Alone Yet Not Alone: Frankia Lives Under the Same Roof With Other Bacteria in Actinorhizal Nodules

Actinorhizal plants host mutualistic symbionts of the nitrogen-fixing actinobacterial genus Frankia within nodule structures formed on their roots. Several plant-growth-promoting bacteria have also been isolated from actinorhizal root nodules, but little is known about them. We were interested investigating the in planta microbial community composition of actinorhizal root nodules using culture-independent techniques. To address this knowledge gap, 16S rRNA gene amplicon and shotgun metagenomic sequencing was performed on DNA from the nodules of Casuarina glauca. DNA was extracted from C. glauca nodules collected in three different sampling sites in Tunisia, along a gradient of aridity ranging from humid to arid. Sequencing libraries were prepared using Illumina NextEra technology and the Illumina HiSeq 2500 platform. Genome bins extracted from the metagenome were taxonomically and functionally profiled. Community structure based off preliminary 16S rRNA gene amplicon data was analyzed via the QIIME pipeline. Reconstructed genomes were comprised of members of Frankia, Micromonospora, Bacillus, Paenibacillus, Phyllobacterium, and Afipia. Frankia dominated the nodule community at the humid sampling site, while the absolute and relative prevalence of Frankia decreased at the semi-arid and arid sampling locations. Actinorhizal plants harbor similar non-Frankia plant-growth-promoting-bacteria as legumes and other plants. The data suggests that the prevalence of Frankia in the nodule community is influenced by environmental factors, with being less abundant under more arid environments.

Draft Genome Sequence for Frankia sp. Strain BMG5.11, a Nitrogen-Fixing Bacterium Isolated from Elaeagnus angustifolia

ABSTRACT Frankia sp. strain BMG5.11, which was isolated from Elaeagnus angustifolia nodules, is able to infect other actinorhizal plants, including Elaeagnaceae, Rhamnaceae, Colletieae, Gymnostoma, and Myricaceae. Here, we report the 11.3-Mbp draft genome sequence of Frankia sp. strain BMG5.11, with a G+C content of 69.9% and 9,926 candidate protein-encoding genes.

Evolution of NIN and NIN-like Genes in Relation to Nodule Symbiosis

Legumes and actinorhizal plants are capable of forming root nodules symbiosis with rhizobia and Frankia bacteria. All these nodulating species belong to the nitrogen fixation clade. Most likely, nodulation evolved once in the last common ancestor of this clade. NIN (NODULE INCEPTION) is a transcription factor that is essential for nodulation in all studied species. Therefore, it seems probable that it was recruited at the start when nodulation evolved. NIN is the founding member of the NIN-like protein (NLP) family. It arose by duplication, and this occurred before nodulation evolved. Therefore, several plant species outside the nitrogen fixation clade have NLP(s), which is orthologous to NIN. In this review, we discuss how NIN has diverged from the ancestral NLP, what minimal changes would have been essential for it to become a key transcription controlling nodulation, and which adaptations might have evolved later.

The δ15N value of N2 fixing actinorhizal plants and legumes grown with N2 as the only nitrogen source

The aim of this study was to investigate the effects of different plant parts and the age of plants at harvest as well as N2 fixing bacterial strains on the N concentration in symbiotic plant parts, especially on the δ15N signature of the actinorhizal plants and legumes. The 15N natural abundance method was used. Two actinorhizal plants were studied: Alnus incana (L.) infected with the Frankia strains ArI3 or “lsF” (local source of Frankia) and Hippophaë rhamnoides (L.) infected with the Frankia strains T1 or E15b. Two legume species were studied: Hedysarum coronarium (L.), infected with a soil suspension, and Robinia pseudoacacia (L.), infected with a crushed nodule suspension. It was particularly interesting that in A. incana, the two Frankia strains affected not only N concentration and δ15N signature of leaves and roots, but also had an impact on plant growth at first harvest. In Hippophaë rhamnoides plants inoculated with the Frankia strains T1 and E15b, N concentrations and δ15N values did not differ at any harvest time. However, plants nodulated by the Frankia strain T1 showed a higher nitrogen fixation rate and higher plant dry matter at all harvesting times. Based on our results for the quantification of N2 fixation with the “B” value, that is the δ15N value of the N2 fixing plants relying only on N2 fixation, plant parts, ages and strains should be carefully considered.

Metallophore profiling of nitrogen-fixingFrankiaspp. to understand metal management in the rhizosphere of actinorhizal plants

Frankiaspp. are widespread nitrogen-fixing and metallophore releasing soil bacteria, which often live in symbiosis with a broad spectrum of hosts.

FrankiaDiversity in Host Plant Root Nodules Is Independent of Abundance or Relative Diversity ofFrankiaPopulations in Corresponding Rhizosphere Soils

ABSTRACTActinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genusFrankia, and specificFrankiataxonomic 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 ofnifHgene fragments. Two of these sequences characterized frankiae inS. argenteanodules, and three others characterized frankiae inA. glutinosanodules. Frankiae inA. cordatanodules were represented by five sequences, one of which was also found in nodules fromA. glutinosaandC. equisetifolia, while another was detected in nodules fromA. glutinosa. Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e.,nifHor the 23S rRNA gene). Targeted Illumina sequencing ofFrankia-specificnifHgene 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 detectableFrankiapopulations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specificFrankiapopulations in soils.IMPORTANCEThe nitrogen-fixing actinobacteriumFrankiaforms root nodules on actinorhizal plants, with members of specificFrankiataxonomic clusters nodulating plants in corresponding host infection groups. We assessedFrankiadiversity in root nodules of different host plant species, and we related specific populations to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Large differences were observed between detectableFrankiapopulations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specificFrankiapopulations in soils but rather results from plants potentially selecting frankiae from the soil for root nodule formation. These data also highlight the necessity of using a combination of different assessment tools so as to adequately address methodological constraints that could produce contradictory data sets.