Tn-seq profiling reveals that NodS of the beta-rhizobium Paraburkholderia phymatum is detrimental for nodulating soybean

Abstract

The beta-rhizobial strain Paraburkholderia phymatum STM815^T 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.

Fig. 1. Characteristics of the Paraburkholderia phymatum STM815^T transposon sequencing (Tn-seq) library.

a P. phymatum STM815^T 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 FS. b Percentage of essential genes belonging to each COG (Cluster of Orthologous Genes) functional category^,. Genes were annotated with eggNOG mapper v2^,. 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.

Fig. 2. Identification of P. phymatum Tn mutants with nodulation abilities in soybean.

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 STM815^T 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 110^Tspc4 (positive control), P. phymatum STM815^T 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.

Fig. 3. Position of the Tn insertions in the P. phymatum nod genes cluster.

The unique counts approach of the Tn-seq Explorer software was used to identify genes that provided a fitness disadvantage for the colonizing by P. phymatum STM815^T on the non-host plant soybean.

Fig. 4. Symbiotic properties of the P. phymatum nodS mutant in soybean.

Soybean seedlings were inoculated with the P. phymatum STM815^T wild-type (wild type), nodS mutant (nodS), complemented nodS mutant strains (nodS_C) and B. diazoefficiens USDA 110^Tspc4 (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.

Fig. 5. The P. phymatum nodS mutant colonizes intracellularly the central zone cells of soybean nodules.

a Representative soybean root nodules induced by B. diazoefficiens USDA 110^Tspc4 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 110^Tspc4 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 110^Tspc4 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.

Fig. 6. Symbiotic properties of the P. phymatum wild-type, nodS mutant and nodS complemented mutant strains in plant hosts.

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).