La symbiose mycorhizienne à arbuscules Rhizophagus irregularis / Medicago truncatula : données génomiques, transcriptomiques et microtranscriptomiques

Près de 80% des espèces végétales vivent en symbiose au niveau de leurs racines avec des champignons du groupe des Gloméromycètes. Cette symbiose mycorhizienne à arbuscules (MA) assure aux végétaux hôtes un apport minéral et hydrique essentiel à leur vie. Nous nous sommes attachés à définir des outils pour analyser la variabilité des génotypes du champignon MA modèle Rhizophagus irregularis à travers l'étude de son génome mitochondrial. Dans un deuxième volet d'étude, nous avons caractérisé des éléments régulateurs de la symbiose chez la plante hôte modèle Medicago truncatula (Mt). L'étude du génome mitochondrial fongique a permis de caractériser in silico les gènes présents, permettant la description de la chaine respiratoire chez ce champignon. Nous confirmons la présence d'une voie alternative de production d'énergie et mettons en évidence la probable capacité de ce champignon à réaliser une respiration cellulaire en condition anoxique. Par comparaison de génomes mitochondriaux séquencés à partir de différentes souches de cette espèce, des éléments de variabilité intraspécifique ont été identifiés, aboutissant à la définition de marqueurs de typage moléculaires des souches. L'analyse du microtranscriptome de Mt en condition symbiotique a été réalisée dans le cadre du projet Génoscope MIRMED. L'analyse des microtranscriptomes obtenus durant les étapes présymbiotiques et symbiotiques de la mycorhization a permis d'identifier des microARN différentiellement exprimés. Des phénotypes non-mycorhiziens associés à la surexpression de deux d'entre eux ont été obtenus soulignant l'intérêt de ces nombreux éléments régulateurs dans l'établissement de la symbiose.The arbuscular mycorrhizal symbiosis (AM) is an association between the roots of around 80% of plant species and fungi of the Glomeromycota group. This symbiosis ensures mineral and water intakes for the host plants. The main objectives of this thesis are the design of typing markers for the model AM fungus Rhizophagus irregularis (Ri) and the characterization of regulatory elements of the symbiosis in the model host plant Medicago truncatula (Mt). The first objective was addressed by the study of fungal mitochondriome and allowed the in silico identification of genes involved in energy production and respiration in the fungus and propose an organizational model of the respiratory chain of Ri. We confirm the presence of an alternative pathway of energy production and highlight the likely ability of the fungus to perform cellular respiration in anoxic conditions. Sequencing of mitochondrial genomes of different strains of this species allowed us to analyze the intraspecific variability and develop markers for molecular typing of the studied strains. The second part was carried out in the Génoscope project MIRMED with a study of the Mt microtranscriptome subjected to various stresses. Analysis of microtranscriptomes obtained during presymbiotic and symbiotic steps of mycorrhization allowed to identify differentially expressed microRNAs. Non-mycorrhizal phenotypes associated with the overexpression of two of them were obtained emphasizing the importance of many of these regulatory elements in the establishment of symbiosis

Identification of miRNAs linked to peanut nodule functional processes

microRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at post-transcriptional level. Thousands of miRNAs have been identified in legumes, but studies about miRNAs linked to peanut nodule functionality are scarce. In this work we analyzed transcriptional changes in peanut nodules to identify miRNAs involved in functional processes of these organs. We found 32 miRNAs precursors differentially expressed in nodules compared with roots, and predicted the potential targets of their corresponding mature miRNAs. Among them, 20 belong to 14 conserved miRNAs families and 12 are Arachis hypogaea-specific miRNAs. Expression levels of 3 miRNAs (ahy-miR399, ahy-miR159 and ahy-miR3508) were confirmed experimentally by qPCR. We also demonstrated that the expression of these miRNAs was not affected by inoculation of a biocontrol bacterium or a fungal pathogen. The catalogue of differentially expressed miRNA precursors and the expression of the corresponding mature miRNA potential targets in the nodules of A. hypogaea obtained in this work is a database of strong candidates, including A. hypogaea-specific miRNAs, for the regulation of the nodule functionality. The analysis of their role in this process will certainly lead to the characterization of essential regulators in these particular aeschynomenoid nodules.Fil: Figueredo, María Soledad. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Formey, Damien. Universidad Nacional Autónoma de México; MéxicoFil: Rodríguez, Johan. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Ibañez, Fernando Julio. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Hernández Delgado, Georgina. Universidad Nacional Autónoma de México; MéxicoFil: Fabra, Adriana Isidora. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentin

Argonaute proteins: Why are they so important for the legume–rhizobia symbiosis?

