Signalling in actinorhizal root nodule symbioses.

Plants able to establish a nitrogen-fixing root nodule symbiosis with the actinobacterium Frankia are called actinorhizal. These interactions lead to the formation of new root organs, called actinorhizal nodules, where the bacteria are hosted intracellularly and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. Like other symbiotic interactions, actinorhizal nodulation involves elaborate signalling between both partners of the symbiosis, leading to specific recognition between the plant and its compatible microbial partner, its accommodation inside plant cells and the development of functional root nodules. Actinorhizal nodulation shares many features with rhizobial nodulation but our knowledge on the molecular mechanisms involved in actinorhizal nodulation remains very scarce. However recent technical achievements for several actinorhizal species are allowing major discoveries in this field. In this review, we provide an outline on signalling molecules involved at different stages of actinorhizal nodule formation and the corresponding signalling pathways and gene networks.

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.

Inhibition of auxin signaling in Frankia species-infected cells in Casuarina glauca nodules leads to increased nodulation.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia spp. that lead to the formation of nitrogen-fixing root nodules. The plant hormone auxin has been suggested to play a role in the mechanisms that control the establishment of this symbiosis in the actinorhizal tree Casuarina glauca. Here, we analyzed the role of auxin signaling in Frankia spp.-infected cells. Using a dominant-negative version of an endogenous auxin-signaling regulator, INDOLE-3-ACETIC ACID7, we established that inhibition of auxin signaling in these cells led to increased nodulation and, as a consequence, to higher nitrogen fixation per plant even if nitrogen fixation per nodule mass was similar to that in the wild type. Our results suggest that auxin signaling in Frankia spp.-infected cells is involved in the long-distance regulation of nodulation in actinorhizal symbioses.

The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals.

Root nodule symbioses (RNS) allow plants to acquire atmospheric nitrogen by establishing an intimate relationship with either rhizobia, the symbionts of legumes or Frankia in the case of actinorhizal plants. In legumes, NIN (Nodule INception) genes encode key transcription factors involved in nodulation. Here we report the characterization of CgNIN, a NIN gene from the actinorhizal tree Casuarina glauca using both phylogenetic analysis and transgenic plants expressing either ProCgNIN::reporter gene fusions or CgNIN RNAi constructs. We have found that CgNIN belongs to the same phylogenetic group as other symbiotic NIN genes and CgNIN is able to complement a legume nin mutant for the early steps of nodule development. CgNIN expression is correlated with infection by Frankia, including preinfection stages in developing root hairs, and is induced by culture supernatants. Knockdown mutants were impaired for nodulation and early root hair deformation responses were severely affected. However, no mycorrhizal phenotype was observed and no induction of CgNIN expression was detected in mycorrhizas. Our results indicate that elements specifically required for nodulation include NIN and possibly related gene networks derived from the nitrate signalling pathways.

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

BACKGROUND: Trees 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. RESULTS: We 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. CONCLUSIONS: We 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.

Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 µM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 µM) and BAP (2,2 µM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Mapping of QTLs Associated with Biological Nitrogen Fixation Traits in Peanuts (Arachis hypogaea L.) Using an Interspecific Population Derived from the Cross between the Cultivated Species and Its Wild Ancestors.

Peanuts (Arachis hypogaea L.) are an allotetraploid grain legume mainly cultivated by poor farmers in Africa, in degraded soil and with low input systems. Further understanding nodulation genetic mechanisms could be a relevant option to facilitate the improvement of yield and lift up soil without synthetic fertilizers. We used a subset of 83 chromosome segment substitution lines (CSSLs) derived from the cross between a wild synthetic tetraploid AiAd (Arachis ipaensis × Arachis duranensis)4× and the cultivated variety Fleur11, and evaluated them for traits related to BNF under shade-house conditions. Three treatments were tested: without nitrogen; with nitrogen; and without nitrogen, but with added0 Bradyrhizobium vignae strain ISRA400. The leaf chlorophyll content and total biomass were used as surrogate traits for BNF. We found significant variations for both traits specially linked to BNF, and four QTLs (quantitative trait loci) were consistently mapped. At all QTLs, the wild alleles decreased the value of the trait, indicating a negative effect on BNF. A detailed characterization of the lines carrying those QTLs in controlled conditions showed that the QTLs affected the nitrogen fixation efficiency, nodule colonization, and development. Our results provide new insights into peanut nodulation mechanisms and could be used to target BNF traits in peanut breeding programs.

Permanent draft genome sequence of Bradyrhizobium vignae, strain ISRA 400, an elite nitrogen-fixing bacterium, isolated from the groundnut growing area in Senegal.

A new Bradyrhizobium vignae strain called ISRA400 was isolated from groundnut (Arachis hypogaea L.) root nodules obtained by trapping the bacteria from soil samples collected in the Senegalese groundnut basin. In this study, we present the draft genome sequence of this strain ISRA400, which spans approximatively 7.9 Mbp and exhibits a G+C content of 63.4%. The genome analysis revealed the presence of 48 tRNA genes and one rRNA operon (16S, 23S, and 5S). The nodulation test revealed that this strain ISRA400 significantly improves the nodulation parameters and chlorophyll content of the Arachis hypogaea variety Fleur11. These findings suggest the potential of Bradyrhizobium vignae strain ISRA400 as an effective symbiotic partner for improving the growth and productivity of groundnut crop.

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.

Auxin carriers localization drives auxin accumulation in plant cells infected by Frankia in Casuarina glauca actinorhizal nodules.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.

Establishment of Actinorhizal Symbiosis in Response to Ethylene, Salicylic Acid, and Jasmonate.

