Molecular analysis of actinorhizal symbiotic systems: Progress to date

The application of molecular tools to questions related to the genetics, ecology and evolution of actinorhizal symbiotic systems has been especially fruitful during the past two years. Host plant phylogenies based on molecular data have revealed markedly different relationships among host plants than have previously been suspected and have contributed to the development of new hypotheses on the origin and evolution of actinorhizal symbiotic systems. Molecular analyses of host plant gene expression in developing nodules have confirmed the occurrence of nodulin proteins and in situ hybridization techniques have been successfully adapted to permit the study of the spatial and temporal patterns of gene expression within actinorhizal nodules. The use of heterologous probes in combination with nucleotide sequence analysis have allowed a number of nif genes to be mapped on the Frankia chromosome which will ultimately contribute to the development of hypotheses related to nif gene regulation in Frankia. The use of both 16S and 23S rDNA nucleotide sequences has allowed the construction of phylogenetic trees that can be tested for congruence with symbiotic characters. In addition the development of Frankia-specific gene probes and amplification primers have contributed to studies on the genetic diversity and distribution of Frankia in the soil.     

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.     

Choosing a reporter for gene expression studies in transgenic actinorhizal plants of the Casuarinaceae family

Transgenic Casuarinaceae and reporter genes provide valuable tools to study gene expression in transgenic actinorhizal nodules. In this paper, we discuss the use of ß-glucuronidase for the histochemical localization and quantification of gene expression in transgenic plants of Allocasuarina verticillata and Casuarina glauca nodulated by the actinomycete Frankia. We also report on the genetic transformation of A. verticillata by the Agrobacterium tumefaciens strain C58C1(pGV2260) containing the 35S-mgfp5-ER construct encoding a modified green fluorescent protein of Aequorea victoria in a binary vector. The evolution of the GFP fluorescence was monitored through all stages of the regeneration process. The data indicate that GFP is not toxic in Casuarinaceae and that this reporter gene can be used for visual screening of transformed calli and transgenic plants. The fluorescence pattern of gfp provides a new tool for monitoring in vivo transgene expression in actinorhizal plants.     

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 N₂-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.     

Nodule distribution on the roots of actinorhizal Discaria trinervis (Rhamnaceae) growing in pots

Roots of actinorhizal plants can develop nitrogen-fixing nodules with actinomycetic bacteria of the genus Frankia. We aimed to know if unrestricted growth of roots in pots could influence the pattern of nodule development that we had previously observed for Discaria trinervis growing in pouches. Growth pouches, although being a space saving device convenient for the analysis of nodule development, do restrict root growth. Thus, the pattern of root nodule development was analysed in actinorhizal D. trinervis growing in pots with inert substrates. Inoculation of axenic seedlings growing in perlite resulted in clustering of nodules in a defined region of the taproot and upper lateral roots. When surface sterilized seeds were sown in pots containing vermiculite that had been previously inoculated with Frankia cells, nodules were again concentrated in defined portions of the main and lateral roots. Potted plants developed comparable numbers of nodules with respect to plants grown in pouches. However, a significant proportion of nodules appeared in lateral roots. As it was first inferred from field grown plants, these results confirm that D. trinervis plants growing in pots display the same autoregulatory mechanism for nodule formation that was previously observed in growth pouches.     

Life in soil by the actinorhizal root nodule endophyte <Emphasis Type="BoldItalic">Frankia</Emphasis>. A review

Frankia is a genus of soil actinomycetes famous for its ability to form N₂-fixing root nodule symbioses with actinorhizal plants. Although Frankia strains display a high diversity in terms of ecological niches in soil, current knowledge about Frankia is dominated by its life as an endophyte in root nodules. Increased use of molecular methods has refined and expanded insights into endophyte-host specificities and Frankia phylogeny. This review has focus on Frankia as a soil organism, including its part of microbial consortia, and how to study Frankia in soil. We highlight the use of nodulation tests and molecular methods to reveal population size and genetic diversity of Frankia in soil and discuss how autoregulation of nodulation and interactions with other soil microorganisms may influence the results. A comprehensive record of published interactions between Frankia and other soil microbes is summarized.     

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.     

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.     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

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.     

