Abstract
The beta-rhizobial strain Paraburkholderia phymatum STM815T is noteworthy for its wide host range in nodulating legumes, primarily mimosoids (over 50 different species) but also some papilionoids. It cannot, however, nodulate soybean (Glycine max [L.] Merr.), one of the world’s most important crops. Here, we constructed a highly saturated genome-wide transposon library of a P. phymatum strain and employed a transposon sequencing (Tn-seq) approach to investigate the underlying genetic mechanisms of symbiotic incompatibility between P. phymatum and soybean. Soybean seedlings inoculated with the P. phymatum Tn-seq library display nodules on the roots that are mainly occupied by different mutants in a gene, nodS, coding for a methyltransferase involved in the biosynthesis of nodulation factors. The construction of a nodS deletion strain and a complemented mutant confirms that nodS is responsible for the nodulation-incompatibility of P. phymatum with soybean. Moreover, infection tests with different host plants reveal that NodS is necessary for optimal nodulation of common bean (Phaseolus vulgaris), but it is not required for nodulation of its natural host Mimosa pudica. In conclusion, our results suggest that NodS is involved in determining nodulation specificity of P. phymatum.
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Introduction
Rhizobia are a polyphyletic group of proteobacteria that engage in a nitrogen-fixing mutualistic relationship with certain plants, but overwhelmingly with legumes1,2,3,4,5. The first rhizobia discovered belonged to the class of alphaproteobacteria, and approximately one century later, some strains belonging to the betaproteobacteria class were also included in the rhizobia group. Accordingly, they were named alpha- and beta-rhizobia1,6,7,8. Rhizobia are of great agricultural importance, as they play a crucial role in enhancing nitrogen availability in nutrient-poor soils through the reduction of atmospheric nitrogen gas (N2) to ammonia (NH3)9,10. This reaction takes place within specialized root organs known as nodules11,12,13,14. The formation of functional nodules is a highly orchestrated process that involves two synchronized events: root-infection and nodule organogenesis15. However, to form a successful symbiosis, the legume and symbiont must be compatible16. The symbiotic interaction begins with the secretion of phenolic compounds (i.e. flavonoids) by legume roots to attract suitable rhizobia present in the rhizosphere. Flavonoids activate the expression of the rhizobial transcriptional regulator NodD, a LysR-type regulator that controls the expression of a group of genes involved in the synthesis and export of nodulation factors (NFs), encoded by the nod genes17,18. The NFs are then recognized by plant membrane proteins called NF receptors (NFRs), which initiate a complex signaling cascade that results in nodule organogenesis13. Rhizobia then attach to and are trapped by root hairs and migrate through infection threads until they are accommodated intracellularly in the nodule primordium where they differentiate into N2-fixing bacteroids11,12,19,20. NFs are lipo-chitooligosaccharides (LCOs) composed of a core structure of two to six N-acetyl-D-glucosamine residues, a fatty acid chain at the non-reducing terminal residue, and species-/strain-specific residues that decorate the core structure, providing specificity16,21,22. The nod genes shared by alpha- and beta-rhizobia are nodD, nodABC (synthesis of the NF backbone), and nodIJT (export of NF)18,23,24. The noe, nol and the remaining nod genes (nodHLPQSUXZ) are strain specific and differ across the two classes of rhizobia, even between species within the same genus. The products of the strain-specific nod genes are involved in modifying the NF core by introducing modifications such as the addition of methyl (nodS), carbamoyl (nodU and nolO) or sulfate (nodH) groups, which determine the host specificity of the NFs16,22. Most rhizobia nodulate a limited range of legume species, and NFs, along with root exudates and polysaccharides, for instance, contribute to this specificity25,26. However, this rule is not followed by Sinorhizobium (Ensifer) fredii NGR234 (alpha-rhizobia)27 or by Paraburkholderia phymatum STM815T (beta-rhizobia)22,28,29,30,31. Sinorhizobium fredii NGR234 has been shown to produce more than 80 structurally different NFs and to nodulate more than 120 legume genera as well as the non-legume Parasponia andersonii (Cannabaceae)27,32,33,34. Given its broad host range, it has been employed as a model organism to understand the genetic basis of promiscuity in the rhizobium-legume symbiosis35,36,37. Paraburkholderia phymatum STM815T, one of the first isolated beta-rhizobia, also stands out among the beta-rhizobia due to its ability to nodulate more than 50 different legume species but also to its high competitiveness against other beta-rhizobia (Paraburkholderia, Cupriavidus, Trinickia) and alpha-rhizobia strains35,36,37,38,39. Paraburkholderia phymatum STM815T nodulates Phaseolus vulgaris (common bean), Macroptilum atropurpureum (siratro), Vigna unguiculata (cowpea), and numerous Mimosa species28,30,31,40,41. On the other hand, it cannot nodulate the agriculturally important soybean (Glycine max)29 and the reason for this failure is unknown29. In fact, most studies on beta-rhizobia have focused on the phylogeny of common nod genes (such as nodA or nodD) to explain their origin, contrary to many alpha-rhizobia wherein the nod genes and their products have been extensively studied and phenotypically characterized8,16,42.
Transposon (Tn) mutagenesis sequencing (Tn-seq) has become a very powerful high-throughput technology that combines Tn mutagenesis and next-generation sequencing43. In this approach, genes conferring a fitness advantage under the given condition are identified by the absence of Tn insertions compared to the standard growth condition, while those causing a disadvantage exhibit a greater number of insertions44,45,46. Tn-seq has been used to identify essential genes (also referred to as core genes) for growth under standard laboratory conditions, genes important after exposure to environmental stressors, and also to better understand the molecular basis of bacteria-host interactions47, including the alpha-rhizobia—legume symbiosis48,49,50,51,52. An example is the Bradyrhizobium diazoefficiens USDA 110Tspc4 (previously B. japonicum USDA110) Tn-seq library, which was used to identify putative essential genes during aerobic growth in yeast extract-arabinose-gluconate medium48. Moreover, a R. johnstonii sv. viciae Rlv3841T Tn-seq library, for example, was used to investigate genes involved in different stages of the colonization process of pea (Pisum sativum) nodules49, whereas in S. fredii CCBAU25509 a Tn-seq library was used to explore genes involved in the rhizoplane colonization of soybean, rice (Oryza sativa) and maize (Zea mays)52. The importance of the C4-dicarboxylate arginine transamination co-catabolism under acidic (H+) conditions to fix N2 (CATCH-N metabolic pathway) in the S. meliloti CL150-Medicago truncatula symbiosis was also discovered using Tn-seq50.
