Contribution of transgenic Casuarinaceae to our knowledge of the actinorhizal symbioses

The Casuarinaceae family is a group of 96 species of trees and shrubs that are tolerant to adverse soil and climatic conditions. In the field, Casuarinaceae bears nitrogen-fixing root nodules (so called actinorhizal nodules) resulting from infection by the soil actinomycete Frankia. The association between Casuarina and Frankia is of tremendous ecological importance in tropical and subtropical areas where these trees contribute to land stabilization and soil reclamation. During differentiation of the actinorhizal nodule, a set of genes called actinorhizal nodulins is activated in the developing nodule. Understanding the molecular basis of actinorhizal nodule ontogenesis requires molecular tools such as genomics together with gene transfer technologies for functional analysis of symbiotic genes. Using the biological vectors Agrobacterium rhizogenes and A. tumefaciens, gene transfer into the two species Allocasuarina verticillata and Casuarina glauca has been successful. Transgenic Casuarinaceae plants proved to be valuable tools for exploring the molecular mechanisms resulting from the infection process of actinorhizal plants by Frankia.     

A Role for auxin during actinorhizal symbioses formation?

The symbiotic interaction between the soil bacteria Frankia and actinorhizal plants leads to the formation of nitrogen-fixing nodules resembling modified lateral roots. Little is known about the signals exchanged between the two partners during the establishment of these endosymbioses. However, a role for plant hormones has been suggested.Recently, we studied the role of auxin influx activity during actinorhizal symbioses. An inhibitor of auxin influx was shown to perturb nodule formation. Moreover we identified a functional auxin influx carrier that is produced specifically in Frankia-infected cells. These results together with previous data showing auxin production by Frankia lead us to propose a model of auxin action during the symbiotic infection process.Key words: lateral roots, nitrogen fixation, Frankia, AUX1, actinorhizal symbioses, phenylacetic acid, auxin influxActinorhizal symbioses result from the interaction between the soil actinomycete Frankia and plants belonging to eight angiosperm families collectively called actinorhizal plants.¹ This symbiotic interaction leads to the formation of a new organ on the root system, the actinorhizal nodule, where the bacteria are hosted and fix nitrogen.² Unlike legume nodules, actinorhizal nodules are structurally and developmentally related to lateral roots.³ Little is known about the signals exchanged between the two partners during the establishment of the symbiosis.² Diffusible signals are emitted by Frankia at early stages of the interaction resulting in root hair deformation.² The chemical nature of these signals remains unknown, however, detailed studies revealed that they are different from rhizobial Nod factors.⁴ Phytohormones are chemicals that control many developmental processes⁵ and have been linked to many plant-microbe interactions. Recently, we studied the role of auxin influx in actinorhizal nodule formation in the tropical tree Casuarina glauca.⁶     

The Actinorhizal Symbiosis

Abstract The term ``actinorhiza' refers both to the filamentous bacteria Frankia, an actinomycete, and to the root location of nitrogen-fixing nodules. Actinorhizal plants are classified into four subclasses, eight families, and 25 genera comprising more than 220 species. Although ontogenically related to lateral roots, actinorhizal nodules are characterized by differentially expressed genes, supporting the idea of the uniqueness of this new organ. Two pathways for root infection have been described for compatible Frankia interactions: root hair infection or intercellular penetration. Molecular phylogeny groupings of host plants correlate with morphologic and anatomic features of actinorhizal nodules. Four clades of actinorhizal plants have been defined, whereas Frankia bacteria are classified into three major phylogenetic groups. Although the phylogenies of the symbionts are not fully congruent, a close relationship exists between plant and bacterial groups. A model for actinorhizal specificity is proposed that includes different levels or degrees of specificity of host-symbiont interactions, from fully compatible to incompatible. Intermediate, compatible, but delayed or limited interactions are also discussed. Actinorhizal plants undergo feedback regulation of symbiosis involving at least two different and consecutive signals that lead to a mechanism controlling root nodulation. These signals mediate the opening or closing of the window of susceptibility for infection and inhibit infection and nodule development in the growing root, independently of infection mechanism. The requirement for at least two molecular recognition steps in the development of actinorhizal symbioses is discussed.     

Diversity and Specificity of Frankia Strains in Nodules of Sympatric Myrica gale, Alnus incana, and Shepherdia canadensis Determined by rrs Gene Polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).     

Symbiosis between Frankia and actinorhizal plants: root nodules of non-legumes

In actinorhizal symbioses, filamentous nitrogen-fixing soil bacteria of the genus Frankia induce the formation of nodules on the roots of a diverse group of dicotyledonous plants representing trees or woody shrubs, with one exception, Datisca glomerata. In the nodules, Frankia fixes nitrogen and exports the products to the plant cytoplasm, while being supplied with carbon sources by the host. Possibly due to the diversity of the host plants, actinorhizal nodules show considerable variability with regard to structure, oxygen protection mechanisms and physiology. Actinorhizal and legume-rhizobia symbioses are evolutionary related and share several features.     

