A Stringent-Response-Defective Bradyrhizobium diazoefficiens Strain Does Not Activate the Type 3 Secretion System, Elicits an Early Plant Defense Response, and Circumvents NH4NO3-Induced Inhibition of Nodulation

Abstract

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N₂ in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N₂-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH₄NO₃, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system.IMPORTANCE The symbiotic N₂ fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N₂ fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.

Keywords: plant defense; regulation of nodulation; stringent response; type 3 secretion system.

FIG 1

Scheme of rsh and its mutant derivative. (A) Domain structure of various rsh, relA or spoT analogs from different species, obtained from Phylome DB-Tree Explorer on 16 August 2019. Blue squares indicate speciation events; red squares indicate duplication events. The green dot indicates the target sequence. Since the species name was not updated in this database, it still appears as “Bradyrhizobium japonicum USDA 110,” but it should be referred to as “Bradyrhizobium diazoefficiens USDA 110.” The following domains (according to Pfam) are shown: the metal-dependent phosphohydrolase (HD_4 [green diamonds]); the characteristic RelA/SpoT protein domain (RelA_SpoT [blue-green rectangles]); ThrRS, GTPase, and SpoT, which is a possible nucleotide-binding region (TGS [blue diamonds]); and the ACT domain, which binds to amino acids and regulates associated enzyme domains (ACT_4 [violet and yellow rectangles]). (B) Comparison of the wild type (upper row) and rsh insertional mutant (lower row) from B. diazoefficiens. The positions and directions of the primers used to obtain and validate the mutation are indicated by green arrows. The sizes of the fragments used for the mutation are indicated below the blue lines. The insertion occurred between the XhoI sites at bases 5619575 and 5619590 in reverse orientation.

FIG 2

Growth and survival of B. diazoefficiens USDA 110 (wild type) and its rsh mutant derivative. (A) Growth as evaluated by optical density at 500 nm (OD₅₀₀) in PSY. (B) Remnant viable bacteria (as CFU) from a suspension in MOPS shaken at 28°C after the indicated times.

FIG 3

Plant defense response in soybean. Plants inoculated with B. diazoefficiens USDA 110 (wild type) or its rsh mutant derivative were cultivated for 12 or 48 h postinoculation (hpi). As controls, a group of plants were kept uninoculated. The significance of the differences between wild-type and rsh mutant strains was evaluated by ANOVA and is indicated as follows: NS, nonsignificant (P > 0.05); **, significant (P < 0.01). Data were collected from three independent experiments, each with at least four different plants for each condition. Black bars show relative expression of the plant defense marker GmPR1 in the rsh mutant with respect to the wild type (fold change). GmEF1 was used as an internal constitutive control. Gray bars show cytoplasmic reactive oxygen species (Cyt-ROS) measured in the root hair tips of soybean plants with a H₂DCF-DA probe.

FIG 4

Nodules from soybean inoculated with B. diazoefficiens USDA 110 in the absence (A to D) or the presence (E to J) of 10 mM NH₄NO₃. The wild type (A, C, E, and G) and its rsh mutant derivative (B, D, F, and H to J) are compared. Entire nodules were viewed under a stereomicroscope (A, B, E, and F). Ultrathin cuts from these nodules were observed with a transmission electron microscope (C, D, and G to J). To confirm the role of rsh in the phenotypes observed, the rsh mutant was complemented with the wild-type rsh complete gene carried in the replicative vector pFAJ1708 (I) or the empty vector (J). The micrograph set is representative of results from three independent experiments.

FIG 5

Auxins released to the culture medium by B. diazoefficiens USDA 110 (wild type) or its rsh mutant derivative. Auxins were measured by a colorimetric method using indoleacetic acid (IAA) as a standard. Error bars indicate standard deviation (SD). Statistical analysis was carried out by ANOVA (****, significant difference at P < 0.0001). Representative results from three independent experiments are shown.