In this study, we constructed a highly saturated Tn-seq library of the beta-rhizobium P. phymatum STM815T, which was then used to investigate its plant-host specificity. To identify genetic elements involved in host incompatibility we selected for mutants that, in contrast to the wild-type strain, were able to nodulate soybean. The analysis of mutants proficient at nodulating soybean revealed that they harbored a Tn insertion in the nodS gene, suggesting that this methyltransferase suppresses the capacity of P. phymatum to nodulate this plant. The absence of nodS in P. phymatum also affected its nodulation efficacy on other legume hosts, implying that NodS is a key player for determining nodulation specificity.
Results
Construction of the P. phymatum transposon mutant library and determination of its core essential genes
To identify the core essential genes of P. phymatum STM815T and to screen for mutants with gain of nodulation in soybean, we created a saturated P. phymatum Tn library as detailed in the Methods section. The pool of P. phymatum mutants was grown on AB minimal medium supplemented with succinate (15 mM) at 28 °C. Our Tn-seq analysis revealed a total of 1,263,342 unique insertions in non-essential coding regions, which covered the full length of the P. phymatum STM815T genome (8,676,562 bp) with a frequency of approximately one insertion every 7 bp (Supplementary Table 1). The unique insertion density (UID) was calculated using Tn-seq Explorer (see Methods section) and a cutoff value of 0.012 was chosen according to the binomial distribution of the frequencies of the UID values. This means that genes with a UID lower than 0.012 (Supplementary Fig. 1) were considered to be part of the core genome. With this definition, 7.9% of the genome was essential (Supplementary Data 1). Of these 589 essential genes, 575 genes were annotated in at least one COG category (Fig. 1a). The categories of translation (J) (to which 98 essential genes were assigned), cell cycle control and mitosis (D) (21 essential genes), and nucleotide metabolism and transport (F) (48 essential genes) were the categories containing the highest percentage of essential genes (Fig. 1b). No essential genes belonged to the categories of chromatin structure and dynamics (B), cell motility (N), and extracellular structures (W), and only 33 genes were assigned to the category unknown function (S). According to our analysis, 89.1 % of the essential genes were located on chromosome 1 (NC_010622.1, 525 genes), 7.6% on chromosome 2 (NC_010623.1, 45 genes), 2.5% on plasmid pBPHY01 (NC_010625.1, 15 genes) and 0.7% on the symbiotic plasmid pBPHY02 (NC_010627.1, 4 genes) (Fig. 1b).
a P. phymatum STM815T essential genes distributed among the two chromosomes and two plasmids. From the outside in, the backbone of each replicon, the plus strand of CDS, the minus strand of CDS and location of essential genes (showed in blue). The segment brackets are displayed at 100 kb intervals. Image performed with Clico FS112. b Percentage of essential genes belonging to each COG (Cluster of Orthologous Genes) functional category106,113. Genes were annotated with eggNOG mapper v2107,108. Asterisks (*) indicate statistical significance of over-represented genes in a specific category (p-value < 0.01). The number of essential genes belonging to each COG category are annotated on top of its corresponding bar. B: Chromatin Structure and dynamics; C: energy production and conversion; D: cell cycle control and mitosis; E: amino acid metabolism and transport; F: nucleotide metabolism and transport; G: carbohydrate metabolism and transport; H: coenzyme metabolism; I: lipid metabolism; J: translation, ribosomal structure, and biogenesis; K: transcription; L: replication, recombination, and repair; M: cell wall, membrane, and envelop biogenesis; N: cell motility; O: post-translational modification, protein turnover, and chaperon; P: inorganic ion transport and metabolism; Q: secondary structure; S: function unknown; T: signal transduction mechanisms; U: intracellular trafficking, secretion, and vesicular transport; V: defense mechanisms; W: extracellular structures.
Inoculation of the P. phymatum Tn-seq library induced nodule formation on the non-host plant soybean
We employed our P. phymatum Tn-seq library to discover genetic elements that might impede nodulation of soybean (Fig. 2a). Paraburkholderia phymatum STM815T is able to nodulate more than 50 different types of legumes, most of them belonging to the genus Mimosa28,30,40. However, it cannot form nodules on the agriculturally important legume soybean29, thus it could be considered a non-host plant of this beta-rhizobium (Fig. 2b). We inoculated soybean seedlings (Glycine max cv. Williams 82) with either 107 cells of the saturated Tn-seq library or with the P. phymatum STM815T wild-type strain as a negative control and with the known soybean symbiont B. diazoefficiens USDA 110Tspc4 as a positive control (Fig. 2b). While inoculation with the P. phymatum wild-type failed to induce nodule formation by 21 days after inoculation, we obtained 48 nodules from seven plants inoculated with the P. phymatum Tn-seq library (Fig. 2b). Next, genomic DNA (gDNA) from these nodules was extracted and sequenced (see Methods section). The Tn-seq Explorer Software mapped the highest number of Tn insertions (59 unique insertion counts or UICs, and a UID of 0.094) at different sites of the gene Bphy_7716, which is also annotated as nodS coding for a S-adenosyl-L-methionine (SAM)-dependent methyltransferase (Fig. 3, Supplementary Data 2). As nodules are generally occupied by clonal populations derived from a single bacterium, we concluded that in most cases, each of the soybean nodules were induced by a single P. phymatum nodS-Tn mutant. However, the presence of different nodS-Tn mutants in the same nodule cannot be excluded, which is why we obtained 59 unique insertion mutants from 48 nodules (Fig. 3). Apart from nodS, Tn-seq found other genes with a maximum of two Tn insertions, but with a UID lower than 0.005 (Supplementary Data 2).
