Pascale Fournier

Transcriptomics of Actinorhizal Symbioses Reveals Homologs of the Whole Common Symbiotic Signaling Cascade

Comparative transcriptomics of two actinorhizal symbiotic plants, Casuarina glauca and Alnus glutinosa, was used to gain insight into their symbiotic programs triggered following contact with the nitrogen-fixing actinobacterium Frankia. Approximately 14,000 unigenes were recovered in roots and 3-week-old nodules of each of the two species. A transcriptomic array was designed to monitor changes in expression levels between roots and nodules, enabling the identification of up- and down-regulated genes as well as root- and nodule-specific genes. The expression levels of several genes emblematic of symbiosis were confirmed by quantitative polymerase chain reaction. As expected, several genes related to carbon and nitrogen exchange, defense against pathogens, or stress resistance were strongly regulated. Furthermore, homolog genes of the common and nodule-specific signaling pathways known in legumes were identified in the two actinorhizal symbiotic plants. The conservation of the host plant signaling pathway is all the more surprising in light of the lack of canonical nod genes in the genomes of its bacterial symbiont, Frankia. The evolutionary pattern emerging from these studies reinforces the hypothesis of a common genetic ancestor of the Fabid (Eurosid I) nodulating clade with a genetic predisposition for nodulation.

The Frankia alni symbiotic transcriptome.

The actinobacteria Frankia spp. are able to induce the formation of nodules on the roots of a large spectrum of actinorhizal plants, where they convert dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living Frankia alni cells and on Alnus glutinosa nodule bacteria, using whole-genome microarrays. Distribution of nodule-induced genes on the genome was found to be mostly over regions with high synteny between three Frankia spp. genomes, while nodule-repressed genes, which were mostly hypothetical and not conserved, were spread around the genome. Genes known to be related to nitrogen fixation were highly induced, nif (nitrogenase), hup2 (hydrogenase uptake), suf (sulfur-iron cluster), and shc (hopanoids synthesis). The expression of genes involved in ammonium assimilation and transport was strongly modified, suggesting that bacteria ammonium assimilation was limited. Genes involved in particular in transcriptional regulation, signaling processes, protein drug export, protein secretion, lipopolysaccharide, and peptidoglycan biosynthesis that may play a role in symbiosis were also identified. We also showed that this Frankia symbiotic transcriptome was highly similar among phylogenetically distant plant families Betulaceae and Myricaceae. Finally, comparison with rhizobia transcriptome suggested that F. alni is metabolically more active in symbiosis than rhizobia.

Differential Effects of Rare Specific Flavonoids on Compatible and Incompatible Strains in the Myrica gale-Frankia Actinorhizal Symbiosis

ABSTRACT Plant secondary metabolites, and specifically phenolics, play important roles when plants interact with their environment and can act as weapons or positive signals during biotic interactions. One such interaction, the establishment of mutualistic nitrogen-fixing symbioses, typically involves phenolic-based recognition mechanisms between host plants and bacterial symbionts during the early stages of interaction. While these mechanisms are well studied in the rhizobia-legume symbiosis, little is known about the role of plant phenolics in the symbiosis between actinorhizal plants and Frankia genus strains. In this study, the responsiveness of Frankia strains to plant phenolics was correlated with their symbiotic compatibility. We used Myrica gale, a host species with narrow symbiont specificity, and a set of compatible and noncompatible Frankia strains. M. gale fruit exudate phenolics were extracted, and 8 dominant molecules were purified and identified as flavonoids by high-resolution spectroscopic techniques. Total fruit exudates, along with two purified dihydrochalcone molecules, induced modifications of bacterial growth and nitrogen fixation according to the symbiotic specificity of strains, enhancing compatible strains and inhibiting incompatible ones. Candidate genes involved in these effects were identified by a global transcriptomic approach using ACN14a strain whole-genome microarrays. Fruit exudates induced differential expression of 22 genes involved mostly in oxidative stress response and drug resistance, along with the overexpression of a whiB transcriptional regulator. This work provides evidence for the involvement of plant secondary metabolites in determining symbiotic specificity and expands our understanding of the mechanisms, leading to the establishment of actinorhizal symbioses.

Candidatus Frankia Datiscae Dg1, the Actinobacterial Microsymbiont of Datisca glomerata, Expresses the Canonical nod Genes nodABC in Symbiosis with Its Host Plant

Frankia strains are nitrogen-fixing soil actinobacteria that can form root symbioses with actinorhizal plants. Phylogenetically, symbiotic frankiae can be divided into three clusters, and this division also corresponds to host specificity groups. The strains of cluster II which form symbioses with actinorhizal Rosales and Cucurbitales, thus displaying a broad host range, show suprisingly low genetic diversity and to date can not be cultured. The genome of the first representative of this cluster, Candidatus Frankia datiscae Dg1 (Dg1), a microsymbiont of Datisca glomerata, was recently sequenced. A phylogenetic analysis of 50 different housekeeping genes of Dg1 and three published Frankia genomes showed that cluster II is basal among the symbiotic Frankia clusters. Detailed analysis showed that nodules of D. glomerata, independent of the origin of the inoculum, contain several closely related cluster II Frankia operational taxonomic units. Actinorhizal plants and legumes both belong to the nitrogen-fixing plant clade, and bacterial signaling in both groups involves the common symbiotic pathway also used by arbuscular mycorrhizal fungi. However, so far, no molecules resembling rhizobial Nod factors could be isolated from Frankia cultures. Alone among Frankia genomes available to date, the genome of Dg1 contains the canonical nod genes nodA, nodB and nodC known from rhizobia, and these genes are arranged in two operons which are expressed in D. glomerata nodules. Furthermore, Frankia Dg1 nodC was able to partially complement a Rhizobium leguminosarum A34 nodC::Tn5 mutant. Phylogenetic analysis showed that Dg1 Nod proteins are positioned at the root of both α- and β-rhizobial NodABC proteins. NodA-like acyl transferases were found across the phylum Actinobacteria, but among Proteobacteria only in nodulators. Taken together, our evidence indicates an Actinobacterial origin of rhizobial Nod factors.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

