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Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Plants within the Nitrogen-fixing Clade (NFC) of Angiosperms form root nodule symbioses with nitrogen-fixing bacteria. Actinorhizal plants (in Cucurbitales, Fagales, Rosales) form symbioses with the actinobacteria Frankia while legumes (Fabales) form symbioses with proteobacterial rhizobia. Flavonoids, secondary metabolites of the phenylpropanoid pathway, have been shown to play major roles in legume root nodule symbioses: as signal molecules that in turn trigger rhizobial nodulation initiation signals and acting as polar auxin transport inhibitors, enabling a key step in nodule organogenesis. To explore a potentially broader role for flavonoids in root nodule symbioses across the NFC, we combined metabolomic and transcriptomic analyses of roots and nodules of the actinorhizal host Datisca glomerata and legumes of the genus Medicago. Patterns of biosynthetic pathways were inferred from flavonoid metabolite profiles and phenylpropanoid gene expression patterns in the two hosts to identify similarities and differences. Similar classes of flavonoids were represented in both hosts, and an increase in flavonoids generally in the nodules was observed, with differences in flavonoids prominent in each host. While both hosts produced derivatives of naringenin, the metabolite profile in D. glomerata indicated an emphasis on the pinocembrin biosynthetic pathway, and an abundance of flavonols with potential roles in symbiosis. Additionally, the gene expression profile indicated a decrease in expression in the lignin/monolignol pathway. In Medicago sativa, by contrast, isoflavonoids were highly abundant featuring more diverse and derived isoflavonoids than D. glomerata. Gene expression patterns supported these differences in metabolic pathways, especially evident in a difference in expression of cinnamic acid 4-hydroxylase (C4H), which was expressed at substantially lower levels in D. glomerata than in a Medicago truncatula transcriptome where it was highly expressed. C4H is a major rate-limiting step in phenylpropanoid biosynthesis that separates the pinocembrin pathway from the lignin/monolignol and naringenin-based flavonoid branches. Shikimate O-hydroxycinnamoyltransferase, the link between flavonoid biosynthesis and the lignin/monolignol pathway, was also expressed at much lower levels in D. glomerata than in M. truncatula. Our results indicate (a) a likely major role for flavonoids in actinorhizal nodules, and (b) differences in metabolic flux in flavonoid and phenylpropanoid biosynthesis between the different hosts in symbiosis.

Two types of nitrogen-fixing root nodule symbioses are known, rhizobial and actinorhizal symbioses. The latter involve plants of three orders, Fagales, Rosales, and Cucurbitales. To understand the diversity of plant symbiotic adaptation, we compared the nodule transcriptomes of Datisca glomerata (Datiscaceae, Cucurbitales) and Ceanothus thyrsiflorus (Rhamnaceae, Rosales); both species are nodulated by members of the uncultured Frankia clade, cluster II. The analysis focused on various features. In both species, the expression of orthologs of legume Nod factor receptor genes was elevated in nodules compared to roots. Since arginine has been postulated as export form of fixed nitrogen from symbiotic Frankia in nodules of D. glomerata, the question was whether the nitrogen metabolism was similar in nodules of C. thyrsiflorus. Analysis of the expression levels of key genes encoding enzymes involved in arginine metabolism revealed up-regulation of arginine catabolism, but no up-regulation of arginine biosynthesis, in nodules compared to roots of D. glomerata, while arginine degradation was not upregulated in nodules of C. thyrsiflorus. This new information corroborated an arginine-based metabolic exchange between host and microsymbiont for D. glomerata, but not for C. thyrsiflorus. Oxygen protection systems for nitrogenase differ dramatically between both species. Analysis of the antioxidant system suggested that the system in the nodules of D. glomerata leads to greater oxidative stress than the one in the nodules of C. thyrsiflorus, while no differences were found for the defense against nitrosative stress. However, induction of nitrite reductase in nodules of C. thyrsiflorus indicated that here, nitrite produced from nitric oxide had to be detoxified. Additional shared features were identified: genes encoding enzymes involved in thiamine biosynthesis were found to be upregulated in the nodules of both species. Orthologous nodule-specific subtilisin-like proteases that have been linked to the infection process in actinorhizal Fagales, were also upregulated in the nodules of D. glomerata and C. thyrsiflorus. Nodule-specific defensin genes known from actinorhizal Fagales and Cucurbitales, were also found in C. thyrsiflorus. In summary, the results underline the variability of nodule metabolism in different groups of symbiotic plants while pointing at conserved features involved in the infection process.