a Methodology used to inoculate and process the P. phymatum Tn-seq library on soybean seedlings. After 21 days, nodules containing Tn-seq mutants were collected, DNA from the nodules was extracted, and libraries were generated for sequencing using the circularization method. The obtained reads were mapped to the genome of P. phymatum STM815T and the exact position of the transposon (Tn) in the genome was determined. This figure was created anew by the authors using PowerPoint 2013 (Microsoft 365). b Soybean plants inoculated with B. diazoefficiens USDA 110Tspc4 (positive control), P. phymatum STM815T wild-type (negative control), and seven soybean plants inoculated with P. phymatum Tn-seq library with developed nodules (Tn-seq plants). Scale bar: 2 mm.
The unique counts approach of the Tn-seq Explorer software105 was used to identify genes that provided a fitness disadvantage for the colonizing by P. phymatum STM815T on the non-host plant soybean.
The absence of P. phymatum nodS induced nodule formation in soybean
The analysis of the Tn-mutants able to colonize soybean nodules indicated that nodS is counteracting soybean nodulation. We validated these Tn-seq data by constructing a deletion mutant strain, in which the nodS gene was replaced by a trimethoprim resistance cassette (Tm), and a complemented strain where nodS is provided in trans on the plasmid pBBR1MCS2. The P. phymatum nodS mutant as well as the complemented mutant strain showed no phenotypic difference from the wild-type strain in terms of growth in standard media, exopolysaccharide production, motility, and resistance to hydrogen peroxide and salt stress (Supplementary Table 2). To validate the results obtained by Tn-Seq, three biological replicates of the P. phymatum wild-type, nodS mutant and complemented strains were inoculated independently onto surface-sterilized germinated soybean seeds. The B. diazoefficiens USDA 110Tspc4 was used as a positive control, as was the P. phymatum wild-type and nodS mutant carrying the empty pBBR1MCS2 vector, which acted as controls to verify that the presence of pBBR1MCS2 did not affect the phenotype of the P. phymatum strains in planta. Twenty-one days after inoculation, only the P. phymatum nodS mutant, the nodS mutant carrying the empty pBBR1MCS2 and B. diazoefficiens induced nodules on soybean (Supplementary Fig. 2, Supplementary Fig. 3). Plants inoculated with the P. phymatum wild-type with and without the empty pBBR1MCS2 and the complemented nodS mutant failed to form nodules (Fig. 4a, Supplementary Fig. 3). The nodS mutant carrying the empty pBBR1MCS2 presented a significantly higher number of nodules than the nodS mutant, however, the dry weight of the nodules and the nitrogenase activity was not significantly different (Supplementary Fig. 3). The fact that the P. phymatum nodS mutant formed nodules on soybean and the complemented strain did not, confirmed that the presence of an intact nodS gene in P. phymatum is detrimental for soybean nodulation. Furthermore, the shoot dry weight (SDW) of the inoculated and uninoculated plants was not significantly different (Supplementary Fig. 4). Accordingly, the number of nodules induced by the P. phymatum nodS mutant as well as the nodule dry weight did not differ significantly to those induced by B. diazoefficiens (Fig. 4b). The N2-fixation efficiency of the P. phymatum nodS mutant and B. diazoefficiens nodules was then assessed. Nodules induced by the P. phymatum nodS mutant presented nitrogenase activity, albeit at a significantly lower rate (approximately 80% less) than B. diazoefficiens (Fig. 4c).
Soybean seedlings were inoculated with the P. phymatum STM815T wild-type (wild type), nodS mutant (nodS), complemented nodS mutant strains (nodS_C) and B. diazoefficiens USDA 110Tspc4 (110spc4). (a) The number of nodules per plant, (b) the dry weight per nodule and (c) the nitrogenase activity were characterized 21 days after inoculation. Three biological replicates per strain were tested, consisting of a total number of plants equal to or higher than eight plants. Error bars indicate the standard error of the mean (SEM). Significant difference between strains was analyzed with unpaired t test (p-value ≤ 0.05). n.s.: not significant; ****; p-value ≤ 0.0001.
A closer examination of the nodules induced by the P. phymatum nodS mutant revealed no significant histological variations from B. diazoefficiens nodules, with the exception of decreased leghemoglobin production in the nodS mutant nodules (Fig. 5a-d). Light microscopy of transversal sections of mature nodules revealed an internal anatomy typical of a determinate nodule, with P. phymatum nodS mutant bacteroids occupying intracellularly the plant cells in a comparable manner to B. diazoefficiens (Fig. 5e-f). The only discernible difference between the plant cells colonized by B. diazoefficiens and those colonized by the P. phymatum nodS mutant was that the latter appeared to occupy more plant cells. However, re-isolation of bacteroids from the nodules on three different selective media showed no significant differences between nodule occupancy among the strains (Supplementary Fig. 5 and Supplementary Fig. 6). When the nodules were examined under higher magnification light microscopy (Fig. 5g, h) it was apparent that the P. phymatum nodS-occupied cells were smaller than those occupied by B. diazoefficiens, but also less densely colonized by bacteroids (Fig. 5g,h). Transmission electron microscopy (TEM) further revealed that the P. phymatum nodS mutant bacteroids appeared to be degrading compared to the B. diazoefficiens bacteroids (Fig. 5i, j), with their symbiosomes coalescing into lytic vacuoles, suggesting premature senescence (Fig. 5j). Nevertheless, these findings still demonstrate that the absence of nodS allows P. phymatum to establish a viable, albeit functionally short-lived, symbiosis with the non-host plant soybean.
a Representative soybean root nodules induced by B. diazoefficiens USDA 110Tspc4 and (b) by the P. phymatum nodS mutant. c After cutting open the nodules in (a) and (b) the interior of (a) suggested a higher leghemoglobin content (red area in the nodule centre) in the B. diazoefficiens-colonized nodules (c) compared to those colonized by the P. phymatum nodS mutant (d). e Light micrograph of a transverse section of a nodule formed by B. diazoefficiens USDA 110Tspc4 and (f) of a nodule formed by the P. phymatum nodS mutant. g Semi-thin section of the nodule in e. h Semi-thin section of the nodule in f. i TEM of a nodule formed by B. diazoefficiens USDA 110Tspc4 and j of a nodule formed by the P. phymatum nodS mutant showing bacteroids being degraded (indicated with a black arrow) and lytic vacuoles enlarging (*). Scale bar (a–f): 500 µm; (g, h): 10 µm; (i, j): 500 nm.