Genome Sequence of the Atypical Symbiotic Frankia R43 Strain, a Nitrogen-Fixing and Hydrogen-Producing Actinobacterium

ABSTRACT Frankia strain R43 is a nitrogen-fixing and hydrogen-producing symbiotic actinobacterium that was isolated from nodules of Casuarina cunninghamiana but infects only Elaeagnaceae. This communication reports the genome of the strain R43 and provides insights into the microbe genomics and physiological potentials.

In‐planta sporulation phenotype: a major life history trait to understand the evolution of Alnus‐infective Frankia strains

Two major types of Frankia strains are usually recognized, based on the ability to sporulate in-planta: spore-positive (Sp+) and spore-negative (Sp-). We carried out a study of Sp+ and Sp- Frankia strains based on nodules collected on Alnus glutinosa, Alnus incana and Alnus viridis. The nodules were phenotyped using improved histology methods, and endophytic Frankia strain genotype was determined using a multilocus sequence analysis approach. An additional sampling was done to assess the relation between Sp+ phenotype frequency and genetic diversity of Frankia strains at the alder stand scale. Our results revealed that (i) Sp+ and Sp- Alnus-infective Frankia strains are genetically different even when sampled from the same alder stand and the same host-plant species; (ii) there are at least two distinct phylogenetic lineages of Sp+ Frankia that cluster according to the host-plant species and without regard of geographic distance and (iii) genetic diversity of Sp+ strains is very low at the alder stand scale compared with Sp- strains. Difference in evolutionary history and genetic diversity between Sp+ and Sp- Frankia allows us to discuss the possible ecological role of in-planta sporulation.

Identification of potential transcriptional regulators of actinorhizal symbioses in Casuarina glauca and Alnus glutinosa

BackgroundTrees belonging to the Casuarinaceae and Betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. These functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus Frankia. However, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. The aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses.ResultsWe identified 202 putative transcription factors by in silico analysis in 40 families in Casuarina glauca (Casuarinaceae) and 195 in 35 families in Alnus glutinosa (Betulaceae) EST databases. Based on published transcriptome datasets and quantitative PCR analysis, we found that 39% and 26% of these transcription factors were regulated during C. glauca and A. glutinosa-Frankia interactions, respectively. Phylogenetic studies confirmed the presence of common key transcription factors such as NSP, NF-YA and ERN-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. We also identified an actinorhizal-specific transcription factor belonging to the zinc finger C1-2i subfamily we named CgZF1 in C. glauca and AgZF1 in A. glutinosa.ConclusionsWe identified putative nodulation-associated transcription factors with particular emphasis on members of the GRAS, NF-YA, ERF and C2H2 families. Interestingly, comparison of the non-legume and legume TF with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific C2H2 TF that could be specifically involved in actinorhizal symbioses. In silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to Frankia infection.

Lectin genes in the Frankia alni genome

Frankia alni strain ACN14a’s genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind specifically to sugar motifs. The genome of F. alni was found to contain 7 such lectin-coding genes, five of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations.

In-planta sporulation capacity enhances infectivity and rhizospheric competitiveness of Frankia strains

Frankia Sp+ strains maintain their ability to sporulate in symbiosis with actinorhizal plants, producing abundant sporangia inside host plant cells, in contrast to Sp− strains, which are unable to perform in-planta sporulation. We herein examined the role of in-planta sporulation in Frankia infectivity and competitiveness for root infection. Fifteen strains belonging to different Sp+ and Sp− phylogenetic lineages were inoculated on seedlings of Alnus glutinosa (Ag) and A. incana (Ai). Strain competitiveness was investigated by performing Sp−/Sp+ co-inoculations. Plant inoculations were standardized using crushed nodules obtained under laboratory-controlled conditions (same plant species, age, and environmental factors). Specific oligonucleotide primers were developed to identify Frankia Sp+ and/or Sp− strains in the resulting nodules. Single inoculation experiments showed that (i) infectivity by Sp+ strains was significantly greater than that by Sp− strains, (ii) genetically divergent Sp+ strains exhibited different infective abilities, and (iii) Sp+ and Sp− strains showed different host preferences according to the origin (host species) of the inocula. Co-inoculations of Sp+ and Sp− strains revealed the greater competitiveness of Sp+ strains (98.3 to 100% of Sp+ nodules, with up to 15.6% nodules containing both Sp+ and Sp− strains). The results of the present study highlight differences in Sp+/Sp− strain ecological behaviors and provide new insights to strengthen the obligate symbiont hypothesis for Sp+ strains.