Root nodule symbiosis (RNS) is a symbiotic interaction established between angiosperm hosts and nitrogen-fixing soil bacteria in specialized organs called root nodules. The host plants provide photosynthate and the microsymbionts supply fixed nitrogen. The origin of RNS represents a major evolutionary event in the angiosperms, and understanding the genetic underpinnings of this event is of major economic and agricultural importance. Plants that engage in RNS are restricted to a single angiosperm clade known as the nitrogen-fixing clade (NFC), yet occur in multiple lineages scattered within the NFC. It has been postulated that RNS evolved in two steps: a gain-of-predisposition event occurring at the base of the NFC, followed by a gain-of-function event in each host plant lineage. Here, we first explore the premise that RNS has evolved from a single common background, and then we explore whether a two-step process better explains the evolutionary origin of RNS than either a single-step process, or multiple origins. We assembled the transcriptomes of root and nodule of two actinorhizal plants, Ceanothus thyrsiflorus and Datisca glomerata. Together with the corresponding published transcriptomes of the model legume Medicago truncatula, the gene expression patterns in roots and nodules were compared across the three lineages. We found that orthologs of many genes essential for RNS in the model legumes are expressed in all three lineages, and that the overall nodule gene expression patterns were more similar to each other than expected by random chance, a finding that supports a common evolutionary background for RNS shared by the three lineages. Moreover, phylogenetic analyses suggested that a substantial portion of the genes experiencing selection pressure changes at the base of the NFC also experienced additional changes at the base of each host plant lineage. Our results (1) support the occurrence of an event that led to RNS at the base of the NFC, and (2) suggest a subsequent change in each lineage, most consistent with a two-step origin of RNS. Among several conserved functions identified, strigolactone-related genes were down-regulated in nodules of all three species, suggesting a shared function similar to that shown for arbuscular mycorrhizal symbioses.

BackgroundIdentifying orthologous genes is an initial step required for phylogenetics, and it is also a common strategy employed in functional genetics to find candidates for functionally equivalent genes across multiple species. At the same time, in silico orthology prediction tools often require large computational resources only available on computing clusters. Here we present OrthoReD, an open-source orthology prediction tool with accuracy comparable to published tools that requires only a desktop computer. The low computational resource requirement of OrthoReD is achieved by repeating orthology searches on one gene of interest at a time, thereby generating a reduced dataset to limit the scope of orthology search for each gene of interest.ResultsThe output of OrthoReD was highly similar to the outputs of two other published orthology prediction tools, OrthologID and/or OrthoDB, for the three dataset tested, which represented three phyla with different ranges of species diversity and different number of genomes included. Median CPU time for ortholog prediction per gene by OrthoReD executed on a desktop computer was