NodS is important for nodulation of common bean, but it does not affect nodulation of Mimosa pudica
Common bean cv. Negro Jamapa and Mimosa pudica are both known hosts of P. phymatum STM815T22,28,30,31,38,39,41. To study the role of NodS in nodulating these two hosts, we inoculated seedlings of both with the P. phymatum wild-type, the nodS mutant, and the complemented nodS mutant, and then quantified the number of nodules, the dry weight per nodule and the nitrogenase activity. At 21 days after inoculation, the absence of the nodS gene reduced the number of nodules on common bean. Moreover, nodules colonized by the nodS mutant had a significantly higher dry weight and nitrogenase activity compared to those infected with the P. phymatum wild-type and nodS complemented strains. Specifically, the plants inoculated by the nodS mutant produced approximately a third less nodules and displayed roughly 2.5 times more nitrogenase activity when compared with those inoculated with the wild-type strain (Fig. 6a). Accordingly, the plants inoculated with the nodS complemented strain, showed a similar number of nodules and dry weight to those inoculated with the wild-type strain with similar levels of nitrogenase activity (Fig. 6a). Moreover, the presence of the empty pBBR1MCS2 vector in P. phymatum did not influence the outcome (Supplementary Fig. 7). In the case of M. pudica, by 28 days after inoculation, plants inoculated with the nodS mutant and the complemented nodS strains did not show significant differences in the number, in the dry weight of the nodules or in their nitrogenase activity compared to those inoculated with the P. phymatum wild-type strain (Fig. 6b), indicating that nodS is dispensable for establishing the P. phymatum-M. pudica symbiosis. We also determined the nodule occupancy by re-isolating the bacteroids from nodules. There was no significant difference between the strains in either host in all selective media we used for culturing (Supplementary Fig. 8). These findings indicate that i) P. phymatum nodS is important for nodulation of common bean and ii) the absence of nodS enhances their nitrogenase activity when inoculated.
Number of nodules, dry weight per nodule and relative normalized nitrogenase activity to the wild-type nitrogenase activity induced by the strains is illustrated for a common bean and b Mimosa pudica. At least three biological replicates per strain, a minimum of four plants per replicate were assayed. Error bars indicate the standard error of the mean (SEM). Significant difference between the mutant or complemented strain in comparison with the P. phymatum wild-type was analyzed with one-way ANOVA (p-value ≤ 0.05). Statistically significant differences are indicated with different letters (a–c).
Discussion
Paraburkholderia phymatum STM815T forms a functional symbiosis with a broad range of legumes, especially mimosoids like M. pudica, from which it most likely originated28,40,53. It has an outstanding ability to outcompete other beta-rhizobia in the genera Paraburkholderia, Cupriavidus, and Trinickia as well as alpha-rhizobial strains, and, moreover, is able to nodulate a very wide range of legumes, including several Mimosa spp., cowpea and common bean30,31,38,39. However, other important crops like soybean cannot be nodulated by P. phymatum STM815T for reasons that were unclear. In this study we constructed a P. phymatum Tn-seq library and used a gain-of-function approach to identify the genetic determinants that prevent nodulation of soybean by P. phymatum. The P. phymatum Tn-seq library presented in this work is based on the Tn5-derived transposon (Tn23). Although it should be noted that Tn5 has a preference for high GC regions, which could lead to inaccurate fitness assignments, we did not observe a location bias of the Tn in this P. phymatum Tn-seq library54. Tn5-based Tn-seq libraries present a very high coverage as evidenced by this P. phymatum Tn-seq library with Tn insertions in 97.4% of the genes and a frequency of one insertion every 7 bp (Supplementary Table 1). The P. phymatum STM815T genome has 7951 annotated CDS, of which 7.9% (equivalent to 589 genes) were considered by our Tn-seq analysis essential (with no insertions) for growth on minimal media with succinate as the sole carbon source (Fig. 1, Supplementary Data 1). This percentage of essential genes in the genome was consistent with prior Tn-seq studies on different Burkholderiaceae family members, which range from 5 to 10% of the genes under laboratory growth conditions51,55,56,57,58,59. The fact that categories N (cell motility) is found non-essential is in accordance with the settings of the experiment as motility has been reported as being unnecessary for E. coli strains growing in liquid medium with shaking60.
