In the rhizosphere, strigolactones not only act as crucial signalling molecules in the communication of plants with parasitic weeds and arbuscular mycorrhiza, but they also play a key role in regulating different aspects of the root system. Here we investigated how strigolactones influence the root architecture of Medicago truncatula. We provide evidence that addition of the synthetic strigolactone analogue GR24 has an inhibitory effect on the lateral root density. Moreover, treatment with GR24 of Sinorhizobium meliloti-inoculated M. truncatula plants affects the nodule number both positively and negatively, depending on the concentration. Plants treated with 0.1 µM GR24 had a slightly increased number of nodules, whereas concentrations of 2 and 5 µM strongly reduced it. This effect was independent of the autoregulation of nodulation mechanism that is controlled by SUPER NUMERIC NODULE. Furthermore, we demonstrate that GR24 controls the nodule number through crosstalk with SICKLE-dependent ethylene signalling. Additionally, because the expression of the nodulation marker EARLY NODULATION11 was strongly reduced in GR24-treated plants, we concluded that strigolactones influence nodulation at a very early stage of the symbiotic interaction.

We provide new insights into the autoregulation of nodulation that allow us to better understand how the legume Medicago truncatula restricts further nodulation once enough nodules have been formed.

Strigolactones are plant metabolites that act as phytohormones and rhizosphere signals. Whereas most research on unraveling the action mechanisms of strigolactones is focused on plant shoots, we investigated proteome adaptation during strigolactone signaling in the roots of Arabidopsis thaliana. Through large-scale, time-resolved, and quantitative proteomics, the impact of the strigolactone analog rac-GR24 was elucidated on the root proteome of the wild type and the signaling mutant more axillary growth 2 (max2). Our study revealed a clear MAX2-dependent rac-GR24 response: an increase in abundance of enzymes involved in flavonol biosynthesis, which was reduced in the max2-1 mutant. Mass spectrometry-driven metabolite profiling and thin-layer chromatography experiments demonstrated that these changes in protein expression lead to the accumulation of specific flavonols. Moreover, quantitative RT-PCR revealed that the flavonolrelated protein expression profile was caused by rac-GR24-induced changes in transcript levels of the corresponding genes. This induction of flavonol production was shown to be activated by the two pure enantiomers that together make up rac-GR24. Finally, our data provide much needed clues concerning the multiple roles played by MAX2 in the roots and a comprehensive view of the rac-GR24-induced

SummaryA plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti.Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts.Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor.The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.

Running title: Strigolactones and root architecture in M. truncatula Date of submission: Number of figures: 6 Number of black and white figures: 5 Number of colour figures: 1 Number of tables: 0 Total word count: 7592 3 For the first time, strigolactones are shown to influence lateral root development and nodulation in Medicago truncatula. Nodulation is affected at early stages and crosstalks with ethylene signalling are involved.

Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. MtEFD (Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION) encodes an AP2/ERF (APETALA2/ETHYLENE RESPONSIVE FACTOR) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the MtRR4 (RESPONSE REGULATOR 4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a non-legume homologue of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralogue MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, CRISPR-Cas9 mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with down-regulation of a set of genes, including notably MtNF-YA1 (NUCLEAR FACTOR-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We therefore conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.