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Abstract Frankia sp. NRRL B-16219 was directly isolated from a soil sample obtained from the rhizosphere of Ceanothus jepsonii growing in the USA. Its host plant range includes members of Elaeagnaceae species. Phylogenetically, strain NRRL B-16219 is closely related to “Frankia discariae” with a 16S rRNA gene similarity of 99.78%. Because of the lack of genetic tools for Frankia, our understanding of the bacterial signals involved during the plant infection process and the development of actinorhizal root nodules is very limited. Since the first three Frankia genomes were sequenced, additional genome sequences covering more diverse strains have helped provide insight into the depth of the pangenome and attempts to identify bacterial signaling molecules like the rhizobial canonical nod genes. The genome sequence of Frankia sp. strain NRRL B-16219 was generated and assembled into 289 contigs containing 8,032,739 bp with 71.7% GC content. Annotation of the genome identified 6211 protein-coding genes, 561 pseudogenes, 1758 hypothetical proteins and 53 RNA genes including 4 rRNA genes. The NRRL B-16219 draft genome contained genes homologous to the rhizobial common nodulation genes clustered in two areas. The first cluster contains nodACIJH genes whereas the second has nodAB and nodH genes in the upstream region. Phylogenetic analysis shows that Frankia nod genes are more deeply rooted than their sister groups from rhizobia. PCR-sequencing suggested the widespread occurrence of highly homologous nodA and nodB genes in microsymbionts of field collected Ceanothus americanus.

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes that are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. The corresponding endosymbionts, Frankia and the rhizobia, however, are distantly related groups of bacteria, leading to questions about their symbiotic mechanisms and evolutionary history. To date, a stable system of electrotransformation has been lacking in Frankia despite numerous attempts by research groups worldwide. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation. Here we report the successful electrotransformation of the model strain F. alni ACN14a with an unmethylated, broad host-range replicating plasmid, expressing chloramphenicol-resistance for selection and GFP as a marker of gene expression. This system circumvented the type IV restriction barrier and allowed the stable maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the expression of egfp under the control of the nif gene cluster promoter was localized using fluorescence imaging, the expression of nitrogen fixation in nitrogen-limited culture was localized in Frankia vesicles but not in hyphae. The ability to separate gene expression patterns between Frankia hyphae and vesicles will enable deeper comparisons of molecular signaling and metabolic exchange between Frankia-actinorhizal and rhizobia-legume symbioses to be made, and may broaden potential applications in agriculture. Further downstream applications are possible, including gene knock-outs and complementation, to open up a range of experiments in Frankia and its symbioses. Additionally, in the transcriptome of F. alni ACN14a, type IV restriction enzymes were highly expressed in nitrogen-replete culture but their expression strongly decreased during symbiosis. The down-regulation of type IV restriction enzymes in symbiosis suggests that horizontal gene transfer may occur more frequently inside the nodule, with possible new implications for the evolution of Frankia.

Alnus spp. (alder) are typical nonleguminous nitrogen-fixing trees that have a symbiotic relationship with Frankia . To explore the differences in nitrogen-fixing microorganisms between three alders ( A. cremastogyne , A. glutinosa , and A. formosana ) with different chromosome ploidies, the community structure and compositional diversity of potential nitrogen-fixing microorganism in root nodules and rhizosphere soil were comparatively analyzed using 16S rRNA and nitrogenase ( nifH ) gene sequencing. The nitrogen contents in the root nodules and rhizosphere soil were also determined. The results showed that the contents of total nitrogen and nitrate nitrogen in the root nodules of the three alders are significantly higher than those in the rhizosphere soils, while the ammonium nitrogen content show the opposite trend. The family, genus, and species levels showed obviously differences between root nodules and rhizosphere soils, while there were no significant differences at the classification level between the three alders. At the phylum level, the dominant phyla from 16S rRNA and nifH gene data in the root nodules and rhizosphere soil of the three alders are phylum Actinomycetota and phylum Pseudomonadota, respectively. The LEfSe results showed that there are significant differences in the dominant groups in the root nodules and rhizosphere oil of the three alders. The relative abundances of dominant groups also showed obvious differences between the root nodules and rhizosphere soils of three alders. The relative abundances of Frankia and unclassified_ Frankia in root nodules are obviously higher than those in rhizosphere soils, and their relative abundances in A. glutinosa root nodules are significantly higher than those in A. cremastogyne and A. formosana at the genus and species levels. The diversity of potential nitrogen-fixing microorganism from 16S rRNA and nifH gene data in the A. glutinosa root nodules and rhizosphere soils are all higher than those in A. cremastogyne and A. formosana . The results of functional prediction also showed that the OTUs for nitrogen fixation, nitrate respiration, and ureolysis in A. glutinosa root nodules are higher than those in the other two alders. Redundancy analysis revealed that the total nitrogen content mostly affects the Frankia community. Overall, there are significant differences in the community composition and structure of potential nitrogen-fixing microorganism in the root nodules and rhizosphere soils between the three alders. A. glutinosa showed a relatively stronger nitrogen fixation capacity than A. formosana and A. cremastogyne . The results help elucidates how the community structure and nitrogen-fixing ability of potential nitrogen-fixing microorganism differ between alder species and serve as a reference for applying Frankia to alder plantations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yuan, Chen, Huang, Wang, Guo, Huang and Yang.)