We successfully obtained soybean nodules colonized by P. phymatum Tn mutants. The analysis of these mutants identified nodS (Bphy_7716), which encodes a putative SAM-dependent methyltransferase that is presumably involved in the methylation of P. phymatum NFs. In this study we show that the presence of nodS prevents P. phymatum from nodulating soybean (Fig. 2b, Supplementary Data 2). In Bradyrhizobium sp. WM9 the three-dimensional structure of NodS has been elucidated61 and it was shown to methylate chitooligosaccharides (COS), specifically the N-deacetylated COS (metabolic product of NodBC) at their non-reducing end, before NodA acetylates the NF that is being synthesized62,63,64. Homologs of the nodS gene have been described and characterized in several Bradyrhizobium, Sinorhizobium, Rhizobium and Azorhizobium strains65,66,67. The NodS homolog of B. diazoefficiens USDA 110T, one of the most studied rhizobial partners of soybean, shares 56% identities with P. phymatum STM815T NodS. Previous studies have hypothesized that, despite the divergence between amino acid sequences of NodS in different rhizobia, NodS maintains its specific enzymatic activity. For instance, Azorhizobium caulinodans ORS571T produces N-methylated NFs although its NodS amino acid sequence shares few similarities with Bradyrhizobium sp. WM9 NodS61 (36% identities). In B. diazoefficiens, a nodS mutant had no effect on symbiosis with soybean, wild soybean (Glycine soja), cowpea (Vigna unguiculata), siratro and mung bean (Vigna radiata)65. However, we show here that in P. phymatum, a nodS mutant induced fewer nodules on common bean but they displayed a higher nitrogenase activity (Fig. 6). Previous studies have described this phenomenon in the rhizobia-legume symbiosis as resulting from a trade-off between the metabolic benefits of the symbiosis and the costs of nodulation68. However, in M. pudica, nodS is dispensable for M. pudica nodulation and its absence does not affect the efficiency of the symbiosis (Fig. 6). This phenotype in M. pudica contrasts with previous RNA-sequencing studies in P. phymatum grown in liquid culture showing that P. phymatum nodS is up-regulated in the presence of M. pudica root exudates69. Notably, rhizobia that have been reported to nodulate common bean, generally produce N-methylated NFs70,71,72, but the specific role of the NodS in rhizobial symbiosis seems to depend on both the rhizobial strain and the host legume. In the broad-host range legume nodulator S. fredii NGR234, NodS is important for the formation of functional nodules on the host legume Leucaena leucocephala. Moreover, a NGR234 nodS mutant shows reduced nodulation of cowpea compared to the wild-type strain and a delayed nodulation in cowpea and in Desmodium intortum66. It is important to mention that the nodS mutant of S. fredii NGR234 was not tested on non-host plants such as soybean as has been done in this study. In Rhizobium tropici CIAT899T NodS is essential for nodulation of the host plants L. leucocephala and common bean73. In A. caulinodans strain ORS571T, the mutation of the nodS gene results in a reduction in nodulation of its host Sesbania rostrata74. Interestingly, while the impairment to nodulate L. leucocephala by a S. fredii NGR234 nodSU mutant (nodU codes for a carbamoyl transferase) could not be complemented with the B. diazoefficiens homologs65,67, the addition of the S. fredii NGR234 nodABCSU operon to the R. etli CE-3 wild-type strain (lacking a nodS homologous gene) extended its host range to Leucaena species64,66,67. In B. diazoefficiens USDA110T, nodS is expressed at low levels in the presence of daidzein (an isoflavone known to induce the expression of B. diazoefficiens nod genes)65 and, as a consequence, NFs of B. diazoefficiens are expected to be non N-methylated. In fact, B. diazoefficiens USDA110T NFs have been characterized and described as a pentameric chitin backbone with C18:1, C16:0 or C16:1 fatty acid chains at the non-reducing end and a 2-O-methylfucose (MeFuc) at the reducing end. Specifically, the most abundant NF in the presence of genistein or soybean seed extract is NodBj-V(C18:1,MeFuc) while only an O-acetylated form (NodBj-V(Ac,C18:1,MeFuc)) is produced in trace amounts75. Similar to USDA110 NFs B. japonicum USDA 135 (also a nodulator of soybean) NFs are not methylated76, despite the presence of a NodS encoding gene in their genome75,77. However, in B. elkanii strain USDA 61T, another soybean symbiont, but which is known for nodulating a wide range of legumes such as black gram (Vigna mungo), mung bean, groundnut (Arachis hypogaea) and Aeschynomene spp.78,79, three out of the eight detected NF were N-methylated77. The absence of methylated NFs in strain USDA 110T is in accordance with the fact that a B. diazoefficiens USDA 110T nodS mutant fails to exhibit a different phenotype to the wild-type on all of its hosts74,75,80. To date, with the exception of the Papilionoid-nodulating P. tuberum STM678T7,81, the structure of NFs produced by P. phymatum and other legume-nodulating Paraburkholderia symbionts has not been elucidated and reported. Indeed, for Mimosa-nodulating beta-rhizobia, the main NF has been identified only for Cupriavidus taiwanensis LMG19424T; it contains vaccenic acid (C18:1), a N-methyl (NMe) and O-carbamoyl (Cb) group at the terminal non-reducing sugar and it is sulfated at the reducing end (S) (LCO-V (C18:1, NMe, Cb, S))82,83. The challenge inherent for the elucidation of the NF structure relates to the high degree of polymerization of the LCOs. Like chito and chitin oligosaccharides, LCOs are a chain of repeated units of N-acetyl-D-glucosamine that are difficult to separate due to their structural and molecular weight similarities. Deciphering the chemical structure of the NF produced by the P. phymatum STM815T wild type and its nodS mutant will contribute to unravelling the role of NodS in rhizobia-legume compatibility. It is plausible that plant-bacteria specificity does not rely on the presence/absence of NodS itself, but on how NodS could influence other NF modifications or N-methylations. In B. diazoefficiens USDA110T for example, the acetylation of C-6 at the non-reducing end of a N-glucosamine interferes with the NodS-dependent N-methylation. Beyond the elucidation of the NF structure of the P. phymatum wild-type and nodS mutant strains, it would be equally critical to determine at which level of the plant-bacteria signaling these different NFs trigger different reactions on host and non-host plants. Nod factors have been shown to induce root hair deformation, morphogenesis of the nodule structure and cortical cell division when they bind to the plant NFR84. This binding induces calcium spiking in the plant cells which activates the Calcium/Calmodulin-Dependent Kinase (CCaMK) inside the nucleus. CCaMK then activates transcriptional regulators involved in the symbiosis development, such as NIN, ERN1 and ERN2, as well as regulators of infection thread growth and nodule primordia, such as the nodulin genes ENOD11 and ENOD1284,85,86,87. In addition, NFs promote nodulation by reducing salicylic acid levels in the roots, which might aid to suppress the host defense response88. Despite the fact that soybean is not a host legume for P. phymatum STM815T, the nodS mutant was able to differentiate into N2-fixing bacteroids inside fully developed nodules, although with a lower N2-fixing efficiency than B. diazoefficiens (Fig. 4, Fig. 5). This lower activity is most likely due to limited compatibility, and hence the infected cells and the bacteroids therein senesce prematurely, as evidenced by the merging of symbiosomes into large lytic vacuoles89. Such symptoms of premature senescence are typical for nodules with incompatible partners, such as in leghemoglobin-deficient nodules90, or as observed in the specific case of B. diazoefficiens USDA110T when the rhizobial symbiont is lacking in alkane sulfate monooxygenase91.