Unlike most other land plants, legumes can fulfill their nitrogen needs through the establishment of symbioses with nitrogen-fixing soil bacteria (rhizobia). Through this symbiosis, fixed nitrogen is incorporated into the food chain. Because of this ecological relevance, the genetic mechanisms underlying the establishment of the legume–rhizobia symbiosis (LRS) have been extensively studied over the past decades. During this time, different types of regulators of this symbiosis have been discovered and characterized. A growing number of studies have demonstrated the participation of different types of small RNAs, including microRNAs, in the different stages of this symbiosis. The involvement of small RNAs also indicates that Argonaute (AGO) proteins participate in the regulation of the LRS. However, despite this obvious role, the relevance of AGO proteins in the LRS has been overlooked and understudied. Here, we discuss and hypothesize the likely participation of AGO proteins in the regulation of the different steps that enable the establishment of the LRS. We also briefly review and discuss whether rhizobial symbiosis induces DNA damages in the legume host. Understanding the different levels of LRS regulation could lead to the development of improved nitrogen fixation efficiency to enhance sustainable agriculture, thereby reducing dependence on inorganic fertilizers.Fil: Valdés-López, Oswaldo. Universidad Nacional Autónoma de México, Escuela Nacional de Estudios Profesionales Iztacala; MéxicoFil: Formey, Damien. Universidad Nacional Autónoma de México; MéxicoFil: Isidra-Arellano, Mariel C.. Universidad Nacional Autónoma de México, Escuela Nacional de Estudios Profesionales Iztacala; México. Universidad Nacional Autónoma de México; MéxicoFil: Reyero-Saavedra, Maria del Rocio. Universidad Nacional Autónoma de México, Escuela Nacional de Estudios Profesionales Iztacala; MéxicoFil: Fernandez Göbel, Tadeo Francisco. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigaciones Agropecuarias. Unidad de Estudios Agropecuarios. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Unidad de Estudios Agropecuarios.; ArgentinaFil: Sánchez-Correa, Maria del Socorro. Universidad Nacional Autónoma de México, Escuela Nacional de Estudios Profesionales Iztacala; Méxic

Compounds Released by the Biocontrol Yeast Hanseniaspora opuntiae Protect Plants Against Corynespora cassiicola and Botrytis cinerea

Plant diseases induced by fungi are among the most important limiting factors during pre- and post-harvest food production. For decades, synthetic chemical fungicides have been used to control these diseases, however, increase on worldwide regulatory policies and the demand to reduce their application, have led to searching for new ecofriendly alternatives such as the biostimulants. The commercial application of yeasts as biocontrol agents, has shown low efficacy compared to synthetic fungicides, mostly due to the limited knowledge of the molecular mechanisms of yeast-induced responses. To date, only two genome-wide transcriptomic analyses have characterized the mode of action of biocontrols using the plant model Arabidopsis thaliana, missing, in our point of view, all its molecular and genomic potential. Here we describe that compounds released by the biocontrol yeast Hanseniaspora opuntiae (HoFs) can protect Glycine max and Arabidopsis thaliana plants against the broad host-range necrotrophic fungi Corynespora cassiicola and Botrytis cinerea. We show that HoFs have a long-lasting, dose-dependent local, and systemic effect against Botrytis cinerea. Additionally, we performed a genome-wide transcriptomic analysis to identify genes differentially expressed after application of HoFs in Arabidopsis thaliana. Our work provides novel and valuable information that can help researchers to improve HoFs efficacy in order for it to become an ecofriendly alternative to synthetic fungicides

Amphibian skin bacteria display antifungal activity and induce plant defense mechanisms against Botrytis cinerea