The evolution of actinorhizal symbioses: Evidence for multiple origins of the symbiotic association

According to morphologically based classification systems, actinorhizal plants, engaged in nitrogen-fixing symbioses with Frankia bacteria, are considered to be only distantly related. However, recent phylogenetic analyses of seed plants based on chloroplast rbcL gene sequences have suggested closer relationships among actinorhizal plants. A more thorough sampling of chloroplast rbcL gene sequences from actinorhizal plants and their nonsymbiotic close relatives was conducted in an effort to better understand the phylogenetic relationships of these plants, and ultimately, to assess the homology of the different actinorhizal symbioses. Sequence data from 70 taxa were analyzed using parsimony analysis. Strict consensus trees based on 24 equally parsimonious trees revealed evolutionary divergence between groups of actinorhizal species suggesting that not all symbioses are homologous. The arrangement of actinorhizal species, interspersed with nonactinorhizal taxa, is suggestive of multiple origins of the actinorhizal symbiosis. Morphological and anatomical characteristics of nodules from different actinorhizal hosts were mapped onto the rbclL-based consensus tree to further assess homology among rbcL-based actinorhizal groups. The morphological and anatomical features provide additional support for the rbcL-based groupings, and thus, together, suggest that actinorhizal symbioses have originated more than once in evolutionary history.     

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Identification by Suppression Subtractive Hybridization of Frankia Genes Induced under Nitrogen-Fixing Conditions

Frankia is an actinobacterium that fixes nitrogen under both symbiotic and free-living conditions. We identified genes upregulated in free-living nitrogen-fixing cells by using suppression subtractive hybridization. They included genes with predicted functions related to nitrogen fixation, as well as with unknown function. Their upregulation was a novel finding in Frankia.Frankia is a Gram-positive actinobacterium that establishes symbiosis with several angiosperms termed actinorhizal plants and forms nitrogen-fixing nodules on their roots (20). Frankia also fixes nitrogen in free-living culture under nitrogen-free conditions (19). Induction of the nitrogen-fixing ability is accompanied by differentiation of vesicles (19). Vesicles are spherical cells specialized to nitrogen fixation and are surrounded by multilayered lipid envelopes by which nitrogenase is protected from oxygen (3). Frankia plays an important role in the global nitrogen cycle, yet little is known about the genes involved in the induction of nitrogen-fixing activity. Recently, three Frankia genome sequences were determined (15), which facilitates the genetic dissection of Frankia biology. In this study, we identified Frankia genes induced in nitrogen-fixing cells under free-living conditions by using suppression subtractive hybridization (SSH) (4).     

Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia

Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

Molecular phylogeny of the actinorhizal Hamamelidae and relationships with host promiscuity towards Frankia

Several of the most studied actinorhizal symbioses involve associations between host plants in the subclass Hamamelidae of the dicots and actinomycetes of the genus Frankia. These actinorhizal plants comprise eight genera distributed among three families of ‘higher’ Hamamelidae, the Betulaceae, Myricaceae, and Casuarinaceae. Contrasting promiscuity towards Frankia is encountered among the different actinorhizal members of these families, and a better assessment of the evolutionary history of these actinorhizal taxa could help to understand the observed contrasts and their implications for the ecology and evolution of the actinorhizal symbiosis. Complete DNA sequences of the chloroplast gene coding for the large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) were obtained from taxa representative of these families and the Fagaceae. The phylogenetic relationships among and within these families were estimated using parsimony and distance-matrix approaches. All families appeared monophyletic. The Myricaceae appeared to derive first before the Betulaceae and the Casuarinaceae. In the Casuarinaceae, the genus Gymnostoma derived before the genera Casuarina and Allocasuarina, which were found closely related. The analysis of character-state changes in promiscuity along the consensus tree topology suggested a strong relationship between the evolutionary history of host plants and their promiscuity toward Frankia. Indeed, the actinorhizal taxa that diverged more recently in this group of plants were shown to be susceptible to a narrower spectrum of Frankia strains. The results also suggest that the ancestor of this group of plant was highly promiscuous, and that evolution has proceeded toward narrower promiscuity and greater specialization. These results imply that a tight relationship between the phytogenies of both symbiotic partners should not be expected, and that host promiscuity is likely to be a key determinant in the establishment of an effective symbiosis.     

Auxin influx activity is associated with Frankia infection during actinorhizal nodule formation in Casuarina glauca

Plants from the Casuarinaceae family enter symbiosis with the actinomycete Frankia leading to the formation of nitrogen-fixing root nodules. We observed that application of the auxin influx inhibitor 1-naphtoxyacetic acid perturbs actinorhizal nodule formation. This suggests a potential role for auxin influx carriers in the infection process. We therefore isolated and characterized homologs of the auxin influx carrier (AUX1-LAX) genes in Casuarina glauca. Two members of this family were found to share high levels of deduced protein sequence identity with Arabidopsis (Arabidopsis thaliana) AUX-LAX proteins. Complementation of the Arabidopsis aux1 mutant revealed that one of them is functionally equivalent to AUX1 and was named CgAUX1. The spatial and temporal expression pattern of CgAUX1 promoter:beta-glucuronidase reporter was analyzed in Casuarinaceae. We observed that CgAUX1 was expressed in plant cells infected by Frankia throughout the course of actinorhizal nodule formation. Our data suggest that auxin plays an important role during plant cell infection in actinorhizal symbioses.