Assessing the phylogeny of Frankia-actinorhizal plant nitrogen-fixing root nodule symbioses with Frankia 16S rRNA and glutamine synthetase gene sequences

Actinomycetes from the genus Frankia induce nitrogen-fixing root nodules on actinorhizal plants in the "core rosid" clade of eudicots. Reported here are nine partial Frankia 16S rRNA gene sequences including the first from host plants of the rosaceous genera Cercocarpus and Chamaebatia, 24 partial glutamine synthetase (GSI; glnA) sequences from Frankia in nodules of 17 of the 23 actinorhizal genera, and the partial glnA sequence of Acidothermus cellulolyticus. Phylogenetic analyses of combined Frankia 16S rDNA and glnA sequences indicate that infective strains belong to three major clades (I-III) and that Clade I strains consisting of unisolated symbionts from the Coriariaceae, Datiscaceae, Rosaceae, and Ceanothus of the Rhamnaceae are basal to the other clades. Clock-like mutation rates in glnA sequence alignments indicate that all three major Frankia clades diverged early during the emergence of eudicots in the Cretaceous period, and suggest that present-day symbioses are the result of an ancestral symbiosis that emerged before the divergence of extant actinorhizal plants.     

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.     

The diversity of actinorhizal symbiosis

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.     

Molecular phylogenies of plants and Frankia support multiple origins of actinorhizal symbioses

Molecular phylogenetic trees were reconstructed from nucleotide sequences of nifH and 16S rDNA for Frankia and of rbcL for actinorhizal plants. Comparison of Frankia phylogenetic trees reconstructed using nifH and 16S rDNA sequences indicated that subgroupings of both trees correspond with each other in terms of plant origins of Frankia strains. The results suggested that 16S rDNAs can be utilized for coevolution analysis of actinorhizal symbioses. Frankia and plant phylogenetic trees reconstructed using 16S rDNA and rbcL sequences were compared. The comparison by tree matching and likelihood ratio tests indicated that although branching orders of both trees do not strictly correspond with each other, subgroupings of Frankia and their host plants correspond with each other in terms of symbiotic partnership. Estimated divergence times among Frankia and plant clades indicated that Frankia clades diverged more recently than plant clades. Taken together, actinorhizal symbioses originated more than three times after the four plant clades diverged.     

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).     

Natural diversity of Frankia strains in actinorhizal root nodules from promiscuous hosts in the family Myricaceae.

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N(2)-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

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.     

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)

Actinomycetes from the genus Frankia are able to form symbiotic associations with more than 200 different species of woody angiosperms, so called actinorhizal plants. Many actinorhizal plants are infected via deformed root hairs. Factor(s) eliciting root hair deformation in actinorhizal symbioses have been found to be released into the culture medium, but the factor(s) has (have) not yet been characterized. In the present work, we describe the constitutive production of factor(s) by Frankia strain ArI3 causing root hair deformation on Alnus glutinosa . Deformation was detected after 4–5 h of incubation with both Frankia cultures and their cell-free culture filtrates. When culture filtrate was used, deformation was concentration dependent. A contact time of 2 min between culture filtrate and host roots was sufficient to induce subsequent root hair deformation. No root hair deformation on A. glutinosa could be detected with purified Nod factors from Rhizobium meliloti or R. leguminosarum biovar viciae . No correlation was found between Frankia strains belonging to different host specificity groups and their ability to deform root hairs on A. glutinosa. However, strains not able to deform root hairs on A. glutinosa were also unable to nodulate.     

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

Regulation of nodulation in the absence of N2 is different in actinorhizal plants with different infection pathways

