symbiose Légumineuse-Rhizobium avec des bactéries fixatrices d'azote, et la symbiose Mycorhizienne à Arbuscules avec des champignons (Gloméromycètes). Les symbiontes rhizobien et mycorhiziens de M. truncatula produisent des lipo-chitooligosaccharides (LCOs), respectivement appelés facteurs Nod et Myc-LCOs, qui stimulent la formation des racines latérales (RL) chez cette plante. Il a été suggéré que ces molécules interfèrent avec le programme développemental de la plante pour former les RL. Afin de déterminer la manière dont ces molécules interviennent dans ce programme, il faut comprendre les mécanismes physiologiques et moléculaires régissant l'action des facteurs Nod et des Myc-LCOs sur la formation des RL. Le projet de thèse a pour objectif de caractériser la stimulation du développement des RL par ces facteurs symbiotiques chez M. truncatula, et en particulier de comprendre s'ils agissent sur un stade de développement particulier, et s'ils interagissent avec des voies hormonales. Nos travaux ont permis de mieux comprendre les caractéristiques du développement des RL chez M. truncatula. Ce développement présente des similarités et des différences avec la RL chez d'autres espèces, mais aussi avec la nodosité fixatrice d'azote. Nous avons mis en évidence une action précoce des facteurs Nod sur la formation de la RL, ainsi que le rôle prépondérant de l'auxine dans cette stimulation. Cette thèse ouvre des perspectives sur la compréhension de l'effet des molécules d'origine microbienne sur le développement des organes racinaires.The model legume Medicago truncatula can be associated with two types of soil microorganisms to form root endosymbioses: a Legume-Rhizobium symbiosis with nitrogen-fixing bacteria and an Arbuscular Mycorrhizal symbiosis with fungi (Glomeromycota). The rhizobial and mycorrhizal symbionts of M. truncatula produce lipo-chitooligosaccharides (LCOs), respectively called Nod factors and Myc-LCOs, which stimulate the formation of lateral roots (LR) in this plant. It has been suggested that these molecules interfere with the developmental program of the plant to form LRs. In order to determine how these molecules are involved in this program, it is necessary to understand the physiological and molecular mechanisms governing the action of Nod factors and Myc-LCOs on LR formation. The aim of the thesis is to characterize the stimulation of LR formation by these symbiotic factors in M. truncatula, and in particular to understand whether they act on a particular developmental stage and whether they interact with hormone pathways. Our work led to a better understanding of the developmental characteristics of the LR in M. truncatula. This development has similarities and differences compared to the LR in other species, but also compared to the nitrogen-fixing root nodules. We highlighted early action of Nod factors on LR formation and the leading role of auxin in this stimulation. This thesis opens up perspectives to understand the effect of microbial molecules on the development of root organs

Adapting the Lateral Root-Inducible System to Medicago truncatula

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TableS1_primers

Table S1: Primer pairs used for Q-PCR on the Medicago truncatula gene

Table S5. List of genes from the first synergistic group

List of genes in the first synergistic group. The first column corresponds to the NimbleGen probe ID, the second to the equivalent Mt4 coding sequence reference when available. Affy ID corresponds to the more accurate medicago affymetrix probe, "other affy ID" to some related affymetrix probes. LFC: Log2 fold change.pval BH= p-value obtained using a Benjamini Hochberg FDR test. CT=control condition. NAA= 10-6M NAA; NF=10-7M NF; NAA+NF=10-6M NAA + 10-7M NF. All treatments are for 10h

Nod factors potentiate auxin signaling for transcriptional regulation and lateral root formation in Medicago truncatula

Nodulation (Nod) factors (NFs) are symbiotic molecules produced by rhizobia that are essential for establishment of the rhizobium-legume endosymbiosis. Purified NFs can stimulate lateral root formation (LRF) in Medicago truncatula, but little is known about the molecular mechanisms involved. Using a combination of reporter constructs, pharmacological and genetic approaches, we show that NFs act on early steps of LRF in M. truncatula, independently of the ethylene signaling pathway and of the cytokinin receptor MtCRE1, but in interaction with auxin. We conducted a whole-genome transcriptomic study upon NF and/or auxin treatments, using a lateral root inducible system adapted for M. truncatula. This revealed a large overlap between NF and auxin signaling and, more interestingly, synergistic interactions between these molecules. Three groups showing interaction effects were defined: group 1 contained more than 1500 genes responding specifically to the combinatorial treatment of NFs and auxin; group 2 comprised auxin-regulated genes whose expression was enhanced or antagonized by NFs; and in group 3 the expression of NF regulated genes was antagonized by auxin. Groups 1 and 2 were enriched in signaling and metabolic functions, which highlights important crosstalk between NF and auxin signaling for both developmental and symbiotic processes

Table S7. List of genes from the second synergistic group

List of genes in the second synergistic group. The first column corresponds to the NimbleGen probe ID, the second to the equivalent Mt4 coding sequence reference when available. Affy ID corresponds to the more accurate medicago affymetrix probe, "other affy ID" to some related affymetrix probes. LFC: Log2 fold change.pval BH= p-value obtained using a Benjamini Hochberg FDR test. CT=control condition. NAA= 10-6M NAA; NF=10-7M NF; NAA+NF=10-6M NAA + 10-7M NF. All treatments are for 10h

Table S6. List of genes differentially regulated by NAA+NF compared to NAA

List of genes differently expressed in the NF+NAA treatment compared to NAA condition. The first column corresponds to the NimbleGen probe ID, the second to the equivalent Mt4 coding sequence reference when available. Affy ID corresponds to the more accurate medicago affymetrix probe. pval BH= p-value obtained using a Benjamini Hochberg FDR test. LFC: Log2 fold change. CT=control condition. NAA= 10-6M NAA; NF=10-7M NF; NAA+NF=10-6M NAA + 10-7M NF. All treatments are for 10h

Table S8. List of genes with a potential hormonal or LRF function

List of differentially expressed genes with a potential hormonal function. The first column corresponds to the NimbleGen probe ID, the second to the equivalent Mt4 coding sequence reference when available. Affy ID corresponds to the more accurate medicago affymetrix probe. All genes had a significant adjusted pvalue in at least one of the conditions tested. LFC: Log2 fold change. CT=control condition. NAA= 10-6M NAA; NF=10-7M NF; NAA+NF=10-6M NAA + 10-7M NF. All treatments are for 10h

Table S3. List of genes differentially regulated by Nod Factors

Table S3. List of genes differently expressed in the NF treatment compared to control conditions. The first column corresponds to the NimbleGen probe ID, the second to the equivalent Mt4 coding sequence reference when available. Affy ID corresponds to the more accurate medicago affymetrix probe. LFC: Log2 fold change. CT=control condition. NAA= 10-6M NAA; NF=10-7M NF; NAA+NF=10-6M NAA + 10-7M NF. All treatments are for 10h