Strains CN4 T , CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus . The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N 2 -fixing vesicles and do not fix nitrogen in vitro . The peptidoglycan of strain CN4 T contains meso -diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H 4 ) and MK-9(H 6 ) are the predominant menaquinones (>15 %), and iso-C 16 : 0 and C 17 : 1 ω8 c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4 T is most closely related to Frankia saprophytica CN 3 T . An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3 T , Frankia asymbiotica M16386 T and Frankia inefficax EuI1c T with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4 T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4 T , CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4 T has a 10.76 Mb genome. CN4 T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4 T (=DSM 114740 T = LMG 32595 T ) to a novel species, with CN4 T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

The taxonomic assignment of uncultured prokaryotes to known taxa is a major challenge in microbial systematics. This relies usually on the phylogenetic analysis of the ribosomal small subunit RNA or a few housekeeping genes. Recent works have disclosed ribosomal proteins as valuable markers for systematics and, due to the boom in complete genome sequencing, their use has become widespread. Yet, in the case of uncultured strains, for which complete genome sequences cannot be easily obtained, sequencing many markers is complicated and time consuming. Taking the advantage of the organization of ribosomal protein coding genes in large gene clusters, we amplified a 32 kb conserved region encompassing the spectinomycin (spc) operon using long range PCR from isolated and from uncultured nodular endophytic Frankia strains. The phylogenetic analysis of the 27 ribosomal protein genes contained in this region provided a robust phylogenetic tree consistent with phylogenies based on larger set of markers, indicating that this subset of ribosomal proteins contains enough phylogenetic signal to address systematic issues. This work shows that using long range PCR could break down the barrier preventing the use of ribosomal proteins as phylogenetic markers when complete genome sequences cannot be easily obtained. [ABSTRACT FROM AUTHOR]

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as "blooms". The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe–4S] cluster containing ferredoxins (eight), a single [2Fe–2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria. Image 1 • Frankia sp. EuI1c (Frankia inefficax sp.), contains 68 CYP encoding genes. • The CYP189 family (21 members) has undergone gene duplication events classified as a "bloom". • Genes for eight [3Fe–4S] and one [2Fe–2S] cluster containing ferredoxins were identified. • Three CYP family from Frankia sp. EuI1c , were produced and purified using Escherichia coli. • Substrates including steroids and terpenoids which bind to all three CYPs were identified. [ABSTRACT FROM AUTHOR]

Nitrogen fixation by symbiotic bacteria associated with different plant species is a key process of natural ecosystems. To better understand the role of native N2-fixing species in the N economy of riparian ecosystems in northwest Patagonia (Argentina), we evaluated: 1) foliar δ15N and N concentrations of actinorhizal Coriaria ruscifolia, Discaria chacaye, and Colletia hystrix, several non-actinorhizal plants (including Gunnera chilensis), and associated soils in riparian forest sites; 2) the proportion of N derived from the atmosphere (Ndfa) of the actinorhizal plant species from riparian forest and of D. chacaye and Ochetophila trinervis from steppe sites; and 3) trends of foliar and soil δ15N and N concentration with mean annual precipitation (MAP). Although soil N concentrations and δ15N did not vary among plant species within any of the sites, foliar N concentration and δ15N differed among species. In general, N2-fixing species had higher foliar N concentrations and δ15N values closer to 0, the atmospheric value. Both variables separated the groups of N2-fixing and non-N2-fixing plant species. Foliar and soil δ15N correlated positively for non-N2-fixing species but not for N2-fixing species. Across all sites, the Ndfa of C. ruscifolia and C. hystrix was ~100%, ~75% for D. chacaye, and ~50% for G. chilensis. For all species, foliar N concentration and soil δ15N was negatively correlated with MAP, but only non-N2-fixing species showed a significant correlation of foliar δ15N with MAP. These data suggest that plant available N decreases as MAP increases but with no effect on N2-fixing species. [ABSTRACT FROM AUTHOR]

