NodD1 and NodD2 Are Not Required for the Symbiotic Interaction of Bradyrhizobium ORS285 with Nod-Factor-Independent Aeschynomene Legumes

Photosynthetic Bradyrhizobium strain ORS285 forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. Depending on the Aeschynomene species, this symbiotic interaction does or does not rely on the synthesis of Nod-factors (NFs). However, whether during the interaction of Bradyrhizobium ORS285 with NF-independent Aeschynomene species the nod genes are expressed and if the general regulator NodD plays a symbiotic role is unknown. Expression studies showed that in contrast to the interaction with the NF-dependent Aeschynomene species, A. afraspera, the Bradyrhizobium ORS285 nod genes are not induced upon contact with the NF-independent host plant A. indica. Mutational analysis of the two nodD genes present in ORS285, showed that deletion of nodD1 and nodD2 did not affect the symbiotic interaction between Bradyrhizobium ORS285 and A. indica whereas the deletions had an effect on the symbiotic interaction with A. afraspera plants. In addition, when the expression of nod genes was artificially induced by adding naringenin to the plant growth medium, the nodulation of A. indica by Bradyrhizobium ORS285 is delayed and resulted in lower nodule numbers.


Introduction
Legume plants have developed symbiotic associations with specific soil bacteria, collectively referred to as rhizobia, which allow the plants to grow in nitrogen poor soils. This symbiosis results in the formation of a new plant organ, the nodule, cells that are intracellularly infected by the rhizobia which differentiate into nitrogen-fixing bacteroids. Some Aeschynomene spp. belonging to the Dalbergioid clade, a pantropical large group of papilionoid legumes, symbiotically interact with photosynthetic bradyrhizobia [1,2] and have the unusual property of forming nodules on both the root and the stem [3]. This stem nodulation can be very profuse resulting in high nitrogen fixation rates. This particularity renders some Aeschynomene spp. ideal candidate as "green manure" for rice production [4]. Among the photosynthetic bradyrhizobia two groups of host specificity can be distinguished [5]. Group I contains strains with a broad host range that extends to all stem nodulating Aeschynomene species, whereas group II contains strains that are only able to nodulate a few stem nodulating Aeschynomene species, including A. indica, A. evenia and A. sensitiva. Sequencing the genomes of two photosynthethic bradyrhizobia belonging to host specificity group II, i.e. ORS278 and BTAi1, showed that the genes coding for enzymes involved in synthesis of the core structure of Nodfactors (NFs; nodABC) were absent [6]. This indicates that some photosynthetic Bradyrhizobium spp. use a novel mechanism, independent of the long-time considered universal NFs, to symbiotically interact with Aeschynomene plants.
The absence of nod genes is not a general rule among photosynthetic bradyrhizobia. Group I strains, such as the model strain Bradyrhizobium ORS285, do contain nodABC genes [7]. Interestingly, these nod genes are essential for the symbiosis with some Aeschynomene spp., such as A. afraspera, but they are dispensable for symbiosis with Aeschynomene spp. nodulated by group II strains, such as A. indica [6]. Thus, two types of interactions can be considered in Aeschynomene-photosynthetic Bradyrhizobium symbiosis: 1) a classical one which is NFdependent and 2) an atypical one which is NF-independent and it is the Aeschynomene species that determines the modus operandi.
In the classical NF-dependent symbiosis, initiation of nodule development involves the exchange of molecular signals between both partners. Flavonoids exuded by plant roots are supposed to be the first signals received by the rhizobia. Flavonoids likely diffuse into the rhizobial cytoplasm where they interact with NodD proteins that belong to the LysR family of transcriptional regulators, and trigger a signal transduction cascade that plays a role in the infection process [8]. Studies of genomes of rhizobia indicate that, depending on the rhizobial species, there are one to five copies of nodD. In species that possess only one nodD copy a mutation usually results in the loss of nodulation, whereas, in the presence of multiple copies, mutations do not always affect nodulation [9][10][11]. In the genome of Bradyrhizobium ORS285 two genes have been annotated as nodD (nodD1 and nodD2) [12]. NodD proteins have been shown to binds to conserved 49 bp motifs (so called nod-boxes) that are found in the promoters of nodulation (nod, nol and noe) genes [13,14]. Most nodulation genes that are expressed in a flavonoid-and NodD-dependent manner are involved in the synthesis and secretion of NFs, rhizobial lipochito-oligosaccharide signal molecules (LCOs). NFs are recognized by plant kinases of the LysM-RLKs family, which, upon activation, initiate a developmental program in the legume host leading to the formation of the nodule structure [15]. Besides induction of genes involved in the synthesis of NFs, flavonoid-and NodD-dependent expression has been demonstrated for genes involved in the synthesis of hopanoids, rhizopine catabolism, nitrogen fixation, synthesis and/or modification of polysaccharides and expression of transcriptional regulators (TtsI) involved in the expression of Type III secretion systems [16]. As all these processes have been suggested to play a role in the establishment of a successful nodulation, the NodD protein(s) can be designed as the central regulator(s) in rhizobial nodulation.