In conclusion, in this study we used our P. phymatum Tn-seq library in a gain-of-function screen, to isolate mutant strains that are able to nodulate soybean. Our results open new research avenues for bioengineering competitive bioinoculants and contribute to the still largely unknown mechanisms underlying rhizobium—legume incompatibility92. Finally, our findings valorize the use of rhizobial Tn-seq libraries as a gain-of-function approach to identify bacterial traits that could be genetically modified with the aim of obtaining plant-growth promoting strains with a broader host range.
Methods
Bacterial strains, plants and growth conditions
All bacterial strains, plasmids, and primers used in this study are listed in Supplementary Table 3. Escherichia coli strains were routinely grown in LB medium93 at 37 °C with 220 rpm shaking with the following antibiotic concentrations as required: trimethoprim (Tm) at 50 µg·mL-1, chloramphenicol (Cm) at 20 µg·mL-1 and kanamycin (Km) at 25 µg·mL-1. Peptone-salts-yeast extract (PSY) medium supplemented with 0.1% L-arabinose and 100 µg·mL-1 spectinomycin was used to grow B. diazoefficiens USDA 110Tspc494,95 at 28 °C with 180 rpm. Paraburkholderia phymatum strains were grown on modified LB medium without salt (LB-NaCl)96 at 28 °C with 180 rpm shaking, while the P. phymatum transposon (Tn) mutant library was cultivated on AB minimal media supplemented with 15 mM succinate (ABS)96 and 0.2% L-rhamnose and, when applied, P. phymatum cultures were supplemented with Tm at 100 µg·mL-1 and Km at 50 µg·mL-1.
Soybean (Glycine max) cv. Williams 82 seeds were surface-sterilized with absolute ethanol for 5 min, followed by 15 min with H2O2 (35 %) and washed 10-fold with sterile deionized water97, whereas for common bean (Phaseolus vulgaris) cv. Negro Jamapa, seeds were surface-sterilized similarly except for a 5 min incubation in H2O2 (35 %)29 instead of 15 min. Mimosa pudica (Samen Mauser AG commercial seeds) seeds were submerged for 15 min in 96% H2SO4 and 15 min in 3% NaClO, each step followed by ten washes with sterile water53. Surface sterilized seeds were germinated on 0.8% agarose plates for 48 h at 28 °C and, once sprouted, they were incubated in yogurt jars filled with vermiculite (VTT-Group, Muttenz, Switzerland) and nitrogen-free Jensen medium98. Soybean and common bean were incubated for 21 and M. pudica for 28 days, with a temperature of 22 °C at night and 25 °C during the day and a light intensity of 200 µM for 16 h at a constant humidity of 60%99.
Construction of a P. phymatum STM815T Tn mutant library
To generate a P. phymatum Tn mutant library, biparental matings were set up with E. coli sm10λpir containing the Tn23-delivery plasmid pLG9946 (Supplementary Table 3) as the donor strain and P. phymatum STM815T as the recipient. Previously, 48 precultures of E. coli and 48 of P. phymatum were grown overnight, washed with 10 mM MgSO4 and 124 random combinations were mixed at a 1:1 ratio with a final OD600 of 1 per strain. The matings were spotted and incubated on LB-NaCl plates for 24 h at 30 °C, collected with 1 mL of MgSO4 (10 mM) and transconjugants were selected on ABS plates with Tm 150 µg·mL-1. After three days of incubation at 28 °C, random colonies were selected to verify P. phymatum transconjugation by PCR using the specific primers pLG99_insert_Fw and pLG99_insert_Rv100 (Supplementary Table 3). Approximately 1,300,000 colonies were recovered in ABS liquid media containing Tm 150 µg·mL-1 and 25% glycerol, and then aliquoted into one hundred 1.5 mL tubes to store at -80 °C.
Growth and inoculation of the Tn mutant library
To screen for essentiality, 100 µL of the Tn mutant library stock (OD600 of ~31.45) was washed twice with ABS supplemented with 0.2% L-rhamnose and 150 µg·mL-1 of Tm (ABS + rh + Tm). The addition of rhamnose avoids polar effects that could be caused by the insertion of a Tn in an operon. In fact, Tn23 contains a rhamnose inducible promoter that would allow the expression of downstream genes46. Then, the final OD600 was adjusted to 0.02 in a 250 mL flask containing 50 mL of ABS + rh + Tm. This preculture was then incubated for 14 h at 28 °C with 200 rpm shaking. Six mL of the grown preculture were washed with ABS + rh and adjusted to an OD600 of 0.05 (corresponding to ~107 colony forming units (CFU) per mL) in 50 mL of ABS + rh. The adjusted culture was grown at 28 °C, the standard growth temperature used for P. phymatum, and shaken at 200 rpm; this was stopped before entering stationary phase at OD600 of 0.8 (control sample with approximately 5 generations) and cells were pelleted and stored at -20 °C for DNA extraction.
For screening experiments with soybean seedlings, 50 µL of the Tn mutant library stock was used to start the preculture as described above and was incubated at 28 °C with 180 rpm shaking overnight. After incubation, 12 mL of the Tn mutant library preculture was washed thrice with a variation of the AB medium without a nitrogen source ((A)B)96 supplemented with 0.2% L-rhamnose ((A)B + rh). The preculture was adjusted to a starting OD600 of 0.025 (roughly 107 CFU·mL-1) in (A)B + rh and 1 mL was inoculated onto each seedling.
