An overview of Parafrankia (Nod+/Fix+) and Pseudofrankia (Nod+/Fix-) interactions through genome mining and experimental modeling in co-culture and co-inoculation of Elaeagnus angustifolia.

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix- phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogen-depleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. IMPORTANCE: While Pseudofrankia strains typically lack the common traits of ability to nodulate the host plant (Nod-) and/or fix nitrogen (Fix-), they are still recovered from actinorhizal nodules. The enigmatic question of how and why these unconventional strains establish themselves within nodule tissue, thriving either alongside symbiotic strains (Nod+/Fix+) or independently, while considering potential metabolic costs to the host plant, remains a perplexing puzzle. This study endeavors to unravel the competitive dynamics between Pseudofrankia inefficax strain EuI1c (Nod+/Fix-) and Parafrankia strain EU1Nf (Nod+/Fix+) through a comprehensive exploration of genomic data and empirical modeling, conducted both in controlled laboratory settings and within the host plant environment.

Cyclic diarylheptanoids as potential signal compounds during actinorhizal symbiosis between Alnus sieboldiana and Frankia.

The nitrogen-fixing actinomycete Frankia coexists with actinorhizal plants via nodules and supplies nitrogen compounds to the plants. Although communication has been suggested to exist through chemical substances in this nodule symbiosis, the details underlying this mechanism remain elusive. The biphenyl-type diarylheptanoids (BP-CDHs), alnusonol, and alnusdione, previously isolated from the actinorhizal plant A. sieboldiana branch wood, are secondary metabolites that accumulate in a limited number of plant species. However, since relatively widely distributed in actinorhizal plants, we investigated whether adding A. sieboldiana root extracts and these BP-CDHs could affect plant seedlings inoculated with Frankia. The results showed that the addition of root extract or alnusonol significantly increased the number of nodules and lobes more than two times compared with that upon Frankia supplementation only. We also proved that the extracted components of this plant affected nodule symbiosis. Finally, we confirmed through LC-MS that the root extract component contained BP-CDH, alnusonol. The above-described results indicate that BP-CDHs, at leaset alnusonol, might function as signal compounds from the plant side of the actinorhizal symbiosis between A. sieboldiana and Frankia.

Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules.

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing "ecometabolome" approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

Detoxification and reduction of selenite to elemental red selenium by Frankia.

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.

Transcription factors network in root endosymbiosis establishment and development.

Root endosymbioses are mutualistic interactions between plants and the soil microorganisms (Fungus, Frankia or Rhizobium) that lead to the formation of nitrogen-fixing root nodules and/or arbuscular mycorrhiza. These interactions enable many species to survive in different marginal lands to overcome the nitrogen-and/or phosphorus deficient environment and can potentially reduce the chemical fertilizers used in agriculture which gives them an economic, social and environmental importance. The formation and the development of these structures require the mediation of specific gene products among which the transcription factors play a key role. Three of these transcription factors, viz., CYCLOPS, NSP1 and NSP2 are well conserved between actinorhizal, legume, non-legume and mycorrhizal symbioses. They interact with DELLA proteins to induce the expression of NIN in nitrogen fixing symbiosis or RAM1 in mycorrhizal symbiosis. Recently, the small non coding RNA including micro RNAs (miRNAs) have emerged as major regulators of root endosymbioses. Among them, miRNA171 targets NSP2, a TF conserved in actinorhizal, legume, non-legume and mycorrhizal symbioses. This review will also focus on the recent advances carried out on the biological function of others transcription factors during the root pre-infection/pre-contact, infection or colonization. Their role in nodule formation and AM development will also be described.

Identification of potential transcriptional regulators of actinorhizal symbioses in Casuarina glauca and Alnus glutinosa.

BACKGROUND: Trees belonging to the Casuarinaceae and Betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. These functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus Frankia. However, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. The aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses. RESULTS: We identified 202 putative transcription factors by in silico analysis in 40 families in Casuarina glauca (Casuarinaceae) and 195 in 35 families in Alnus glutinosa (Betulaceae) EST databases. Based on published transcriptome datasets and quantitative PCR analysis, we found that 39% and 26% of these transcription factors were regulated during C. glauca and A. glutinosa-Frankia interactions, respectively. Phylogenetic studies confirmed the presence of common key transcription factors such as NSP, NF-YA and ERN-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. We also identified an actinorhizal-specific transcription factor belonging to the zinc finger C1-2i subfamily we named CgZF1 in C. glauca and AgZF1 in A. glutinosa. CONCLUSIONS: We identified putative nodulation-associated transcription factors with particular emphasis on members of the GRAS, NF-YA, ERF and C2H2 families. Interestingly, comparison of the non-legume and legume TF with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific C2H2 TF that could be specifically involved in actinorhizal symbioses. In silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to Frankia infection.

Regulation of nitrogen metabolism in salt tolerant and salt sensitive Frankia strains.

Effect of salinity (0, 50, 100, 250, 500 and 750 mM NaCI) was observed on some important physiological parameters of nitrogen metabolism such as nitrate uptake, intracellular and extracellular ammonium status and activities of nitrogenase, nitrate reductase, nitrite reductase and glutamine synthetase among Frankia strains differing in their salt tolerance capacity. Nitrogenase activity closely followed the growth pattern with regular decline on NaCI supplementation. All the other enzymes showed optimum activity at 100 mM and declined further. Co-regulation of the nitrate uptake system and sequential enzyme activities plays a crucial role in governing the nitrogen status of strains during salt stress. HsIil0 experiencing minimum decline in enzyme activities and best possible nitrogen regulation under NaC1 replete condition showed adequate nutritional management. Among all the strains, HsIil0 proved to be salt tolerant on account of above features while the salt sensitive strain HsIi8 lacked the ability to regulate various steps of nitrogen metabolism during salinity, and thus Frankia strain HsIil0 can potentially serve as a potential biofertilizer in the saline soil.

Influence of iron and chelator on siderophore production in Frankia strains nodulating Hippophae salicifolia D. Don.

Effect of iron and chelator on the growth and siderophore production in the ten newly Frankia strains isolated from the root nodules of Hippophae salicifolia D. Don and the two reference strains were studied. Growth of the strains was greatly affected when grown in the iron and EDTA deprived conditions. All the strains were capable of producing both the hydroxamate and catecholate type siderophore that was detected using the Csaky and Arnow assays. Production of siderophore was enhanced in the EDTA replenish condition in contrast to the iron supplemented medium suggesting that EDTA reduces the availability of other free metals and hence creates the stress condition for which the secretion of siderophore is enhanced. A decrease in siderophore production was observed with an increase in iron concentration. Strains HsIi2 and HsIi10 were found to be producing more siderophore than the other strains.