Root nodulation in actinorhizal plants, like Discaria trinervis and Alnus incana, is subject to feedback regulatory mechanisms that control infection by Frankia and nodule development. Nodule pattern in the root system is controlled by an autoregulatory process that is induced soon after inoculation with Frankia. The final number of nodules, as well as nodule biomass in relation to plant biomass, are both modulated by a second mechanism which seems to be related to the N status of the plant. Mature nodules are, in part, involved in the latter process, since nodule excision from the root system releases the inhibition of infection and nodule development. To study the effect of N(2) fixation in this process, nodulated D. trinervis and A. incana plants were incubated under a N(2)-free atmosphere. Discaria trinervis is an intercellularly infected species while A. incana is infected intracellularly, via root hairs. Both symbioses responded with an increment in nodule biomass, but with different strategies. Discaria trinervis increased the biomass of existing nodules without significant development of new nodules, while in A. incana nodule biomass increased due to the development of nodules from new infections, but also from the release of arrested infections. It appears that in D. trinervis nodules there is an additional source for inhibition of new infections and nodule development that is independent of N(2) fixation and nitrogen assimilation. It is proposed here that the intercellular Frankia filaments commonly present in the D. trinervis nodule apex, is the origin for the autoregulatory signals that sustain the blockage of initiated nodule primordia and prevent new roots from infections. When turning to A. incana plants, it seems likely that this signal is related to the early autoregulation of nodulation in A. incana seedlings and is no longer present in mature nodules. Thus, actinorhizal symbioses belonging to relatively distant phylogenetic groups and displaying different infection pathways, show different feedback regulatory processes that control root nodulation by Frankia.     

Phylogenetic relationships among actinorhizal plants. The impact of molecular systematics and implications for the evolution of actinorhizal symbioses

A review of recent molecular systematic studies of actinorhizal plants and their Frankia endosymbionts is presented. For comparative purposes, a discussion of recent studies pertaining to the evolution of nodulation in the legume-rhizobium system is included. Molecular systematic studies have revealed that actinorhizal plants are more closely related than current taxonomic schemes imply. Broad-based analyses of the chloroplast gene rbcL indicate that all symbiotic root-nodulating higher plants belong to a single large clade. More focused molecular analyses of both legume and actinorhizal hosts within this large clade indicate that symbioses have probably arisen more than once. By comparing host phylogenies and recently published bacterial phylogenies, we consider the coevolution of bacterial symbionts with their actinorhizal hosts.     

Endophyte sporulation in root nodules of actinorhizal plants

All strains of isolated Frankia possess the genetic capacity to form sporangia since, when grown in vitro, they usually sporulate freely, depending on the physical and chemical environment in which they are cultured. Endophytic sporulation involving Frankia differentiation of sporangia within root nodules has been described in only 16 host species in 9 genera within six families of actinorhizal plants. From studies published to date, endophytic sporulation cannot be correlated with specific environmental conditions surrounding the host plants. Based on the literature and on previously unpublished observations from field and greenhouse studies, an account is given of the occurrence of sporulation in actinorhizal plants with emphasis on Alnus, Casuarina, Comptonia, Elaeagnus and Myrica . The possible role of the host plant in controlling Frankia sporulation as contrasted to the control exerted by the genetic constitution of the microbial symbiont is explored.     

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.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

Characterization of a Casuarina glauca nodule-specific subtilisin-like protease gene, a homolog of Alnus glutinosa ag12

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.     

Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) west of the Sierra Nevada (California)

Frankia spp. strains typically induce N2-fixing root nodules on actinorhizal plants. The majority of host plant taxa associated with the uncultured Group 1 Frankia strains, i.e., Ceanothus of the Rhamnaceae, Datisca glomerata (Datiscaceae), and all actinorhizal members of the Rosaceae except Dryas, are found in California. A study was conducted to determine the distribution of Frankia strains among root nodules collected from both sympatric and solitary stands of hosts. Three DNA regions were examined, the 5' end of the 16S rRNA gene, the internal transcribed spacer region between the 16S and 23S rRNA genes, and a portion of the glutamine synthetase gene (glnA). The results suggest that a narrow range of Group 1 Frankia spp. strains dominate in root nodules collected over a large area of California west of the Sierra Nevada crest with no apparent host-specificity. Comparisons with Group 2 Frankia strain diversity from Alnus and Myrica within the study range suggest that the observed low diversity is peculiar to Group 1 Frankia strains only. Factors that may account for the observed lack of genetic variability and host specificity include strain dominance over a large geographical area, current environmental selection, and (or) a past evolutionary bottleneck.     

Genome sequence of "Candidatus Frankia datiscae" Dg1, the uncultured microsymbiont from nitrogen-fixing root nodules of the dicot Datisca glomerata

Members of the noncultured clade of Frankia enter into root nodule symbioses with actinorhizal species from the orders Cucurbitales and Rosales. We report the genome sequence of a member of this clade originally from Pakistan but obtained from root nodules of the American plant Datisca glomerata without isolation in culture.     

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