Composite Actinorhizal Plants with Transgenic Roots for the Study of Symbiotic Associations with Frankia

More than 200 species of dicotyledonous plants belonging to eight different families and 24 genera can establish actinorhizal symbiosis with the nitrogen-fixing soil actinomycete Frankia. Compared to the symbiotic interaction between legumes and rhizobia, little is known about the molecular basis of the infection process and nodule formation in actinorhizal plants. Here, we review a gene transfer system based on Agrobacterium rhizogenes that opens the possibility to rapidly analyze the function of candidate symbiotic genes. The transformation protocol generates ?composite plants? that consist of a nontransgenic aerial part with transformed hairy roots. Composite plants have already been obtained in three different species of actinorhizal plants, including the tropical tree species Casuarina glauca, the Patagonian shrub Discaria trinervis, and the nonwoody plant Datisca glomerata. The potential of this technique to advancing our understanding of the molecular mechanisms underlying infection by Frankia is demonstrated by functional analyses of symbiotic genes.Fil: Meriem Benabdoun, Faiza. Université Mentouri; ArgeliaFil: Nambiar Veetil, Mathish. No especifíca;Fil: Imanishi, Leandro Ezequiel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Svistoonoff, Sergio. No especifíca;Fil: Ykhlef, Nadia. Université Mentouri; ArgeliaFil: Gherbi, Hassen. No especifíca;Fil: Franche, Claudine. No especifíca

Transformed Hairy Roots of Discaria trinervis: A Valuable Tool for Studying Actinorhizal Symbiosis in the Context of Intercellular Infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.Fil: Imanishi, Leandro Ezequiel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vayssières, Alice. Institut de Recherche Pour Le Developpement; FranciaFil: Franche, Claudine. Institut de Recherche Pour Le Developpement; FranciaFil: Bogusz, Didier. Institut de Recherche Pour Le Developpement; FranciaFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Svistoonoff, Sergio. Institut de Recherche Pour Le Developpement; Franci

Role of auxin during intercellular infection of Discaria trinervis by Frankia

ogen-fixing nodules induced by Frankia in the actinorhizal plant Discaria trinervis result from a primitive intercellular root invasion pathway that does not involve root hair deformation and infection threads. Here, we analyzed the role of auxin in this intercellular infection pathway at the molecular level and compared it with our previous work in the intracellular infected actinorhizal plant Casuarina glauca. Immunolocalisation experiments showed that auxin accumulated in Frankia-infected cells in both systems. We then characterized the expression of auxin transporters in D. trinervis nodules. No activation of the heterologous CgAUX1 promoter was detected in infected cells in D. trinervis. These results were confirmed with the endogenous D. trinervis gene, DtAUX1. However, DtAUX1 was expressed in the nodule meristem. Consistently, transgenic D. trinervis plants containing the auxin response marker DR5:VENUS showed expression of the reporter gene in the meristem. Immunolocalisation experiments using an antibody against the auxin efflux carrier PIN1, revealed the presence of this transporter in the plasma membrane of infected cells. Finally, we used in silico cellular models to analyse auxin fluxes in D. trinervis nodules. Our results point to the existence of divergent roles of auxin in intercellularly- and intracellularly-infected actinorhizal plants, an ancestral infection pathways leading to root nodule symbioses

Symbiotic signaling in actinorhizal symbioses

Actinorhizal symbioses are mutualistic associations between plants belonging to eight angiosperm families and soil bacteria of the genus Frankia. These interactions lead to the formation of new root organs, actinorhizal nodules, where the bacteria are hosted and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. It involves an elaborate signaling between both partners of the symbiosis. In recent years, our knowledge of this signaling pathway has increased tremendously thanks to a series of technical breakthroughs including the sequencing of three Frankia genomes [1] and the implementation of RNA silencing technology for two actinorhizal species. In this review, we describe all these recent advances, current researches on symbiotic signaling in actinorhizal symbioses and give some potential future research directions