The actinobacterial genus Frankia establishes nitrogen-fixing root nodule symbioses with specific hosts within the nitrogen-fixing plant clade. Of four genetically distinct subgroups of Frankia, cluster I, II, and III strains are capable of forming effective nitrogen-fixing symbiotic associations, while cluster IV strains generally do not. Cluster II Frankia strains have rarely been detected in soil devoid of host plants, unlike cluster I or III strains, suggesting a stronger association with their host. To investigate the degree of host influence, we characterized the cluster II Frankia strain distribution in rhizosphere soil in three locations in northern California. The presence/absence of cluster II Frankia strains at a given site correlated significantly with the presence/absence of host plants on the site, as determined by glutamine synthetase (glnA) gene sequence analysis, and by microbiome analysis (16S rRNA gene) of a subset of host/nonhost rhizosphere soils. However, the distribution of cluster II Frankia strains was not significantly affected by other potential determinants such as host-plant species, geographical location, climate, soil pH, or soil type. Rhizosphere soil microbiome analysis showed that cluster II Frankia strains occupied only a minute fraction of the microbiome even in the host-plant-present site and further revealed no statistically significant difference in the α-diversity or in the microbiome composition between the host-plant-present or -absent sites. Taken together, these data suggest that host plants provide a factor that is specific for cluster II Frankia strains, not a general growth-promoting factor. Further, the factor accumulates or is transported at the site level, i.e., beyond the host rhizosphere. Biological nitrogen fixation is a bacterial process that accounts for a major fraction of net new nitrogen input in terrestrial ecosystems. Transfer of fixed nitrogen to plant biomass is especially efficient via root nodule symbioses, which represent evolutionarily and ecologically specialized mutualistic associations. Frankia spp. (Actinobacteria), especially cluster II Frankia spp., have an extremely broad host range, yet comparatively little is known about the soil ecology of these organisms in relation to the host plants and their rhizosphere microbiomes. This study reveals a strong influence of the host plant on soil distribution of cluster II Frankia spp.

BackgroundThe ability to establish root nodule symbioses is restricted to four different plant orders. Soil actinobacteria of the genus Frankia can establish a symbiotic relationship with a diverse group of plants within eight different families from three different orders, the Cucurbitales, Fagales and Rosales. Phylogenetically, Frankia strains can be divided into four clusters, three of which (I, II, III) contain symbiotic strains. Members of Cluster II nodulate the broadest range of host plants with species from four families from two different orders, growing on six continents. Two Cluster II genomes were sequenced thus far, both from Asia.ResultsIn this paper we present the first Frankia cluster II genome from North America (California), Dg2, which represents a metagenome of two major and one minor strains. A phylogenetic analysis of the core genomes of 16 Frankia strains shows that Cluster II the ancestral group in the genus, also ancestral to the non-symbiotic Cluster IV. Dg2 contains the canonical nod genes nodABC for the production of lipochitooligosaccharide Nod factors, but also two copies of the sulfotransferase gene nodH. In rhizobial systems, sulfation of Nod factors affects their host specificity and their stability.ConclusionsA comparison with the nod gene region of the previously sequenced Dg1 genome from a Cluster II strain from Pakistan shows that the common ancestor of both strains should have contained nodABC and nodH. Phylogenetically, Dg2 NodH proteins are sister to rhizobial NodH proteins. A glnA-based phylogenetic analysis of all Cluster II strains sampled thus far supports the hypothesis that Cluster II Frankia strains came to North America with Datisca glomerata following the Madrean-Tethyan pattern.

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.

Background: The ability to establish root nodule symbioses is restricted to four different plant orders. Soil actinobacteria of the genus Frankia can establish a symbiotic relationship with a diverse group of plants within eight different families from three different orders, the Cucurbitales, Fagales and Rosales. Phylogenetically, Frankia strains can be divided into four clusters, three of which (I, II, III) contain symbiotic strains. Members of Cluster II nodulate the broadest range of host plants with species from four families from two different orders, growing on six continents. Two Cluster II genomes were sequenced thus far, both from Asia. Results: In this paper we present the first Frankia cluster II genome from North America (California), Dg2, which represents a metagenome of two major and one minor strains. A phylogenetic analysis of the core genomes of 16 Frankia strains shows that Cluster II the ancestral group in the genus, also ancestral to the non-symbiotic Cluster IV. Dg2 contains the canonical nod genes nodABC for the production of lipochitooligosaccharide Nod factors, but also two copies of the sulfotransferase gene nodH. In rhizobial systems, sulfation of Nod factors affects their host specificity and their stability. Conclusions: A comparison with the nod gene region of the previously sequenced Dg1 genome from a Cluster II strain from Pakistan shows that the common ancestor of both strains should have contained nodABC and nodH. Phylogenetically, Dg2 NodH proteins are sister to rhizobial NodH proteins. A glnA-based phylogenetic analysis of all Cluster II strains sampled thus far supports the hypothesis that Cluster II Frankia strains came to North America with Datisca glomerata following the Madrean-Tethyan pattern. [ABSTRACT FROM AUTHOR]