Aims: Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains originally isolated from Casuarina species, however, have been found to be non-infective on Casuarina species. The goal of this study was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of these non-infective Frankia strains in root nodule formation on Casuarina equisetifolia.Methods: Soil microcosms were established with plants of C. equisetifolia and inoculated with Frankia casuarinae strain CcI3 or cluster 3 strain R43, or combinations of both at different densities. Basic plant growth characteristics, root nodule formation and localization of both Frankia strains in nodule periderm and cortex, as well as population development in soils were monitored.Results: The presence of strain R43 did not affect plant growth performance nor root nodule formation, while inoculation with strain CcI3 enhanced plant growth and resulted in root nodule formation. qPCR analyses on selected nodule lobes revealed the presence of strain CcI3 in cortex samples in all treatments, while strain R43 was not detected in any cortex samples but in 40% of the periderm samples from lobes from treatments with highest inoculation values. In situ hybridization detected cells of strain R43 on the outside of the nodules, i.e. on the periderm only.Conclusions: These results demonstrate that the cluster 3 Frankia strain R43 is not co-infecting root nodules formed by the F. casuarinae strain CcI3 on C. equisetifolia, but has likely been isolated as a surface contaminant from Casuarina nodules. [ABSTRACT FROM AUTHOR]

Root nodule endosymbiosis with nitrogen-fixing bacteria provides plants with unlimited access to fixed nitrogen, but at a significant energetic cost. Nodulation is generally considered to have originated in parallel in different lineages, but this hypothesis downplays the genetic complexity of nodulation and requires independent recruitment of many common features across lineages. Recent phylogenomic studies revealed that genes that function in establishing or maintaining nitrogen-fixing nodules are independently lost in non-nodulating relatives of nitrogen-fixing plants. In our opinion, these data are best explained by a scenario of a single gain followed by massively parallel loss of nitrogen-fixing root nodules triggered by events at geological scale. Highlights N 2 -fixing nodulation symbiosis is a complex and important agronomic trait. It occurs in phylogenetically separated lineages, and its evolution may be explained by two alternative hypotheses: (i) single gain followed by massively parallel loss, or (ii) parallel evolution and fewer losses. The latter hypothesis is widely accepted, but the first hypothesis is supported by recent phylogenomic data. Molecular and developmental commonalities across distinct lineages support a common origin of nodulation. Moreover, recent comparative genomics studies revealed parallel loss of key nodulation genes in non-nodulating species. These findings support a single gain of nodulation followed by massively parallel loss in most descendant lineages. Such massive loss may have been triggered by reductions in global atmospheric CO 2 levels. [ABSTRACT FROM AUTHOR]

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation. [ABSTRACT FROM AUTHOR]

Carbohydrate active enzymes (CAZymes) are capable of breaking complex polysaccharides into simpler form. In plant-host-associated microorganisms CAZymes are known to be involved in plant cell wall degradation. However, the biology and evolution of Frankia CAZymes are largely unknown. In the present study, we took a genomic approach to evaluate the presence and putative roles of CAZymes in Frankia. The CAZymes were found to be potentially highly expressed (PHX) proteins and contained more aromatic amino acids, which increased their biosynthetic energy cost. These energy rich amino acids were present in the active sites of CAZymes aiding in their carbohydrate binding capacity. Phylogenetic and evolutionary analyses showed that, in Frankia strains with the capacity to nodulate host plants, CAZymes were evolving slower than the other PHX genes, whereas similar genes from non-nodulating (or ineffectively nodulating) Frankia strains showed little variation in their evolutionary constraints compared to other PHX genes. Thus, the present study revealed the persistence of a strong purifying selection on CAZymes of Frankia indicating their crucial role. [ABSTRACT FROM AUTHOR]

