Didier Reinhardt

Titular Prof.

photo

 didier.reinhardt@unifr.ch
 +41 26 300 8818
 https://orcid.org/0000-0003-3495-6783

Principle Investigator (PI); Radiosafety Officer; Biosafety Officer

Research and publications

  • Publications Didier Reinhardt
    66 publications

    Chromosome-level phased genome assembly of the argan treeSideroxylon spinosumL
    Laurent Falquet, Mohamed Hijri, Ahmed Qaddoury, Didier Reinhardt, Ivan D. Mateus, Ivan B Mateus, Abdellatif Essahibi, Pamela Nicholson, (2025) | Preprint

  • Research projects

    Role of leghemoglobin phosphorylation in nitrogen fixation in legumes during root-nodule symbiosis (RNS)
    Status: Ongoing

    Start 01.04.2025
    End 31.03.2029
    Funding SNSF  Open project sheet
    The systematic use of plant symbioses is an indispensable element of strategies towards sustainable agriculture. The symbiosis of legumes with N2-fixing bacteria (rhizobia) is an important contribution to crop production and natural soil fertility. A central element of symbiotic nitrogen fixation (SNF) is oxygen (O2) which is required for bacterial respiration but, paradoxically, is also toxic to the N2-fixing nitrogenase complex. Free O2 levels in nodules are regulated by leghemoglobins (Lbs) which function as a buffer to maintain a steadily low O2 supply for bacterial metabolism. Besides its function in controlling bacterial O2 supply, Lb also creates the microoxic conditions that are required to activate symbiotic gene expression in the host; therefore, O2 also acts as a signal in nodules. Recent evidence has shown that Lb can be phosphorylated during nodule symbiosis in several legumes. Also, we have observed characteristic phosphorylation patterns under conditions that are associated with inhibition of SNF. Hence, the question arises whether Lb phosphorylation is functionally relevant. In this project, we will examine various Lb versions with mutations that render them either phosphorylation-defective (substitution of Ser by Ala) or that transform them into a constitutive phosphomimetic state (substitution of Ser by Asp). We will do this for a representative Lb (Lb2) of Medicago truncatula (model legume of indeterminate nodulation) and for a representative Lb (Lb2) of Lotus japonicus (model legume of determinate nodulation). The biological function of altered Lb versions (non-phosphorylatable and phosphomimetic) in nodulation will be assessed by complementa-tion of an Lb-defective L. japonicus triple mutant (lb123). Altered Lbs under the control of an endogenous Lb promoter will be expressed in transgenic hairy roots, and nodulation, SNF, and plant performance, as well as nodule morphology, will be assessed. Finally, the stability of the mutant Lb versions will be tested during nodulation in transgenic hairy roots. Lb versions that result in an interesting phenotype in the hairy root assay with rhizobia will be biochemically characterized in a recombinant form by purification and assessing the effects of phosphorylation on O2 affinity, O2 binding capacity, and Lb protein stability. Ultimately, this project will increase our basic understanding of Lb function in symbiosis and will highlight possible points of control that allow plants to reduce O2 supply to the bacterial endosymbiont. In addition, it will result in the identification of new Lb-versions with improved characteristics for modern agriculture, relative to natural Lbs that evolved for optimal functioning under conditions in natural environments. Finally, our work may also shed light on functionality of hemoglobins in general, with potential relevance for human health.

    Role of hydrolases and N-acetylglucosamine transporters as modulators of symbiotic communication in nodulation and arbuscular mycorrhiza
    Status: Ongoing