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

The genetic diversity of Frankia populations in soil and in root nodules of sympatrically grown Alnus taxa was evaluated by rep-polymerase chain reaction (PCR) and nifH gene sequence analyses. Rep-PCR analyses of uncultured Frankia populations in root nodules of 12 Alnus taxa (n?=?10 nodules each) growing sympatrically in the Morton Arboretum near Chicago revealed identical patterns for nodules from each Alnus taxon, including replicate trees of the same host taxon, and low diversity overall with only three profiles retrieved. One profile was retrieved from all nodules of nine taxa (Alnus incana subsp. incana, Alnus japonica, Alnus glutinosa, Alnus incana subsp. tenuifolia, Alnus incana subsp. rugosa, Alnus rhombifolia, Alnus mandshurica, Alnus maritima, and Alnus serrulata), the second was found in all nodules of two plant taxa (A. incana subsp. hirsuta and A. glutinosa var. pyramidalis), and the third was unique for all Frankia populations in nodules of A. incana subsp. rugosa var. americana. Comparative sequence analyses of nifH gene fragments in nodules representing these three profiles assigned these frankiae to different subgroups within the Alnus host infection group. None of these sequences, however, represented frankiae detectable in soil as determined by sequence analysis of 73 clones from a Frankia-specific nifH gene clone library. Additional analyses of nodule populations from selected alders growing on different soils demonstrated the presence of different Frankia populations in nodules for each soil, with populations showing identical sequences in nodules from the same soil, but differences between plant taxa. These results suggest that soil environmental conditions and host plant genotype both have a role in the selection of Frankia strains by a host plant for root nodule formation, and that this selection is not merely a function of the abundance of a Frankia strain in soil.     

Is the Legume Nodule a Modified Root or Stem or an Organ sui generis?

The legume nodule, which houses nitrogen-fixing rhizobia, is a unique plant organ. Its homology with lateral roots has been inferred by a comparison with other nitrogen-fixing nodules, especially those formed on actinorhizal plants in response to Frankia inoculation or on Parasponia roots following inoculation with Bradyrhizobium species. These nodules are clearly modified lateral roots in terms of their structure and development. However, legume nodules differ from lateral roots and these other nodules in their developmental origin, anatomy, and patterns of gene expression, and, consequently, several other evolutionary derivations, including from stems, wound or defense responses, or the more ancient vesicular-arbuscular mycorrhizal symbiosis, have been postulated for the legume nodule. In this review, we first present a broad view of the legume family showing the diversity of nodulation occurrence and types in the different subfamilies and particularly within the subfamily Papilionoideae. We then define the typological and molecular criteria used to discriminate the basic organs — root, stem, leaf— of the plant. Finally, we discuss the possible origins of the legume nodule in terms of these typological and molecular bases.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Variation in Frankia Populations of the Elaeagnus Host Infection Group in Nodules of Six Host Plant Species after Inoculation with Soil

The potential role of host plant species in the selection of symbiotic, nitrogen-fixing Frankia strains belonging to the Elaeagnus host infection group was assessed in bioassays with two Morella, three Elaeagnus, and one Shepherdia species as capture plants, inoculated with soil slurries made with soil collected from a mixed pine/grassland area in central Wisconsin, USA. Comparative sequence analysis of nifH gene fragments amplified from homogenates of at least 20 individual lobes of root nodules harvested from capture plants of each species confirmed the more promiscuous character of Morella cerifera and Morella pensylvanica that formed nodules with frankiae of the Alnus and the Elaeagnus host infection groups, while frankiae in nodules formed on Elaeagnus umbellata, Elaeagnus angustifolia, Elaeagnus commutata, and Shepherdia argentea generally belonged to the Elaeagnus host infection group. Diversity of frankiae of the Elaeagnus host infection groups was larger in nodules on both Morella species than in nodules formed on the other plant species. None of the plants, however, captured the entire diversity of nodule-forming frankiae. The distribution of clusters of Frankia populations and their abundance in nodules was unique for each of the plant species, with only one cluster being ubiquitous and most abundant while the remaining clusters were only present in nodules of one (six clusters) or two (two clusters) host plant species. These results demonstrate large effects of the host plant species in the selection of Frankia strains from soil for potential nodule formation and thus the significant effect of the choice of capture plant species in bioassays on diversity estimates in soil.     

Effectiveness of Different Frankia Cell Types as Inocula for the Actinorhizal Plant Casuarina

The soil bacterium Frankia of the Actinomycetales, capable of forming N₂-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of Casuarina at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.