Upon naringenin addition to the growth medium, the major NF synthesized by Bradyrhizobium strain ORS285 is a pentameric lipochitooligosaccharide (LCO) with a 2-O-methylfucose at the reducing end [12]. This suggests that the interaction between ORS285 strain and NFdependent Aeschynomene species involves a classical molecular dialogue (i.e. induction by host plant flavonoids and subsequent synthesis of NF). However, whether the ORS285 strain uses the same or a different molecular dialogue when interacting with NF-independent Aeschynomene plants is not known. Therefore, the aim of this study was to determine whether the nodgenes of the ORS285 strain are expressed during the interaction with NF-independent Aeschynomene plants, and to determine the role of the nodulation regulators NodD1 and NodD2 in this interaction.

Root exudate isolation and qualitative analysis by high performance liquid chromatography
A. afraspera and A. indica seeds were surface sterilized and germinated on 0.8% agar plates. After germination, seedlings were grown for 7 days in 48-well plates containing 5.8 ml BNM medium [19]. Root exudate of eight 48-well plates (total volume:~2 liter) was filtered (45 μm filter) and concentrated to~100 ml using a rota evaporator (40°C). The concentrated root exudate was centrifuged (15 minutes Ã g) and flavonoids present in the exudate were isolated by solid phase extraction using a SepPak C18 column (1 column / 25 ml concentrated root exudate). The eluted fractions (in 100% methanol) were combined, dried under vacuum and the weight of the dry pellet was determined. The flavonoids were dissolved in 50% ethanol (~20 mg/ml) and 150 μg of material was analysed on a high-performance liquid chromatography system (Waters 2690 separation module, Waters, Milford, Massachusetts, USA) equipped with a analytic C18 reverse phase column (5 μm, 4.6 mm x 250 mm, Symmetry C18, Waters) using an isocratic gradient of solvent A (water-methanol, 70:30 [vol/vol]) for 5 min, followed by a sixty-minutes linear gradient from solvent A to solvent B (100% methanol) and finally an isocratic gradient of solvent B for 10 min, at a flow rate of 1 ml min-1. The eluent of the HPLC column was monitored at 206-400 nm.

Nod gene inducing capabilities of pure flavonoids and root exudate
The Bradyrhizobium ORS285 nodA-lacZ reporter strain was used to assess the capacities of root exudates from A. indica and A. afraspera, respectively, to induce the expression of the nodA-lacZ transcriptional fusion. Purified flavonoids were purchased from Extrasynthese (Genay, France) or Sigma-Aldrich (St. Louis, USA). Bradyrhizobium ORS285::nodA-lacZ was grown in BNM-B medium till an optical density of OD600~0.4. Subsequently, the bacterial culture was diluted into fresh BNM-B medium (OD600 = 0.1) and supplemented with root exudate or pure flavonoids. After 24 hours of growth at 28°C, the absorbance at 600 nm of the cultures were determined and βgalactosidase activities were measured according to the method of Miller (Miller 1972). Experiments were at least repeated twice with three technical replicates in one experiment.

Plant growth and acetylene reduction assay
Sterilization of seeds, germination, plant growth and inoculation with bacterial strains were as described [12]. At 4, 5, 6, 7, 8, 10, 12 and 14 days after inoculation, the number of nodules on the roots were counted. The acetylene reduction assay (ARA) was used to measure the nitrogenase enzyme activity 14 days after inoculation [12].