Tn-seq library preparation
Genomic DNA (gDNA) from a Tn mutant library grown in standard conditions (28 °C) was isolated using the GeneEluteTM Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA, NA2110). For the gDNA extraction of the soybean nodules colonized by the Tn mutant library, roots were first surface-sterilized for 10 seconds with absolute ethanol, 3 min in 3% NaClO and washed 5 times with sterile deionized water31. All root nodules were collected and flash-frozen in liquid nitrogen. Nodules were then disrupted using 3-mm Tungsten Carbide beads (QIAGEN, Hilden, Germany) with a TissueLyzer 3 times for 1 min at 30 Hz97. Total DNA was extracted using a modified phenol-chloroform extraction protocol101 followed by the utilization of the GeneEluteTM Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA, NA2110) to remove the ribosomal RNA. The presence of gDNA from the P. phymatum Tn mutant library was verified by PCR from the extracted DNA using pLG99_insert_Fw and pLG99_insert_Rv primers100 (Supplementary Table 3).
After DNA isolation from the Tn mutant libraries, Tn-seq libraries were prepared using the circle method45,59,100,102,103,104. Briefly, the gDNA was first sheared to 300 bp using Covaris (E220 focused ultrasonicator, CovarisTM, MA, USA). The sheared fragments obtained were end-repaired, ligated to the adaptors using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs (NEB), Ipswich, MA, USA, E7645S) and digested with BamHI. Then, DNA with size between 180 and 400 bp was selected and circularized, and the non-circularized DNA was degraded using the exonuclease ExoI (NEB, M0293L), T7 Gene 6 Exo (NEB, M0263S) and Lambda Exo (NEB, M0262S). A PCR using T23_SLXA_PAIR_AmpF_3 and SLXA_PAIR_REV_AMP primers (Supplementary Table 3) was carried out to amplify the selected circularized DNA.
Sequencing and data analysis of the Tn-seq library
Tn-seq libraries were sequenced using paired-end MiSeq reagent kit (V2 300 cycles, Illumina, San Diego, CA, USA, MS-102-2002) with the Illumina MiSeq platform. Sequenced DNA libraries were then analyzed100,102,104. Concretely, the adapters and the demultiplexed FASTQ reads were trimmed from the Tn-seq library for the essential genes study. The forward and reverse trimmed reads were mapped to the genome sequence of P. phymatum STM815T30 using the Tn-seq Explorer software105, while only the forward reads (read in the direction of the Tn) were used for the mapping of the Tn-seq library from soybean nodules. The unique insertion counts (UIC) of the transposon for every gene was calculated with the Tn-seq Explorer software for the soybean nodule Tn-seq library and the unique insertion density (UID) was also calculated by dividing the UIC of each gene by the length in base pairs of the gene. In this case, the Tn-seq Explorer suggested a theoretical cutoff of UID of 0.012 for genes to be essential59 and the functional classification to COG categories106 of the genes with a UID below this threshold was performed using eggNOG-Mapper v2107,108.
Construction of P. phymatum STM815T mutant strains
The nodS (Bphy_7716) deletion mutant was created by a double-crossover event. A nodS upstream region of 486 bp and a downstream region of 484 bp were amplified from P. phymatum STM815T gDNA by PCR using Bphy7716_up_F_EcoRI and Bphy7716_up_R_XhoI, and Bphy7716_dn_F_NdeI and Bphy7716_dn_R_EcoRI paired primers (Supplementary Table 3), respectively. The Tm resistance cassette dhfr was obtained by digesting plasmid p34E-TpTer109 with NdeI restriction enzyme and ligated between the amplified flanking regions of the nodS gene. The constructed 1865 bp-long insert was then cloned into the suicide vector pSHAFT2109, resulting in pSHAFT2::Bphy_7716 plasmid, and the insert was confirmed by sequencing. Through a triparental mating, pSHAFT2::Bphy_7716 was transferred into the P. phymatum wild-type strain and the double-crossover recombinant clones that were Tm-resistant, Cm-sensitive were selected. After two purification steps, the obtained deletion mutant was verified by PCR using Bphy7716_veri_F and Bphy7716_veri_R primers (Supplementary Table 3). To construct the nodS complemented strain, nodS was amplified using the Bphy_7716_c_R_XbaI and Bphy_7716_c_F_XhoI primers (Supplementary Table 3) and cloned into pGEM® T-Easy vector. After sequencing the constructed plasmid, the insert was digested with EcoRI and cloned into pBBR1MCS2110 in the opposite direction of the lacZα gene (transcription driven by the T7 promotor), to generate the plasmid pBBR1MCS-2::Bphy_7716, which was finally conjugated into the nodS mutant to create the strain called nodS complemented. In addition, the empty vector pBBR1MCS2 was conjugated into the P. phymatum wild-type and nodS deletion mutant strains by triparental mating.
Phenotypic characterization of P. phymatum strains
P. phymatum strains (wild-type and nodS mutant) were aerobically grown in 25 mL of LB-NaCl liquid medium in 100 mL Erlenmeyer flasks at 28 °C with shaking at 180 rpm for 19 h. Their growth was monitored by measuring OD600 and CFU. For the exopolysaccharide (EPS) production and swimming motility assay, cells were washed with 10 mM MgSO4, and the OD600 was adjusted to a final OD600 of 0.5. EPS production was tested on YEM medium (1% mannitol, 0.06% yeast extract) plates which were incubated for four days at 28 °C41,96. The swimming motility assay was conducted on LB-NaCl plates containing 0.2% agar. The bacteria were inoculated on the center of the plate and were incubated for 48 h at 28 °C. The growth-diameter was measured after 24 and 48 h and compared for each strain111. Resistance to hydrogen peroxide was carried out on soft agar plates of LB-NaCl, where bacteria were inoculated at a final OD600 of 0.05 and discs containing H2O2 (10 M, 5 M and 1 M) or H2O were placed on the plate. The inhibition zone for each strain was measured after 24 h incubation at 28°C111. To conduct the salt stress assay, cell cultures were washed and the OD600 was normalized to 0.05 in 5 mL of LB-NaCl; the NaCl being added to make different final concentrations (0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M). The cultures were incubated for 17 h at 28°C with shaking at 180 rpm, and growth was monitored by measuring OD600 and CFU. Each experiment was performed with three biological replicates per strain.