Four Frankia strains (EuI1c, CN3, ACN14a and CcI3) were tested for selenite tolerance. Frankia inefficax strain EuI1c was resistant to selenite with a MIC value of 518.8 µg ml−1. After 48 h incubation with selenite, a reddish precipitate began to appear in these cultures. The red color suggests the reduction of the toxic, soluble, and colorless sodium selenite (Na2SeO32−) to the nontoxic, insoluble, and red colored elemental selenium (Seº). Analysis showed F. inefficax strain EuI1c cultures exposed to 17.3 and 86.5 µg ml−1selenite completely reduced all of the selenite after 5 and 8 days, respectively. When observed under Scanning Electron Microscopy, selenite-resistant F. inefficax strain EuI1c grown with selenite formed nanosphere particles on the hyphal surface as free deposits or in aggregates and inside the hyphae. EDAX analysis of the nanosphere particles determined that they are composed of selenium with up to 27.3-fold increase in intensity as compared to control cells. FTIR Spectroscopy of selenite-stressed cells showed cell surface changes in fatty acids, polysaccharides, carbohydrates and phosphate groups. This result suggests a mechanism for selenite reduction and nanosphere transport through cell membrane in this strain. Native gel electrophoresis of extracted cell-free protein revealed one band showing activity after staining with selenite and NADH. SDS-PAGE analysis revealed the presence of several bands with one dominant band of 37.8 kDa. Mass spectrometry analysis of the bands determined that the main proteins were a periplasmic-binding protein, sulfate ABC transporter and extracellular ligand-binding receptor. [ABSTRACT FROM AUTHOR]

It has been 40 years since the first meeting dedicated to Frankia and actinorhizal plants, which was held at Petersham, Massachusetts (reported in Torrey and Tjepkema, 1979). Since then biennial meetings have been organised and held in different venues around the globe (Table 1). The most recent meeting, the "19th International Meeting on Frankia and Actinorhizal Plants", organised in Hammamet, Tunisia from 17th to 19th of March, 2018, gathered scientists from Algeria, Argentina, Belgium, China, Egypt, France, India, Portugal, Senegal, Sweden, UK, USA and Tunisia. The event was a stimulating opportunity for active researchers to share many advances since the previous meeting held in Montpellier, France (Franche et al. 2016) and to discuss new perspectives in this research field. [ABSTRACT FROM AUTHOR]

Strain CpI1T was, in 1978, the first isolate of the genus Frankia to be obtained from Comptonia peregrina root nodules. In this study, a polyphasic approach was performed to identify the taxonomic position of strain CpI1T among the members of the genus Frankia. The strain contains meso-diaminopimelic acid as the diagnostic diamino acid and galactose, glucose, mannose, rhamnose, ribose and xylose as cell wall sugars. The polar lipids were found to consist of phosphatidylinositol, diphosphatidylglycerol, glycophospholipids, phosphatidylglycerol, an aminophospholipid and unidentified phospholipids and lipids. The predominant menaquinone was identified as MK-9 (H8), while the major fatty acid are iso-C16:0 and C17:1ω 8c. The 16S rRNA gene sequence identity varies from 97.4 to 99.6% with the type strains of currently described Frankia species. Phylogenetic analyses based on 16S rRNA gene sequences and multi-locus sequence analysis (MLSA) using atp1, ftsZ, dnaK, gyrA and secA gene sequences showed that strain CpI1T is closely related to Frankia alni ACN14aT. The genome size of strain CpI1T is 7.6 Mb with a digital DNA G+C content of 72.4%. Digital DNA:DNA hybridization (values between strain CpI1T and its close phylogenetic relative F. alni ACN14aT was 44.1%, well below the threshold of 70% for distinguishing between bacterial genomic species. Based on the phenotypic, phylogenetic and genomic data, strain CpI1T (= DSM44263T = CECT9035T) warrants classification as the type strain of a novel species, for which the name Frankia torreyi sp. nov. is proposed. [ABSTRACT FROM AUTHOR]