Root exudate of a NF-independent Aeschynomene species does not induce nod gene expression in Bradyrhizobium ORS285
Bradyrhizobium ORS285 interacts with A. afraspera and A. indica using a NF-dependent and NF-independent process, respectively. To determine whether nod genes are induced upon interaction with NF-independent Aeschynomene plants, we extracted and concentrated the root exudate of 7-day old A. indica seedlings using a C18 solid-phase extraction protocol. As control, we also extracted root exudate of the NF-dependent host plant, A. afraspera. Qualitative analysis of the C18-extracted root exudate by reversed phase high-performance liquid chromatography showed clear differences in chromatographic pattern between the two Aeschynomene species (Fig 1A and 1B). The UV-VIS spectra of the majority of peaks present in the chromatograms of both root exudates contained two absorbance bands (one in the 310-385 nm region, band A and one in the 250-295 nm region, band B) which are typical for flavonoids (S1 and S2 Figs). To determine the expression of the nod genes (nodA-J operon) upon root exudate addition, we used a Bradyrhizobium ORS285 reporter strain which contained a nodA-lacZ transcriptional fusion on the chromosome. In the absence or presence of root exudate from A. indica plants, we measured a low (basal) β-glactosidase activity with the reporter strain ( Fig  1C). In contrast, upon addition of the flavonoid naringenin or the root exudate of A. afraspera plants to the bacterial growth medium, we measured a concentration-dependent increase in the β-galactosidase activity (Fig 1C). During the solid phase extraction procedure, flavonoids or other molecules that induce nod gene expression could have been lost. To analyze this possibility, we inoculated 7-day old seedlings with the reporter strain and measured the β-galactosidase activity of the cells present in the plant culture medium after 3 days of incubation. Cells present in the plant culture medium of A. afraspera plants have a 3-fold higher β-galactosidase activity as compared to cells present in plant culture medium of A. indica plants or cells that had been incubated in plant growth medium alone (Fig 1D). To analyze whether A. indica root exudate contains compounds that inhibit nod-gene expression, we grew the ORS285 nodA-lacZ reporter strain with a high concentration of C18-extracted A. indica exudate and increasing amounts of naringenin. In the presence of A. indica root exudate, the naringenin-dependent increase in β-galactosidase -activity was under all conditions slightly higher as observed with cells grown in the presence of naringenin alone (Fig 2). Based on above results, we concluded that A. indica root exudate does not contain molecules that induce or repress nod-geneexpression in Bradyrhizobium ORS285 cells.
NodD1 and NodD2 are not required for NF-independent nodulation of A. indica plants Like B. diazoefficiens USDA110, Bradyrhizobium ORS285 contains two genes (nodD1 and nodD2; accession numbers: FQ790406 and FQ790405, respectively) encoding for the symbiotic regulator NodD [12]. Both nodD genes are flanked by genes involved in C4 dicarboxylate metabolism (nodD1, dctB/D; nodD2, dctA). In addition, nodD2 is found close to a putative nolA gene which encodes a protein involved in the cell density control mechanism of NF biosynthesis in B. diazoefficiens [20], (Fig 3). The nodD1 and nodD2 genes were inactivated by deletion of more than 95% of the coding region and insertion of a chloramphenicol and streptomycine resistance cassette (omega interposon) in the remaining part of the gene, respectively. Bradyrhizobium strain ORS285 forms nodules on A. indica using a NF-independent symbiotic process [6]. To investigate whether nodD1 or nodD2 deletion affects the symbiosis with A. indica, we infected this plant with the ORS285 ΔnodD1 and ORS285 ΔnodD2 mutant, respectively, and followed the kinetics of nodule formation. In both cases, no effect of the mutation could be detected on the kinetics of nodule formation and the nitrogenase activity of the plants (Fig 4A and 4B). Also the size and morphology of the ΔnodD1 and ΔnodD2 nodules were similar as the WT nodules ( Fig 4C). In contrast, when the NF-dependent host plant, A. afraspera, was inoculated with the nodD mutants, no nodules were formed (ΔnodD1) or a delay in nodule formation and reduction in nodule number and nitrogenase enzyme activity was observed (ΔnodD2) (Fig 5A and 5B). In addition, the ORS285 ΔnodD2 nodules lack the atypical superficial outgrowth as observed on nodules of A. afraspera plants inoculated with WT ORS285 (Fig 5C) [21]. These results indicate that in contrast to the interaction with A. afraspera both NodD1 and NodD2 do not play a role in the symbiotic interaction between Bradyrhizobium ORS285 and A. indica plants.