Phytohormones play a crucial role in regulating plant developmental processes. Among them, ethylene and jasmonate are known to be involved in plant defense responses to a wide range of biotic stresses as their levels increase with pathogen infection. In addition, these two phytohormones have been shown to inhibit plant nodulation in legumes. Here, exogenous salicylic acid (SA), jasmonate acid (JA), and ethephon (ET) were applied to the root system of Casuarina glauca plants before Frankia inoculation, in order to analyze their effects on the establishment of actinorhizal symbiosis. This protocol further describes how to identify putative ortholog genes involved in ethylene and jasmonate biosynthesis and/or signaling pathways in plant, using the Arabidopsis Information Resource (TAIR), Legume Information System (LIS), and Genevestigator databases. The expression of these genes in response to the bacterium Frankia was analyzed using the gene atlas for Casuarina-Frankia symbiosis (SESAM web site).

Draft Genome Sequence of the Symbiotic Frankia sp. strain B2 isolated from root nodules of Casuarina cunninghamiana found in Algeria.

Frankia sp. strain B2 was isolated from Casuarina cunninghamiana nodules. Here, we report the 5.3-Mbp draft genome sequence of Frankia sp. strain B2 with a G+C content of 70.1 % and 4,663 candidate protein-encoding genes. Analysis of the genome revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters.

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 intracellular 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 photosynthates. 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 Ca2+ reporter to identify symbiotic signaling responses to AM fungi in roots of both Casuarina glauca and Discaria trinervis, 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 chitotetraose is a more efficient elicitor of CSSP activation compared to AM fungal lipo-chitooligosaccharides. 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.

Cryopreservation by encapsulation-dehydration of plumules of coconut (Cocos nucifera L.).

This study describes the use of an encapsulation-dehydration cryopreservation technique on coconut plumules (apical dome with three or four leaf primordia) excised from embryos. In order to establish a reliable cryopreservation process for plumules, several different key factors were tested: pretreatment duration, sugar concentration, dehydration period and freezing. In parallel, histological studies were performed to describe the structural changes of tissues and plumule cells subjected to dehydration and freezing. A good survival level of around 60% was obtained. However, after 8 months culture regrowth, this level decreased to a maximum of 20 % which was achieved using sucrose treatment. In this paper we report for the first time the regeneration of leafy shoots from coconut plumules after cryopreservation.

Actinorhizal Signaling Molecules: Frankia Root Hair Deforming Factor Shares Properties With NIN Inducing Factor.

Actinorhizal plants are able to establish a symbiotic relationship with Frankia bacteria leading to the formation of root nodules. The symbiotic interaction starts with the exchange of symbiotic signals in the soil between the plant and the bacteria. This molecular dialog involves signaling molecules that are responsible for the specific recognition of the plant host and its endosymbiont. Here we studied two factors potentially involved in signaling between Frankia casuarinae and its actinorhizal host Casuarina glauca: (1) the Root Hair Deforming Factor (CgRHDF) detected using a test based on the characteristic deformation of C. glauca root hairs inoculated with F. casuarinae and (2) a NIN activating factor (CgNINA) which is able to activate the expression of CgNIN, a symbiotic gene expressed during preinfection stages of root hair development. We showed that CgRHDF and CgNINA corresponded to small thermoresistant molecules. Both factors were also hydrophilic and resistant to a chitinase digestion indicating structural differences from rhizobial Nod factors (NFs) or mycorrhizal Myc-LCOs. We also investigated the presence of CgNINA and CgRHDF in 16 Frankia strains representative of Frankia diversity. High levels of root hair deformation (RHD) and activation of ProCgNIN were detected for Casuarina-infective strains from clade Ic and closely related strains from clade Ia unable to nodulate C. glauca. Lower levels were present for distantly related strains belonging to clade III. No CgRHDF or CgNINA could be detected for Frankia coriariae (Clade II) or for uninfective strains from clade IV.

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.

Symbiotic Performance of Diverse Frankia Strains on Salt-Stressed Casuarina glauca and Casuarina equisetifolia Plants.

Symbiotic nitrogen-fixing associations between Casuarina trees and the actinobacteria Frankia are widely used in agroforestry in particular for salinized land reclamation. The aim of this study was to analyze the effects of salinity on the establishment of the actinorhizal symbiosis between C. glauca and two contrasting Frankia strains (salt sensitive; CcI3 vs. salt tolerant; CeD) and the role of these isolates in the salt tolerance of C. glauca and C. equisetifolia plants. We show that the number of root nodules decreased with increasing salinity levels in both plants inoculated with CcI3 and CeD. Nodule formation did not occur in seedlings inoculated with CcI3 and CeD, at NaCl concentrations above 100 and 200 mM, respectively. Salinity also affected the early deformation of plant root hairs and reduced their number and size. In addition, expression of symbiotic marker Cg12 gene, which codes for a subtilase, was reduced at 50 mM NaCl. These data suggest that the reduction of nodulation in C. glauca under salt stress is in part due to inhibition of early mechanisms of infection. We also show that prior inoculation of C. glauca and C. equisetifolia with Frankia strains CcI3 and CeD significantly improved plant height, dry biomass, chlorophyll and proline contents at all levels of salinity tested, depending on the Casuarina-Frankia association. There was no correlation between in vitro salt tolerance of Frankia strains and efficiency in planta under salt-stressed conditions. Our results strongly indicate that increased N nutrition, photosynthesis potential and proline accumulation are important factors responsible for salt tolerance of nodulated C. glauca and C. equisetifolia.

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.