Molecular analysis of the filamentous actinobacteria Frankia is laborious because of the slow growth rate and required biomass needed for these techniques. An efficient and simple colony PCR protocol for Frankia was developed that saved time for analysis of any Frankia strains growing on a plate. Previously, it took 5-6 weeks to get the correct size Frankia colonies on plates and then a minimum of 5 weeks of growth in liquid culture for DNA extraction. With this technique, these colonies could be screened after 5-6 weeks of growth by colony PCR. The procedure used a combination of mechanical and heat treatments and required no added buffers or chemicals. Our results demonstrate rapid and efficient PCR. [ABSTRACT FROM AUTHOR]

Alnus genus forms symbiosis with the actinobacteria Frankia and ectomycorrhizal fungi. Two types of Frankia lineages can be distinguished based on their ability to sporulate in planta. Spore-positive (Sp+) strains are predominant on Alnus incana (Ai) and Alnus viridis (Av) in highlands, while spore-negative (Sp-) strains are mainly associated with Alnus glutinosa (Ag) in lowlands. Here, we investigated whether the Sp+ predominance in nodules is due to host selection of certain Frankia genotypes from soil communities or the result of the ecological history of the alder stand soil, and the effect of the sporulation genotype on the ECM communities. Trapping experiments were conducted using Ag, Ai and Av plantlets on 6 soils, differing in the alder species and the frequency of Sp+ nodules in the field. Higher diversity of Frankia and variation in Sp+ frequencies were observed in the trapping compared to the fields. Both indigenous and trapping species shape Frankia community structure in trapped nodules. Nodulation impediments were observed on several trapping conditions in Sp+ soils supporting a narrower host-range of Sp+ Frankia. Ai and Av were able to associate equally with compatible Sp+ and Sp- in the greenhouse. Additionally, no host shift was observed for Alnus-specific ECM, and the sporulation genotype of Frankia defined the ECM communities on the host roots. The symbiotic association is likely determined by the host-range, the soil history and the type of in planta Frankia. These results provide an insight into the biogeographical drivers of alder symbionts in the Holarctic region. [ABSTRACT FROM AUTHOR]

Nowadays, nitrogen-fixing tree species are often planted in combination with poplar to provide nutrients, though this can reduce the number of host plants and have a negative impact on soil Frankia population. In order to study the effects of mixed plantation of Caucasian alder (Alnus subcordata) and poplar (Populus deltoides) on Frankia nodulation potential, an experiment was carried out within a randomized block design. Five different mixtures of 17–years old P. deltoides (P) and A. subcordata (A) (100A, 70A30p, 50A50P, 30A70 and 100P) were established in Safrabasteh Poplar Research Station in Guilan province of Iran. The results showed that the presence of alder in mixture with poplar positively affected the diameter at breast height (DBH) and height of poplar trees. Mixed plantations with alder can improve Soil pH and the amount of soil nutrients and organic carbon. Furthermore, foliar nutrient concentrations of poplar trees were higher in mixed plantations than in pure plantations. The results of Frankia population analysis showed significant difference between the soils across different species mixture classes. The highest and lowest levels of Frankia population were observed in the rhizosphere soil of pure alder (49.17 g-1 soil) and pure poplar (0.89 g-1 soil), respectively. The Frankia population was negatively correlated with amounts of available P and K, yet it was positively correlated with pH. In addition, Frankia population showed positive and negative correlations with alder DBH and height, respectively. These results confirmed the effects of plant-host density on the viability and population of Frankia.

Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF., (© 2023. Springer Nature Limited.)

Frankia strains isolated from Saudi Arabia, reported for the first time, were identified based on the morphological and molecular tools compared to those isolated from Egypt. All strains displayed typical morphological characterization of Frankia strains represented by branched hyphae, production of vesicles and sporangia. The phylogenetic analysis and relationships among Frankia strains were investigated by comparing 16S rRNA gene sequences. The analysis revealed three genetic groups which formed two clusters. The first cluster was composed of eight Frankia strains subdivided into two genetic groups (one group containing five strains; CgIT3L2, CgIS3N2, CgIS1N1, CgIT7N2, and G5; the other group included of three strains: CgIT5L3, CgIS1N2, and CcI13). The second cluster was composed of only one genetic group of Frankia strain CgIS3N1. The strains in each genetic group exhibited similar genetic distances. All Frankia strains were able to reinfect their host of Casuarina species. For ability of these strains to resist heavy metals, our results proved that all Frankia strains isolated can resist Cu, Co, and Zn at low concentration except Pb which exhibit highly toxic effect at the same concentration used. Frankia strain G5 was proved to be the most resistant strain for heavy metals tested. [ABSTRACT FROM AUTHOR]