Characterization of symbiotic properties in planta
The inoculation was performed immediately after transferring the seedlings from agarose plates into yogurt jars filled with vermiculite. To prepare the inocula, precultures of B. diazoefficiens and the P. phymatum wild-type and P. phymatum mutant strains were washed twice with (A)B liquid media, adjusted to an OD600 of 0.025. One mL was then inoculated onto each germinated soybean, common bean or mimosa seedling. Any nodules developed by each plant after 21 days for soybean and common bean, and 28 days for Mimosa pudica, were picked, counted, and weighed after drying overnight at 55 °C. The nitrogenase activity was determined by the acetylene reduction assay (ARA). Specifically, each root, was incubated with one mL of acetylene gas (PanGas, Zurich, Switzwerland) in 50 mL tubes (Infochroma AG, Zug, Switzerland) for 18 h at room temperature. After incubation, 25 µL of the gas from each tube containing a root was injected into a gas chromatograph (Agilent, Santa Clara, CA, USA) to analyze the percentage of acetylene converted into ethylene. Nitrogenase activity was normalized by the dry weight of all the nodules on the plants and the incubation time of the reaction41. The shoot dry weight was obtained by drying each plant shoot for 48 h at 60 °C and weighed. One nodule per bacterial inoculum was used to reisolate the bacteroids and to determine the nodule occupancy. In short, after roots were surface sterilized as described above (see “Tn-seq library preparation”), nodules were crushed in 100 µL of LB-NaCl containing 25% glycerol and plated on LB-NaCl with and without the corresponding antibiotics to count CFU31.
Histological preparations
To analyze the internal structure of the soybean nodules induced by the P. phymatum nodS mutant in comparison to those induced by B. diazoefficiens, soybean nodules were sectioned transversally under a binocular microscope using sterile blades. The nodule slices were then analyzed under a Leica M165 FC fluorescence stereomicroscope to get images of the entire nodule structure. Images from the transmission electron microscope (TEM) were obtained from nodules sectioned transversally and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7); these slices were then embedded in resin and further sectioned to obtain one µm semithin sections for light microscopy and 80 nm ultrathin sections for TEM28. The acquisition parameters for every experimental set were replicated for each sample. The acquired images were analyzed with Fiji (ImageJ 2.14.0/1.54 f) and Photoshop™.
Statistics and reproducibility
The statistical analysis of the category distribution of the essential genes was performed using a Fisher’s exact test (online calculator for 2×2 contingency table from GraphPad) to assess a possible over-representation of the functional eggNOG categories41. Statistical analyses and graphics were performed using GraphPad Prism® version 6.01. For screening experiments, seven soybean seeds were inoculated with the P. phymatum Tn mutant library. For characterization of the symbiotic properties in planta, three biological replicates of B. diazoefficiens, P. phymatum wild-type and nodS mutant and complemented strains were tested on soybean. Each replicate was inoculated onto four soybean seedlings. For M. pudica, five biological replicates and four plants per replicate were used per strain and, in the case of common bean, four plants per replicate with three independent biological replicates. Differences among each strain (wild-type, nodS and complemented nodS) were analyzed using a two-way ANOVA and Tukey’s post hoc test and were considered significant when the p-value was lower than 0.05. When comparing the mutant and the complemented strain to the wild-type, a one-way ANOVA was used and a p-value lower than 0.05 was considered as significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All data are available in the main text, the supplementary data and materials or upon request to the corresponding authors. The source data for charts/graphs can be found in Supplementary Data 3. The raw FASTQ files created by MiSeq Illumina platform have been deposited into the NCBI Short Reads Archive (SRA) platform and are found under the BioProject entitled “Tn-seq profiling reveals that NodS of the beta-rhizobium Paraburkholderia phymatum is detrimental for nodulating soybean” and under the accession number PRJNA1089526.
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Acknowledgements
We thank Prof. Dr. Hans-Martin Fischer (ETH Zurich, Switzerland) and Dr. Dulce-Nombre Rodríguez-Navarro (IFAPA las Torres, Spain) for providing the B. diazoefficiens 110T spc4 strain and soybean seeds, respectively. We acknowledge Vicente Fco. Bellés-Sospedra and Karl Huwiler for providing common bean seeds. Romy G. Leemann, Giada Ornaghi and Julie Ahrens are acknowledged for their help in performing phenotypic tests and characterizing the inoculated plants, and Dr. Stefano Gualdi for helping with the visualization of Tn-seq data. This work was supported by the Swiss National Science Foundation (SNSF) with project number 31003A_179322 and 310030_215282 to Gabriella Pessi and the Uniscientia Stiftung with project number 211−2023 to Paula Bellés Sancho.
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The following author contributions were made: Conceptualization: G.P. Methodology: P.B.-S and G.P. Investigation: P.B.-S., D.G., S.P., A.V., Y.L., A.B. and E.K.J. Data analysis: P.B.-S., D.G., S.P., A.V., L.E, E.K.J, G.P. Visualization: P.B.-S., D.G., S.P., and E.K.J. Funding acquisition: P.B.-S. and G.P. Writing – original draft: P.B.-S. and G.P. Writing – review & editing: P.B.-S., D.G., S.P., A.V., Y.L., A.B. L.E., E.K.J. and G.P.
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Bellés-Sancho, P., Golaz, D., Paszti, S. et al. Tn-seq profiling reveals that NodS of the beta-rhizobium Paraburkholderia phymatum is detrimental for nodulating soybean. Commun Biol 7, 1706 (2024). https://doi.org/10.1038/s42003-024-07385-x
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DOI: https://doi.org/10.1038/s42003-024-07385-x