    Start 01.04.2022
    End 31.03.2026
    Funding SNSF  Open project sheet
    A central component of symbiotic communication in interactions between plants and mutualistic microbes such as rhizobia and arbuscular mycorrhizal fungi involves lipochitooligosaccharides (LCO) and chitooligosaccharides (CO) produced by the microbial partners. Specificity in the interactions can depend on different LCO constituents and on lysin-motif receptor-like kinases (LysM-RLK) involved in signal perception in the host. An additional and only recently appreciated level of control are enzymes that modify and hydrolyze LCO. Such hydrolases are related to chitinases, and they are thought to contribute to specificity, symbiotic compatibility and nodule morphogenesis in the root nodule symbiosis (RNS) of the legumes. Chitinases have diversified in several major types (I-IV) with multiple subfamilies, of which some are specifically expressed in arbuscular mycorrhizal symbiosis (AMS), suggesting that they may be involved in symbiosis. LCO-degrading enzymes such as NOD FACTOR HYDROLASE1 of Medicago truncatula (MtNFH1) are required for normal nodulation. However, the role of LCO and CO cleavage into monomeric N-acetylglucosamine (GlcNAc) has not been investigated in the past. Here, we propose to characterize the orthologous copies of a ß-N-acetylhexosaminidase (HEXO2) in two symbiosis model species, M. truncatula, and Petunia hybrida, where the former engages in both, RNS and AMS, whereas the latter has only AMS. Preliminary experiments indicated that MtHEXO2 impinges on AMS. We plan to characterize MtHEXO2 and PhHEXO2 at the genetic and biochemical levels in order to explore their function in symbiosis. In addition, we will functionally and biochemically characterize predicted GlcNAc transporters from M. truncatula and P. hybrida, which are induced during symbiosis. GlcNAc is the ultimate hydrolytic degradation product of LCO and CO produced by chitinases and ß-N-acetylhexosaminidases such as HEXO2. GlcNAc has been hypothesized to act as a symbiotic signal in AMS of monocots (maize and rice) in which a first GlcNAc transporter has been identified. This project will establish the role of hydrolytic enzymes in LCO/CO signaling in RNS and AMS. We will address the question of similarity and specificity in these very different symbioses, and we will test whether GlcNAc transporters have a conserved function in the dicot species M. truncatula and P. hybrida. Taken together, this work will provide information on LCO/CO signaling in RNS and AMS, particularly on the fate of LCO/CO in the symbiotic interface and the role of LCO/CO degradation products in symbiosis. Finally, our dual approach with a legume (M. truncatula) and a Solanaceous model species (P. hybrida) will shed light on conservation in symbiotic signaling, and on evolutionary changes associated with the emergence of RNS in the context of AMS.

    How do legume hosts impose selection for mutualist quality on their rhizobial patners during nodulation?
    Status: Ongoing

    Start 01.06.2021
    End 31.05.2025
    Funding SNSF  Open project sheet
    The nitrogen-fixing root nodule symbiosis (RNS) of plants (mostly Fabaceae) with bacteria collectively referred to as rhizobia alleviates nitrogen limitation in agricultural and natural environments, and accounts for approximately 50% of the fixed nitrogen that is introduced in the global nitrogen cycles. Since RNS is facultative for both partners, it is inherently unstable, therefore, the mutualistic bacteria can turn into parasitic cheaters that enjoy carbon supply from the plant without reciprocal supply of fixed nitrogen. Natural populations of rhizobia can exhibit a wide range of mutualistic potential, including cheaters that are completely unable to fix nitrogen. Plants can avoid to become exploited by either selecting good partners (pre-infection mechanisms) or by sanctioning bad mutualists (post-infection mechanisms), but potential mechanisms remain unclear. A special situation arises in nodulation, due to the fact that one or a few bacterial founder cells establish large rhizobial population with millions of individual cells, thus inevitably giving rise to many spontaneous mutations in genes required for N-fixation. Since such non-fixing clones have no metabolic burden from the energy-intensive N-fixation process, they have more resources for growth and proliferation and therefore could potentially outcompete the N-fixing population. Hence, the question arises whether plants can detect such parasitic rhizobial clones and inhibit them from overgrowing the mutualistic original clone. It is presently unknown, how plants may be able to detect cheaters, and how these could be sanctioned. We hypothesize that negative selection by plants of ineffective rhizobia should result in a characteristic signature at the level of the bacterial genome. If bacterial clones with mutations in N-fixation genes are sanctioned, then mutations should be particularly infrequent in these genes in rhizobia of fully established nodules. Such positive selection for functional N-fixation genes would be expected to result in predictable signatures in the rhizobial genome. In a first approach, we have used a publicly available dataset to test this hypothesis. Using the complete transcriptome of nodules of Medicago truncatula and its symbiont Sinorhizobium meliloti (synonym. Ensifer meliloti) we have assessed the level of polymorphisms over the entire bacterial genome, encompassing the chromosome, symbiotic plasmid A (pSymA) and pSymB. We find that a large gene cluster on pSymA shows significantly reduced levels of polymorphisms. This region encompasses the majority of nitrogen fixation (nif- and fix-genes), indicating that they may be under strong purifying selection during nodulation. Here, we propose to use genetic and genomic approaches in combination with cytological, microscopic, and metabolomic analyses, and with mathematical modeling to study how host plants shape their rhizobial populations. Furthermore, we propose forward genetic strategies to identify host factors required for selection of the bacterial community. This work addresses at the same time fundamental questions concerning the biology of nodulation and symbioses in general, as well as applied issues related to agrosystems involving managed symbiotic associations.