Summary: Nitrogen‐fixing filamentous Frankia colonize the root tissues of its actinorhizal host Discaria trinervis via an exclusively intercellular pathway. Here we present studies aimed at uncovering mechanisms associated with this little‐researched mode of root entry, and in particular the extent to which the host plant is an active partner during this process. Detailed characterization of the expression patterns of infection‐associated actinorhizal host genes has provided valuable tools to identify intercellular infection sites, thus allowing in vivo confocal microscopic studies of the early stages of Frankia colonization. The subtilisin‐like serine protease gene Dt12, as well as its Casuarina glauca homolog Cg12, are specifically expressed at sites of Frankia intercellular colonization of D. trinervis outer root tissues. This is accompanied by nucleo‐cytoplasmic reorganization in the adjacent host cells and major remodeling of the intercellular apoplastic compartment. These findings lead us to propose that the actinorhizal host plays a major role in modifying both the size and composition of the intercellular apoplast in order to accommodate the filamentous microsymbiont. The implications of these findings are discussed in the light of the analogies that can be made with the orchestrating role of host legumes during intracellular root hair colonization by nitrogen‐fixing rhizobia. [ABSTRACT FROM AUTHOR]

Summary: We investigated whether the diversity, endemicity and specificity of alder symbionts could be changed by isolation in a Mediterranean glacial refugium. We studied both ectomycorrhizal (EM) fungi and nitrogen‐fixing actinobacteria associated with alders, and compared their communities in Corsica and on the European continent. Nodules and root tips were sampled on the three alder species present in Corsica and continental France and Italy. Phylogenies based on internal transcribed spacer (ITS) and a multilocus sequence analysis approach were used to characterize fungal and Frankia species, respectively. Patterns of diversity, endemism and specialization were compared between hosts and regions for each symbiont community. In Corsica, communities were not generally richer than on the mainland. The species richness per site depended mainly on host identity: Alnus glutinosa and Alnus cordata hosted richer Frankia and EM communities, respectively. Half of the Frankia species were endemic to Corsica against only 4% of EM species. Corsica is not a hotspot of diversity for all alder symbionts but sustains an increased frequency of poor‐dispersers such as hypogeous fungi. Generalist EM fungi and host‐dependent profusely sporulating (Sp+) Frankia were abundantly associated with Corsican A. cordata, a pattern related to a more thermophilic and xerophylic climate and to the co‐occurrence with other host trees. [ABSTRACT FROM AUTHOR]

Diazotrophic Actinobacteria of the genus Frankia represent a challenge to classical bacterial taxonomy as they include many unculturable strains. As a consequence, we still have a poor understanding of their diversity, evolution and biogeography. In this study, a Multi-Locus Sequence Analysis (MLSA) using atpD , dnaA , ftsZ , pgk, and rpoB loci was done on a large set of cultured and uncultured strains, compared to 16S rRNA and correlated to Average Nucleotide Identity (ANI) from available Frankia genomes. MLSA provided a robust resolution of Frankia genus phylogeny and clarified the status of unresolved species and complex of species. The robustness of single-gene topologies and their congruence with the MLSA tree were tested. Lateral Gene Transfers (LGT) were few and scattered, suggesting they had no impact on the concatenate topology. The pgk marker – providing the longest sequence, highest mean genetic divergence and least occurrence of LGT – was used to survey an unequalled number of Alnus -infective Frankia — mainly uncultured strains from a broad range of host-species and geographic origins. This marker allowed reliable Single-Locus Strain Typing (SLST) below the species level, revealed an undiscovered taxonomical diversity, and highlighted the effect of cultivation, sporulation phenotype and host plant species on symbiont richness, diversity and phylogeny. [ABSTRACT FROM AUTHOR]

Actinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genus Frankia, and specific Frankia taxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated (Alnus glutinosa, Alnus cordata, Shepherdia argentea, Casuarina equisetifolia) while others were not (Alnus viridis, Hippophaë rhamnoides). Nodule populations were represented by eight different sequences of nifH gene fragments. Two of these sequences characterized frankiae in S. argentea nodules, and three others characterized frankiae in A. glutinosa nodules. Frankiae in A. cordata nodules were represented by five sequences, one of which was also found in nodules from A. glutinosa and C. equisetifolia, while another was detected in nodules from A. glutinosa. Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e., nifH or the 23S rRNA gene). Targeted Illumina sequencing of Frankia-specific nifH gene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils. [ABSTRACT FROM AUTHOR]