Frankia cluster-2 strains are diazotrophs that engage in root nodule symbiosis with actinorhizal plants of the Cucurbitales and the Rosales. Previous studies have shown that an assimilated nitrogen source, presumably arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), while a different metabolite is exported in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported to the host by cluster-2 strains, and which metabolite would be exported in Ceanothus, we analysed gene expression levels, metabolite profiles, and enzyme activities in nodules. We conclude that the export of assimilated nitrogen in symbiosis seems to be a common feature for Frankia cluster-2 strains, but which source is host-dependent. The export of assimilated ammonium to the host suggests that 2-oxoglutarate is drawn from the TCA cycle at a high rate. This specialised metabolism obviates the need for the reductive branch of the TCA cycle. We found several genes encoding enzymes of the central carbon and nitrogen metabolism were lacking in Frankia cluster-2 genomes: the glyoxylate shunt and succinate semialdehyde dehydrogenase. This led to a linearization of the TCA cycle, and we hypothesize this could explain the low saprotrophic potential of Frankia cluster-2., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Lysin-motif receptor-like kinases (LysM-RLKs) are involved in the recognition of microbe-associated molecular patterns to initiate pattern-triggered immunity (PTI). LysM-RLKs are also required for recognition of microbe-derived symbiotic signal molecules upon establishing mutualistic interactions between plants and microsymbionts. A LysM-RLK CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) plays central roles both in chitin-mediated PTI and in arbuscular mycorrhizal symbiosis, suggesting the overlap between immunity and symbiosis, at least in the signal perception and the activation of downstream signal cascades. In this study, we screened for the interacting proteins of Nod factor Receptor1 (NFR1), a CERK1 homolog in the model legume Lotus japonicus , and obtained a protein orthologous to NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13), a protein involved in the activation of innate immunity in Arabidopsis thaliana , which we named LjNHL13a. LjNHL13a interacted with NFR1 and with the symbiosis receptor kinase SymRK. LjNHL13a also displayed positive effects in nodulation. Our results suggest that NHL13 plays a role both in plant immunity and symbiosis, possibly where they overlap. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Two types of nitrogen-fixing root nodule symbioses are known, rhizobial and actinorhizal symbioses. The latter involve plants of three orders, Fagales, Rosales, and Cucurbitales. To understand the diversity of plant symbiotic adaptation, we compared the nodule transcriptomes of Datisca glomerata (Datiscaceae, Cucurbitales) and Ceanothus thyrsiflorus (Rhamnaceae, Rosales); both species are nodulated by members of the uncultured Frankia clade, cluster II. The analysis focused on various features. In both species, the expression of orthologs of legume Nod factor receptor genes was elevated in nodules compared to roots. Since arginine has been postulated as export form of fixed nitrogen from symbiotic Frankia in nodules of D. glomerata , the question was whether the nitrogen metabolism was similar in nodules of C. thyrsiflorus . Analysis of the expression levels of key genes encoding enzymes involved in arginine metabolism revealed up-regulation of arginine catabolism, but no up-regulation of arginine biosynthesis, in nodules compared to roots of D. glomerata , while arginine degradation was not upregulated in nodules of C. thyrsiflorus . This new information corroborated an arginine-based metabolic exchange between host and microsymbiont for D. glomerata , but not for C. thyrsiflorus. Oxygen protection systems for nitrogenase differ dramatically between both species. Analysis of the antioxidant system suggested that the system in the nodules of D. glomerata leads to greater oxidative stress than the one in the nodules of C. thyrsiflorus , while no differences were found for the defense against nitrosative stress. However, induction of nitrite reductase in nodules of C. thyrsiflorus indicated that here, nitrite produced from nitric oxide had to be detoxified. Additional shared features were identified: genes encoding enzymes involved in thiamine biosynthesis were found to be upregulated in the nodules of both species. Orthologous nodule-specific subtilisin-like proteases that have been linked to the infection process in actinorhizal Fagales, were also upregulated in the nodules of D. glomerata and C. thyrsiflorus . Nodule-specific defensin genes known from actinorhizal Fagales and Cucurbitales, were also found in C. thyrsiflorus. In summary, the results underline the variability of nodule metabolism in different groups of symbiotic plants while pointing at conserved features involved in the infection process.