Botrytis cinerea is the causal agent of gray mold, which affects a wide variety of plant species. Chemical agents have been used to prevent the disease caused by this pathogenic fungus. However, their toxicity and reduced efficacy have encouraged the development of new biological control alternatives. Recent studies have shown that bacteria isolated from amphibian skin display antifungal activity against plant pathogens. However, the mechanisms by which these bacteria act to reduce the effects of B. cinerea are still unclear. From a diverse collection of amphibian skin bacteria, three proved effective in inhibiting the development of B. cinerea under in vitro conditions. Additionally, the individual application of each bacterium on the model plant Arabidopsis thaliana, Solanum lycopersicum and post-harvest blueberries significantly reduced the disease caused by B. cinerea. To understand the effect of bacteria on the host plant, we analyzed the transcriptomic profile of A. thaliana in the presence of the bacterium C32I and the fungus B. cinerea, revealing transcriptional regulation of defense-related hormonal pathways. Our study shows that bacteria from the amphibian skin can counteract the activity of B. cinerea by regulating the plant transcriptional responses

Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing

Background: MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events.Results: We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study.Conclusions: We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N2-fixing symbiotic nodules.Peer reviewedBiochemistry and Molecular Biolog

Comparative analysis of mitochondrial genomes of Rhizophagus irregularis – syn. Glomus irregulare – reveals a polymorphism induced by variability generating elements

Chantier qualité GAInternational audienceArbuscular mycorrhizal (AM) fungi are involved in one of the most widespread plant–fungus interactions. A number of studies on the population dynamics of AM fungi have used mitochondrial (mt) DNA sequences, and yet mt AM fungus genomes are poorly known. To date, four mt genomes of three species of AM fungi are available, among which are two from Rhizophagus irregularis. In order to study intra- and interstrain mt genome variability of R. irregularis, we sequenced and de novo assembled four additional mt genomes of this species. We used 454 pyrosequencing and Illumina technologies to directly sequence mt genomes from total genomic DNA.The mt genomes are unique within each strain. Interstrain divergences in genome size, as a result of highly polymorphic intergenic and intronic sequences, were observed. The polymorphism is brought about by three types of variability generating element (VGE): homing endonucleases, DNA polymerase domain-containing open reading frames and small inverted repeats. Based on VGE positioning, mt sequences and nuclear markers, two subclades of R. irregularis were characterized. The discovery of VGEs highlights the great intraspecific plasticity of the R. irregularis mt genome. VGEs allow the design of powerful mt markers for the typing and monitoring of R. irregularis strains in genetic and population studies

Knockdown of Lotus japonicus ROP3 alters the root symbiotic phenotype and alters the expression of genes involved in nutrient acquisition during both rhizobial and mycorrhizal symbioses

International audienceThe rhizobial and the arbuscular mycorrhizal symbioses are present on legume roots and lead to local and systemic transcriptional changes of common and specific plant genes. Among them, some small GTPase proteins called ROPs (Rho of plants) have been shown to be involved in the establishment of the legume-rhizobia interaction. In this study, we aimed to characterise the effects of LjROP3 knockdown in Lotus japonicus on plant physiology and expression of symbiosis-related genes after single and dual inoculation with rhizobia and arbuscular mycorrhizal fungus. In wild-type (Gifu) plants, the dual inoculation increased the shoot and root dry weight, nitrogen (derived from symbiosis) and phosphate content, and the number of arbuscules or nodules compared with single inoculation treatments. In addition, we observed a decrease in the expression of genes encoding the mycorrhizal transcription factors LjRAM1 and LjRAM2, and the downstream genes involved in ammonium (LjAMT2.2) and phosphate (LjPT4 and LjPT8) uptake by the plant at the arbuscule level when the dual inoculation was compared with fungal inoculation. An alteration in the expression of the Nod factor receptor LjNFR1, but not of LjNFR5, was measured in wild-type (Gifu) L. japonicus plants compared to rop3 plants under dual inoculation. We have also measured a reduction in the expression of genes encoding rhizobial and mycorrhizal transcription factors (LjNIN and LjRAM1), and of the downstream mycorrhizal genes involved in ammonium (LjAMT2.2) and phosphate (LjPT4 and LjPT8) uptake by the plant at the arbuscule level. In addition, the expression of AM fungal genes encoding nutrient transporters (known to be expressed at the arbuscule level) was also altered. In conclusion, despite altered expression of plant genes involved in the functioning of the symbioses, and associated with a reduction in the number of nodules and arbuscules, knockdown of LjROP3 did not alter plant growth and nutrition under dual inoculation, suggesting that the beneficial effects of the dual symbiosis were maintained