Sea buckthorn ( Hippophae rhamnoides L.) is a pioneer plant used for land reclamation and an appropriate material for studying the interactions of symbiotic microorganisms because of its nitrogen-fixing root nodules and mycorrhiza. We used high-throughput sequencing to reveal the diversities and community structures of rhizospheric fungi and their link with nitrogen-fixing Frankia harbored in sea buckthorn collected along an altitude gradient from the Qinghai Tibet Plateau to interior areas. We found that the fungal diversities and compositions varied between different sites. Ascomycota, Basidiomycota, and Zygomycota were the dominant phyla. The distribution of sea buckthorn rhizospheric fungi was driven by both environmental factors and the geographic distance. Among all examined soil characteristics, altitude, AP, and pH were found to have significant ( p < 0.05) effect on the rhizospheric fungal community. The rhizospheric fungal communities became more distinct as the distance increased. Moreover, co-inertia analysis identified significant co-structures between Frankia and AMF communities in the rhizosphere of sea buckthorn. We conclude that at the large scale, there are certain linkages between nitrogen-fixing bacteria and the AMF expressed in the distributional pattern. [ABSTRACT FROM AUTHOR]

Background Recent climatic history has strongly impacted plant populations, but little is known about its effect on microbes. Alders, which host few and specific symbionts, have high genetic diversity in glacial refugia. Here, we tested the prediction that communities of root symbionts survived in refugia with their host populations. We expected to detect endemic symbionts and a higher species richness in refugia as compared to recolonized areas. Methods We sampled ectomycorrhizal (EM) root tips and the nitrogen-fixing actinomycete Frankia communities in eight sites colonized by Alnus glutinosa subsp. barbata close to the Caucasus in Georgia. Three sites were located in the Colchis, one major Eurasian climatic refugia for Arcto-Tertiary flora and alders, and five sites were located in the recolonized zone. Endemic symbionts and plant ITS variants were detected by comparing sequences to published data from Europe and another Tertiary refugium, the Hyrcanian forest. Species richness and community structure were compared between sites from refugia and recolonized areas for each symbionts. Results For both symbionts, most MOTUs present in Georgia had been found previously elsewhere in Europe. Three endemic Frankia strains were detected in the Colchis vs two in the recolonized zone, and the five endemic EM fungi were detected only in the recolonized zone. Frankia species richness was higher in the Colchis while the contrary was observed for EM fungi. Moreover, the genetic diversity of one alder specialist Alnicola xanthophylla was particularly high in the recolonized zone. The EM communities occurring in the Colchis and the Hyrcanian forests shared closely related endemic species. Discussion The Colchis did not have the highest alpha diversity and more endemic species, suggesting that our hypothesis based on alder biogeography may not apply to alder’s symbionts. Our study in the Caucasus brings new clues to understand symbioses biogeography and their survival in Tertiary and ice-age refugia, and reveals that isolated host populations could be of interest for symbiont diversity conservation.

Molecular signaling networks in the actinorhizal rhizosphere select host-compatible Frankia strains, trigger the infection process and eventually the genesis of nitrogen-fixing nodules. The molecular triggers involved remain difficult to ascertain. Root exudates (RE) are highly dynamic substrates that play key roles in establishing the rhizosphere microbiome. RE are known to induce the secretion by rhizobia of Nod factors, polysaccharides, and other proteins in the case of legume symbiosis. Next-generation proteomic approach was here used to decipher the key bacterial signals matching the first-step recognition of host plant stimuli upon treatment of Frankia coriariae strain BMG5.1 with RE derived from compatible (Coriaria myrtifolia), incompatible (Alnus glutinosa), and non-actinorhizal (Cucumis melo) host plants. The Frankia proteome dynamics were mainly driven by host compatibility. Both metabolism and signal transduction were the dominant activities for BMG5.1 under the different RE conditions tested. A second set of proteins that were solely induced by C. myrtifolia RE and were mainly linked to cell wall remodeling, signal transduction and host signal processing activities. These proteins may footprint early steps in receptive recognition of host stimuli before subsequent events of symbiotic recruitment.