    C14.0074: Analysis and manipulation of strigolactone biosynthesis in petunia
    Status: Completed

    Start 01.01.2017
    End 31.01.2017
    Funding SNSF  Open project sheet

    Establishment and functioning of arbuscular mycorrhizal symbiosis
    Status: Completed

    Start 01.10.2016
    End 31.07.2020
    Funding SNSF  Open project sheet
    Most land plants worldwide live in mutualistic associations known as arbuscular mycorrhiza (AM) with soil fungi of the order Glomeromycota. AM improve the fitness of both symbiotic partners, they stabilize the diversity of plant communities, and they enable plants to colonize extremely harsh environments. In recent years, the genetic basis of early symbiotic communication in AM has been deciphered in considerable detail, and AM-associated transcriptional reprogramming has been described in several AM host species. However, little is known about the mechanisms involved in intracellular accommodation of the fungal partner, and it is not clear how persistent compatibility and symbiotic function in AM is ensured. To obtain insight in these mechanisms, we have carried out several forward genetic screens in the host model species Petunia hybrida for mutants defective at the intracellular stages of AM. This approach has led to the identification of two important genes, VAPYRIN and REQUIRED FOR ARBUSCULAR MYCORRHIZA1 (RAM1), that are required for cellular infection, arbuscule development, and symbiotic functioning. VAPYRIN encodes a yet unexplored protein that consists of two protein:protein interaction modules, an N-terminal VAP domain and a C-terminal ankyrin domain with 11 ankyrin repeats. VAPYRIN localizes to a highly mobile subcellular compartment of unknown identity and function (VAPYRIN-bodies). Interestingly, related VAP and ankyrin domains of mammalian proteins are known to interact with membranes, and to be implicated in cellular transport processes and communication. RAM1, on the other hand, encodes a GRAS transcription factor that regulates many of the conserved AM-related genes, in particular the phosphate transporter PT4, the glycerol-3-phosphate acyltransferase (GPAT) RAM2, and two ABC transporters known as STUNTED ARBUSCULE (STR), and STR2, all of them being essential for AM. While the function of VAPYRIN can best be understood by characterizing its resident compartment and its interacting partner proteins, the function of RAM1 has to be explored via its target genes.

    Training in molecular biology Schweizerische Kommission für Molekularbiologie (SKMB)
    Status: Completed

    Start 01.10.2014
    End 31.12.2017
    Funding SNSF  Open project sheet
    Training in molecular biology Swiss Committee for Molecular Biology (SKMB)

    Recognition and intracellular accommodation of arbuscular mycorrhizal fungi in petunia
    Status: Completed

    Start 01.04.2012
    End 30.09.2016
    Funding SNSF  Open project sheet
    Most land plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi, which have profound consequences for plant fitness, and for the composition and stability of wild plant communities. A limited number of plant species (mostly legumes) harbor also rhizobacteria, resulting in root nodule symbiosis (RNS). A wealth of cell biological and genetic evidence documents an active role of plants in the entry and accommodation of AM fungi and rhizobia within their root cells. However, apart from the well characterized common symbiosis signalling pathway (common Sym pathway), that is shared between AM and RNS, little is known about the mechanisms involved in intracellular accommodation of endosymbitic microbes. The first event in the recognition of rhizobia by plants is the perception of bacterial nod factors by a pair of LysM-type nod factor receptors (NFR1 and NFR5). Since NFR homologues occur also in non-nodulating species, it has been hypothesized that they may also function in the perception of an AM fungal signal, the elusive myc factor. We have recently isolated the petunia homologues of NFR1 and NFR5 (PhNFR1 and PhNFR5). Interestingly, PhNFR5 is down-regulated by high phosphate supply, a condition under which AM are suppressed. Hence, PhNFR5 is a good candidate for a positive regulator of AM. These results, and the current petunia genome initiative opens the possibility to systematically assess the role of LysM receptor kinases in AM of petunia. Forward genetic screens for petunia mutants with defects in intracellular accommodation of AM fungi revealed a number of loci, of which the gene PENETRATION AND ARBUSCULE MORPHOGENESIS1 (PAM1) has recently been cloned and characterized in detail. The predicted PAM1 protein has a novel plant-specific structure and associates with the membranes of a cellular sub-compartment of unknown identity. The occurrence of a close homologue in the moss Physcomitrella patens points to an evolutionary ancient origin of PAM1 in early land plants. The study of PpPAM1 function in the non-mycorrhizal moss will provide insights into the ancestral cellular pathways that were recruited for intracellular symbiont accommodation during evolution of AM symbiosis.

