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.     

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     

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.     

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.     

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.     

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 nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.     

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.     

Functional analysis of the metallothionein gene cgMT1 isolated from the actinorhizal tree Casuarina glauca

cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a beta-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.     

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.     

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.     

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.     

PCR-restriction fragment length polymorphism identification and host range of single-spore isolates of the flexible Frankia sp. strain UFI 132715.

Twelve single-spore isolates of the flexible Elaeagnus-Frankia strain UFI 132715 fulfilled the third and the fourth of Koch's postulates on both Alnus and Elaeagnus axenic plants. Seminested nifD-nifK PCR-restriction fragment length polymorphisms provided evidence for the genetic uniformity of the single-spore frankiae with the mother strain and its plant reisolates and allowed their molecular identification directly inside Alnus and Elaeagnus nodules. The clonal nature of these single-spore-purified frankiae should allow safe mutagenesis programs, while their flexible phenotype makes them a powerful tool for understanding the molecular interactions between Frankia strains and actinorhizal plants and for identifying Frankia nodulation genes.     

Divergence in symbiotic interactions between same genotypic PCR–RFLP Frankia strains and different Casuarinaceae species under natural conditions

The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long-term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD – nifK intergenic transcribed spacer (ITS) and 16S–23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N₂ fixation using the δ¹⁵N method. The PCR–RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N₂-fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long-term management of the Casuarinaceae symbiosis.     

Ethylene modulates the susceptibility of the root for nodulation in actinorhizal Discaria trinervis

Ethylene is produced by plants in response to a wide variety of environmental signals and mediates several developmental processes in higher plants. We investigated whether ethylene has a regulatory function in nodulation in the actinorhizal symbiosis between Discaria trinervis and Frankia BCU110501. Roots of axenic D. trinervis seedlings showed aberrant growth and reduced elongation rate in the presence of ethylene donors [i.e. 2-aminocyclopropane carboxylic acid (ACC) and 2-chloroethylphosphonic acid (CEPA)] in growth pouches. By contrast, inhibitors of ethylene synthesis (aminoethoxyvinylglycine, AVG) or perception (Ag⁺) did not modify root growth. This indicates that the development of D. trinervis roots is sensitive to elevated ethylene levels in the absence of symbiotic Frankia . The drastic response to higher ethylene levels did not result in a systemic impairment of root nodule development. Nodulation occurred in seedlings inoculated with Frankia BCU110501 in the presence of ethylene donors or inhibitors. Overall, the ability of the seedlings to shut down nodule formation in the younger portions of the root (i.e. to autoregulate nodulation) was not significantly impaired by a modification of endogenous ethylene levels. In contrast, we detected subtle changes in the nodulation pattern of the taproots. As a result of exposing the roots to CEPA, less nodules developed in older portions of the taproot. In line with this observation, AVG or Ag⁺ caused the opposite effect, i.e. a slight increase in nodulation of the mature regions of the taproot. These results suggest that ethylene is involved in modulating the susceptibility for nodulation of the basal portion of D. trinervis seedling roots.     

Diversity and distribution of Frankia strains symbiotic with Ceanothus in California

Frankia strains symbiotic with Ceanothus present an interesting opportunity to study the patterns and causes of Frankia diversity and distribution within a particular host infectivity group. We intensively sampled Frankia from nodules on Ceanothus plants along an elevational gradient in the southern Sierra Nevada of California, and we also collected nodules from a wider host taxonomic and geographic range throughout California. The two sampling scales comprised 36 samples from eight species of Ceanothus representing six of the seven major biogeographic regions in and around California. The primary objective of this study was to use a quantitative model to test the relative importance of geographic separation, host specificity, and environment in influencing the identity of Ceanothus Frankia symbionts as determined by ribosomal DNA sequence data. At both sampling scales, Frankia strains symbiotic with Ceanothus exhibited a high degree of genetic similarity. Frankia strains symbiotic with Chamaebatia (Rosaceae) were within the same clade as several Ceanothus symbionts. Results from a classification and regression tree model used to quantitatively explain Frankia phylogenetic groupings demonstrated that the only significant variable in distinguishing between phylogenetic groups at the more local sampling scale was host species. At the regional scale, Frankia phylogenetic groupings were explained by host species and the biogeographic province of sample collection. We did not find any significant correspondence between Frankia and Ceanothus phylogenies indicative of coevolution, but we concluded that the identity of Frankia strains inhabiting Ceanothus nodules may involve interactions between host species specificity and geographic isolation.