Expression of nod genes delays nodulation of A. indica plants
Above we have shown that NodD1 and NodD2 are not required for the establishment of the symbiosis between ORS285 and A. indica and that the genes involved in the synthesis of NFs  are not induced during this interaction. One can ask the question what will happen when nod genes are expressed during the interaction with A. indica. Does nod-gene expression interferes positively or negatively on this interaction or are A. indica plants completely "blind" to NFs produced upon nod gene expression? To address this question, we have inoculated 7-day-old A. indica seedlings with Bradyrhizbium ORS285 in the presence of the nod-gene inducing flavonoid naringenin (20 μM). Analysis of the nodulation kinetics showed that the appearance of the first nodules was delayed in the presence of naringenin (Fig 6A). Interestingly, this delay in nodule organogenesis was not observed with the ORS285 ΔnodD1 and ORS285 ΔnodA-J mutant strains suggesting that it is most probably a consequence of NF synthesis (Fig 6B and  6C). Although the addition of naringenin delayed the nodulation of plants by the WT strain (Fig 6A), the nitrogenase activity of the plants at 14 dpi was comparable with that of plants inoculated in the absence of naringenin (Fig 6D). Altogether, these observations suggest that forcing the synthesis of NFs in the ORS285 strain interferes negatively with the establishment of the NF-independent symbiosis but has no effect on the functioning of the nodules.

Discussion
Previously, we have shown that the symbiotic interaction between photosynthetic Bradyrhizobium strain ORS285 and certain tropical aquatic legumes of the Aeschynomene genus, e.g. A. afraspera, involves a classical molecular dialogue (i.e., induction of nod genes by host plant flavonoids and subsequent synthesis of LCO) (12). However, with certain species of the Aeschynomene genus, e.g. A. indica, Bradyrhizobium ORS285 is able to establish a symbiotic interaction in a NF-independent manner [6]. Little is known about the molecular mechanism of the NFindependent symbiosis of ORS285 with A. indica. Are NFs synthesized during this interaction? Do NFs interfere positively or negatively with the establishment of the NF-independent symbiosis? Does the regulator NodD known to control the expression of nod genes but also many other symbiosis related genes play a role in the NF-independent symbiosis?
Using a nodA-lacZ reporter strain, we showed that the nod-genes are not expressed during the interaction of Bradyrhizobium ORS285 with the NF-independent host plant A. indica. Qualitative analysis of the C18-extracted root exudate of A. afraspera and A. indica, respectively, by reversed-phase chromatography showed drastic differences between the two plant species (Fig 1A and 1B). For both root exudates, most compounds isolated after C18 extraction have a typical flavonoid UV-VIS spectrum (S1 and S2 Figs). Moreover, A. indica root exudate does not inhibit the naringenin-stimulated nod-gene expression. The absence of nod-geneinducing flavonoids in A. indica root exudates is thus likely the explanation for the absence of nodA-J gene expression with this plant. The obtained results, however, do not rule out an important role of flavonoids as plant signal and developmental regulator in NF-independent symbiosis. In the NF-independent symbiosis between Frankia and actinorhizal plants, flavonoids have been shown to play an important role [22]. In this context, several pure flavonoids, like kaempherol and quercetein, that do not induce nod gene expression stimulate the growth of the ORS285 strain at micromolar concentrations (S3 Fig). A similar growth stimulation is observed with very low amounts of A. indica root exudate (S3 Fig), suggesting that it contains molecules that are recognized by the ORS285 strain. Currently, we are comparing the transcriptomic profile of ORS285 cells grown in the presence of root exudate from A. afraspera and A. indica plants, respectively, to identify genes that are specifically expressed upon interaction with NF-independent Aeschynomene species. Also silencing the flavonoid biosynthetic pathway in A. indica plants will shine a light on the role of flavonoids in NF-independent Aeschynomene symbiosis.