Chitotetraose activates the fungal-dependent endosymbiotic signaling pathway in actinorhizal plant species

Mutualistic plant-microbe associations are widespread in natural ecosystems and have made major contributions throughout the evolutionary history of terrestrial plants. Amongst the most remarkable of these are the so-called root endosymbioses, resulting from the intra-cellular colonization of host tissues by either arbuscular mycorrhizal (AM) fungi or nitrogen-fixing bacteria that both provide key nutrients to the host in exchange for energy-rich photo-synthates. Actinorhizal host plants, members of the Eurosid 1 clade, are able to associate with both AM fungi and nitrogen-fixing actinomycetes known as Frankia. Currently, little is known about the molecular signaling that allows these plants to recognize their fungal and bacterial partners. In this article, we describe the use of an in vivo Ca 2+ reporter to identify symbiotic signaling responses to AM fungi in roots of both Casuarina glauca and Discaria tri-nervis, actinorhizal species with contrasting modes of Frankia colonization. This approach has revealed that, for both actinorhizal hosts, the short-chain chitin oligomer chitotetraose is able to mimic AM fungal exudates in activating the conserved symbiosis signaling pathway (CSSP) in epidermal root cells targeted by AM fungi. These results mirror findings in other AM host plants including legumes and the monocot rice. In addition, we show that chitote-traose is a more efficient elicitor of CSSP activation compared to AM fungal lipo-chitooligo-saccharides. These findings reinforce the likely role of short-chain chitin oligomers during the initial stages of the AM association, and are discussed in relation to both our current knowledge about molecular signaling during Frankia recognition as well as the different microsymbiont root colonization mechanisms employed by actinorhizal hosts. PLOS ONE | https://doi.org/10.1371/journal.pone

Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization

Nitrogen‐fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little‐researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection‐associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing in vivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin‐like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D. trinervis outer root tissues. This is accompanied by nucleo‐cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen‐fixing rhizobia.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Fournier, Joëlle. Université de Toulouse. LIPM; Francia. INRA-CNRS; FranciaFil: Imanishi, Leandro Ezequiel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chabaud, Mireille. Université de Toulouse. LIPM; Francia. INRA-CNRS; FranciaFil: Abdou‐Pavy, Iltaf. Université de Toulouse. LIPM; Francia. INRA-CNRS; FranciaFil: Genre, Andrea. University of Torino. Department of Life Sciences and Systems Biology; ItaliaFil: Brichet, Lukas. Université de Toulouse. LIPM; Francia. INRA-CNRS; FranciaFil: Lascano, Hernan Ramiro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Muñoz, Nacira Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Vayssières, Alice. IRD-INRA-CIRAD-Université de Montpellier-Supagro. Laboratoire des Symbioses Tropicales et Méditerranéennes; FranciaFil: Pirolles, Elodie. IRD-INRA-CIRAD-Université de Montpellier-Supagro. Laboratoire des Symbioses Tropicales et Méditerranéennes; FranciaFil: Brottier, Laurent. IRD-INRA-CIRAD-Université de Montpellier-Supagro. Laboratoire des Symbioses Tropicales et Méditerranéennes; FranciaFil: Gherbi, Hassen. IRD-INRA-CIRAD-Université de Montpellier-Supagro. Laboratoire des Symbioses Tropicales et Méditerranéennes; FranciaFil: Hocher, Valérie. IRD-INRA-CIRAD-Université de Montpellier-Supagro. Laboratoire des Symbioses Tropicales et Méditerranéennes; FranciaFil: Svistoonoff, Sergio. IRD-INRA-CIRAD-Université de Montpellier-Supagro. Laboratoire des Symbioses Tropicales et Méditerranéennes; Francia. Centre de Recherche de Bel Air. Laboratoire Mixte International Adaptation des Plantes et Microorganismes Associés aux Stress Environnementaux; Senegal. Institut de Recherche pour le Développement-Institut Sénégalais des Recherches Agricoles-Université Cheikh Anta Diop. Laboratoire Commun de Microbiologie; SenegalFil: Barker, David G. Université de Toulouse. LIPM; Francia. INRA-CNRS; FranciaFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis

The root nodule symbiosis of plants with nitrogen-fixing bacteria affects global nitrogen cycles and food production but is restricted to a subset of genera within a single clade of flowering plants. To explore the genetic basis for this scattered occurrence, we sequenced the genomes of 10 plant species covering the diversity of nodule morphotypes, bacterial symbionts, and infection strategies. In a genome-wide comparative analysis of a total of 37 plant species, we discovered signatures of multiple independent loss-of-function events in the indispensable symbiotic regulator NODULE INCEPTION in 10 of 13 genomes of nonnodulating species within this clade. The discovery that multiple independent losses shaped the present-day distribution of nitrogen-fixing root nodule symbiosis in plants reveals a phylogenetically wider distribution in evolutionary history and a so-far-underestimated selection pressure against this symbiosis.Fil: Griesmann, Maximilian. Ludwig Maximilians Universitat; AlemaniaFil: Chang, Yue. No especifíca;Fil: Liu, Xin. No especifíca;Fil: Song, Yue. No especifíca;Fil: Haberer, Georg. Helmholtz Center Munich; AlemaniaFil: Crook, Matthew B.. Weber State University; Estados UnidosFil: Billault-Penneteau, Benjamin. Ludwig Maximilians Universitat; AlemaniaFil: Lauressergues, Dominique. Université de Toulouse; FranciaFil: Keller, Jean. Université de Toulouse; FranciaFil: Imanishi, Leandro Ezequiel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Roswanjaya, Yuda Purwana. University of Agriculture Wageningen; Países BajosFil: Kohlen, Wouter. University of Agriculture Wageningen; Países BajosFil: Pujic, Petar. Université de Lyon; FranciaFil: Battenberg, Kai. University of California at Davis; Estados UnidosFil: Alloisio, Nicole. No especifíca;Fil: Liang, Yuhu. No especifíca;Fil: Hilhorst, Henk. No especifíca;Fil: Salgado, Marco G.. Stockholms Universitet; SueciaFil: Hocher, Valerie. Université Montpellier II; FranciaFil: Gherbi, Hassen. Université Montpellier II; FranciaFil: Svistoonoff, Sergio. Université Montpellier II; FranciaFil: Doyle, Jeff J.. Cornell University; Estados UnidosFil: He, Shixu. No especifíca;Fil: Xu, Yan. China National Genebank; ChinaFil: Xu, Shanyun. China National Genebank; ChinaFil: Qu, Jing. China National Genebank; ChinaFil: Gao, Qiang. No especifíca;Fil: Fang, Xiaodong. No especifíca;Fil: Fu, Yuan. China National Genebank; ChinaFil: Normand, Philippe. Universite Lyon 2; FranciaFil: Berry, Alison M.. University of California at Davis; Estados UnidosFil: Wall, Luis Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ané, Jean Michel. University of Wisconsin; Estados UnidosFil: Pawlowski, Katharina. Stockholms Universitet; SueciaFil: Xu, Xun. China National Genebank; ChinaFil: Yang, Huanming. James D. Watson Institute Of Genome Sciences; ChinaFil: Spannagl, Manuel. Helmholtz Center Munich German Research Center For Environmental Health; AlemaniaFil: Mayer, Klaus F.X.. Helmholtz Center Munich German Research Center For Environmental Health; AlemaniaFil: Wong, Gane Ka-Shu. University of Alberta; CanadáFil: Parniske, Martin. Ludwig Maximilians Universitat; AlemaniaFil: Delaux, Pierre Marc. No especifíca;Fil: Cheng, Shifeng. China National Genebank; Chin