    Dynamics of mycorrhiza formation
    Status: Completed

    Start 01.04.2009
    End 31.03.2013
    Funding SNSF  Open project sheet

    Plant Growth in a Changing Environment
    Status: Completed

    Start 01.06.2008
    End 31.05.2011
    Funding Other 

    Plant Growth in a Changing Environment
    Status: Completed

    Start 01.06.2008
    End 31.05.2011
    Funding Other 
    In diesem Projekt untersuchen PD Dr. Didier Reinhardt, Prof. Jean-Pierre Gabriel und Prof. Christian Mazza (Departement Mathematik), wie Pflanzenzellen das Wachstum des ganzen Organismusses beeinflussen. Das Ziel ist zu verstehen, welche Proteine und Zellorganellen den Wachstumsprozess steuern und vollziehen. Anhand von mathematischen Modellen und Computersimulationen wird die Plausibilität der Hypothesen geprüft und laufend verbessert.

    Genetic and cellular analysis of the AM symbiosis in petunia and tobacco
    Status: Completed

    Start 01.03.2008
    End 31.03.2012
    Funding SNSF  Open project sheet
    The arbuscular mycorrhizal (AM) symbiosis is under genetic control by the plant host. This notion is supported by a large number of legume mutants in which the interaction is blocked early during the infection process, and by cell biological studies that revealed an active cellular program required for fungal entry into rhizodermal cells. This symbiotic program comprises a sequence of events which is shared with the root nodule symbiosis, and hence is referred to as the common symbiosis signaling pathway (common SYM pathway). We have isolated the Petunia homologues of several of the common SYM genes, and we are in the process of silencing them by RNAi and by transposon insertion mutagenesis. The common SYM pathway is well characterized in legumes, and the molecular and cellular events, with calcium signaling at the core of the pathway, are being deciphered in detail. Less is known, however, about the signaling events up- and downstream of the common SYM pathway. We have performed direct genetic screens for mutants with defects in AM development and we have characterized one mutant, penetration and arbuscule morphogenesis (pam1), which has a defect in intracellular accomodation of AM fungi. Since pam1 permits a certain level of intraradical colonization and has a prominent defect in arbuscule formation, PAM1 might function downstream of the common SYM pathway. Additional five mutants with symbiotic defects are being characterized. A central aspect of the AM symbiosis is the intimate contact between the symbiotic partners during the intracellular stages of the interaction. However, their cytoplasm remains separate at all stages of the interaction. This is achieved by membrane systems of the plant that contain the intracellular fungal structures, the perihyphal membrane (PHM) around the hyphal coils in rhizodermal cells, and the periarbuscular membrane (PAM) around the arbuscules in cortical cells. Here, we refer to these membranes collectively as peri-fungal membranes (PFM). The PFM are involved in nutrient exchange and, presumably, in communication between the symbionts. However, little is known about the origin, the identity, the composition, and the dynamics of the PFM. We have established transgenic tobacco lines expressing GFP- and YFP-tagged marker proteins that are localized to different intracellular membrane compartments. Preliminary analysis suggests that root cells have various vacuolar compartments: the large central vacuole, intermediate peripheral vacuoles, and mobile vesicles. During cell invasion, the hyphal coils and arbuscules of the fungus become surrounded by host membranes that are at least partially marked by the fluorescent fusion proteins. The mobile vesicles appear to be associated with invading hyphae and may serve to deliver membrane material to the PFM and/or cargo material to the apoplastic space between the symbionts.

    Grapevine Diseases and Resistance Mechanisms
    Status: Completed

    Start 01.04.2005
    End 31.03.2009
    Funding SNSF  Open project sheet

    Genetic and Nutritional regulation of the AM symbiosis
    Status: Completed

    Start 01.02.2005
    End 29.05.2009
    Funding Unifr 

    Genetic analysis of the arbuscular mycorrhizal symbiosis in Petunia
    Status: Completed

    Start 01.03.2004
    End 29.02.2008
    Funding SNSF  Open project sheet

    Molecular genetics of the arbuscular mycorrhizal symbiosis in a new model system: Petunia hybrida
    Status: Completed

    Start 01.02.2004
    End 24.09.2007
    Funding Unifr 

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