Root nodule symbiosis (RNS) is a symbiotic interaction established between angiosperm hosts and nitrogen-fixing soil bacteria in specialized organs called root nodules. The host plants provide photosynthate and the microsymbionts supply fixed nitrogen. The origin of RNS represents a major evolutionary event in the angiosperms, and understanding the genetic underpinnings of this event is of major economic and agricultural importance. Plants that engage in RNS are restricted to a single angiosperm clade known as the nitrogen-fixing clade (NFC), yet occur in multiple lineages scattered within the NFC. It has been postulated that RNS evolved in two steps: a gain-of-predisposition event occurring at the base of the NFC, followed by a gain-of-function event in each host plant lineage. Here, we first explore the premise that RNS has evolved from a single common background, and then we explore whether a two-step process better explains the evolutionary origin of RNS than either a single-step process, or multiple origins. We assembled the transcriptomes of root and nodule of two actinorhizal plants, Ceanothus thyrsiflorus and Datisca glomerata . Together with the corresponding published transcriptomes of the model legume Medicago truncatula , the gene expression patterns in roots and nodules were compared across the three lineages. We found that orthologs of many genes essential for RNS in the model legumes are expressed in all three lineages, and that the overall nodule gene expression patterns were more similar to each other than expected by random chance, a finding that supports a common evolutionary background for RNS shared by the three lineages. Moreover, phylogenetic analyses suggested that a substantial portion of the genes experiencing selection pressure changes at the base of the NFC also experienced additional changes at the base of each host plant lineage. Our results (1) support the occurrence of an event that led to RNS at the base of the NFC, and (2) suggest a subsequent change in each lineage, most consistent with a two-step origin of RNS. Among several conserved functions identified, strigolactone-related genes were down-regulated in nodules of all three species, suggesting a shared function similar to that shown for arbuscular mycorrhizal symbioses.

The actinobacterial genus Frankia establishes nitrogen-fixing root nodule symbioses with specific hosts within the nitrogen-fixing plant clade. Of four genetically distinct subgroups of Frankia, cluster I, II, and III strains are capable of forming effective nitrogen-fixing symbiotic associations, while cluster IV strains generally do not. Cluster II Frankia strains have rarely been detected in soil devoid of host plants, unlike cluster I or III strains, suggesting a stronger association with their host. To investigate the degree of host influence, we characterized the cluster II Frankia strain distribution in rhizosphere soil in three locations in northern California. The presence/absence of cluster II Frankia strains at a given site correlated significantly with the presence/absence of host plants on the site, as determined by glutamine synthetase (glnA) gene sequence analysis, and by microbiome analysis (16S rRNA gene) of a subset of host/nonhost rhizosphere soils. However, the distribution of cluster II Frankia strains was not significantly affected by other potential determinants such as host-plant species, geographical location, climate, soil pH, or soil type. Rhizosphere soil microbiome analysis showed that cluster II Frankia strains occupied only a minute fraction of the microbiome even in the host-plant-present site and further revealed no statistically significant difference in the α-diversity or in the microbiome composition between the host-plant-present or -absent sites. Taken together, these data suggest that host plants provide a factor that is specific for cluster II Frankia strains, not a general growth-promoting factor. Further, the factor accumulates or is transported at the site level, i.e., beyond the host rhizosphere., Importance: Biological nitrogen fixation is a bacterial process that accounts for a major fraction of net new nitrogen input in terrestrial ecosystems. Transfer of fixed nitrogen to plant biomass is especially efficient via root nodule symbioses, which represent evolutionarily and ecologically specialized mutualistic associations. Frankia spp. (Actinobacteria), especially cluster II Frankia spp., have an extremely broad host range, yet comparatively little is known about the soil ecology of these organisms in relation to the host plants and their rhizosphere microbiomes. This study reveals a strong influence of the host plant on soil distribution of cluster II Frankia spp., (Copyright © 2016 American Society for Microbiology.)