NodD proteins are members of the LysR family of transcriptional regulators [23] and play a key role in the activation of transcription of nod genes. Like B. diazoefficiens USDA110, Bradyrhizobium ORS285 contains two nodD genes, nodD1 and nodD2. NodD1 or nodD2 deletion has no effect on the interaction with the NF-independent Aeschynomene species, A. indica. In contrast, deletion of nodD1 completely abolished nodulation of A. afraspera plants, whereas nodulation of A. afraspera by the nodD2 mutant was retarded.
Nodules of A. afraspera contain an atypical superficial outgrowth containing bacterial infected cells [21]. Recently, it has been shown that A. afraspera nodules formed by an ORS285 Type III secretion mutant (rhcN) lack this superficial outgrowth [24]. Interestingly, nodules formed by the ORS285 ΔnodD2 mutant also lack this outgrowth. In some other rhizobia, NodD1 proteins regulate the expression of ttsI, which encodes for a regulator of Type III secretion functions [16,25]. Future studies, using lacZ/gusA reporter strains should clarify if a similar T3SS regulation-cascade is present in the ORS285 strain and if in this cascade NodD2 plays a role.
Here, we demonstrated that nod(A-J) genes are not expressed upon contact with A. indica plants and that deletions of nodD1 and nodD2, respectively, do not affect the interaction of Bradyrhizobium ORS285 with A. indica. This suggests that in Bradyrhizobium ORS285 there is no overlap between the NF-dependent and NF-independent mechanism to interact with different host plants. To investigate what happens when the nod-gene-dependent pathway is induced during the interaction with a NF-independent host plant, we have infected A. indica plants with Bradyrhizobium ORS285 in the presence of 20 μM of the nod gene-inducing flavonoid naringenin. Naringenin addition to the plant culture medium delays the nodulation of A. indica plants by Bradyrhizobium ORS285 whereas it had no effect on nodulation of A. indica plants by the ΔnodD1 and ΔnodA-J mutant strains. This suggests that the observed delay is due to the induction of nod-gene expression and consequent NF synthesis. Why would nod-gene expression be incompatible with nodulation of NF-independent host plants? One possibility could be that the induction of synthesis of NFs alters the physiology of the ORS285 strain and that this distinct physiology negatively affects the interaction with A. indica plants. Another possibility is that in NF-independent Aeschynomene species (a) dedicated LysM-RLK receptor (s) play a role in the perception of bacterial signals leading to nodule organogenesis. For the classical NF-dependent symbiosis two putative models have been proposed to explain the evolutionary conserved dual function of LysM-RLK receptors in immune response and nodulation. (a) Perception of NFs modulates the balance between different LysM-RLK receptor complexes, favoring a symbiotic complex at the expense of complexes required for immune responses. (b) Early immune responses are co-opted to facilitate symbiont infection. Tight regulation of the receptor complexes at the post-translational level, involving rapid endocytotic turnover, subsequently prevents activation of defense responses (for review, see [26]). Both models can explain how the artificially induced NFs could interfere with the nodulation of NFindependent Aeschynomene species when they are recognized and go into competition with the so far unknown bacterial factor(s) inducing nodulation. Currently, we are analyzing genomic/ transcriptomic data from the model NF-independent Aeschynomene species, A. evenia, for the presence of LysM-RLK receptors with the goal to analyze their role in the nodule organogenesis signaling cascade. In addition, future experiments using pure NFs and transcriptomic analysis will help to identify how NFs are recognized by NF-independent Aeschynomene species and why this interferes with nodule organogenesis.
Here, we showed that the nod(A-J) genes of Bradyrhizobium ORS285 are only expressed upon contact with the NF-dependent Aeschynomene species, A. afraspera. Although the nodD1 containing region is absolutely essential and nodD2 stimulatory for the interaction of Bradyrhizobium ORS285 with A. afraspera, no effect of a nodD1 or nodD2 deletion has been observed for the interaction with the NF-independent Aeschynomene species, A. indica. This suggests that Bradyrhizbium ORS285 uses two completely separated mechanisms to interact with NF-dependent and NF-independent host plants. Bradyrhizobium ORS285 is thus an ideal model strain to study and compare the two symbiotic mechanisms that photosynthetic bradyrhizobia use to interact with their host plants.