Genetic design of soybean hosts and bradyrhizobial endosymbionts reduces N2O emissions from soybean rhizosphere

Abstract

Soybeans fix atmospheric N₂ through symbiosis with rhizobia. The relationship between rhizobia and soybeans, particularly those with high nitrous oxide (N₂O)-reducing (N₂OR) activities, can be leveraged to reduce N₂O emissions from agricultural soils. However, inoculating soybeans with these rhizobia under field conditions often fails because of the competition from indigenous rhizobia that possess low or no N₂OR activity. In this work, we utilize natural incompatibility systems between soybean and rhizobia to address this challenge. Specifically, Rj2 and GmNNL1 inhibit certain rhizobial infections in response to NopP, an effector protein. By combining a soybean line with a hybrid accumulation of the Rj2 and GmNNL1 genes and bradyrhizobia lacking the nopP gene, we develop a soybean-bradyrhizobial symbiosis system in which strains with high N₂OR activity predominantly infect. Our optimize symbiotic system substantially reduces N₂O emissions in field and laboratory tests, presenting a promising approach for sustainable agricultural practices.

Fig. 1. Phylogenetic tree of soybean bradyrhizobial strains based on Internal Transcribed Sequences (ITS) between 16S and 23S ribosomal RNA genes and their N₂O-reducing activities.

The ITS phylogenetic tree was constructed by aligning ITS sequences using ClustalW and applying the neighbor-joining method. The N₂O-reducing activities were determined via gas chromatography. *Strains of B. ottawaense isolated in this study. Boxplots indicate medians (centerlines), interquartile ranges (box edges), individual data points (dots), and mean values (red crosses). Each strain was analyzed with three biological replicates (n = 3). Different letters indicate statistically significant differences (p < 0.05; Multiple comparisons with correction were performed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test). Source data are provided as a Source data file.

Fig. 2. Isolation of incompatibility-bypassing bradyrhizobia strains.

a A large nodule (white arrowhead) formed in the Rj2 soybean cultivar “Hardee” following inoculation with B. ottawaense GMA461. b Positions of endogenous transposon insertions in nopP of FY2-m1, GMA461-m4, and OSA024. Insertions of ISRj2 were detected in nopP of FY2-m1 and GMA461-m4, and ISBj11 in nopP of OSA024. White arrowheads indicate the positions of the four amino acid residues that define the NopP type; corresponding residues and positions are labeled above each arrowhead. c PCR amplification of the nopP region using nopP-specific primers. Bands with increased molecular weight, indicating transposon insertion, are marked with red arrowheads. The experiment was repeated two times independently with similar results. Source data are provided as a Source data file.

Fig. 3. Selection of Rj2/GmNNL1 soybean and its nodulation phenotypes.

a Electrophoresis images of molecular markers for Rj2 and GmNNL1 in soybean lines. Number of mature nodules on the roots of Rj2/GmNNL1-accumulated soybean inoculated with FY2 or FY2-m1 (b), and GMA461 or GMA461-m4 (c). Photographs of each nodulated root were shown below each graph. Boxplots indicate medians (centerlines), interquartile ranges (box edges), individual data points (dots), and mean values (red crosses). Each inoculation combination was performed with n = 4 biological replicates. Asterisks indicate statistically significant differences (Two-sided Wilcoxon rank-sum test, p < 0.05). Source data are provided as a Source data file.

Fig. 4. Mitigation of N₂O emission through predominant infection by incompatibility-bypassing bradyrhizobia strains with high N₂OR activity.

Nodule occupancy rates of USDA6, USDA110, USDA122, and incompatibility-bypassing rhizobial strains in Rj2/GmNNL1 and rj2/Gmnnl1 soybean lines inoculated with FY2-m1 (a; For each inoculation combination, 192 and 189 nodules were collected and analyzed from 6 plants, respectively), GMA461-m4 (b; For each inoculation combination, 190 and 192 nodules were collected and analyzed from 6 plants, respectively), and OSA024 (c; For each inoculation combination, 191 and 190 nodules were collected and analyzed from 5 plants, respectively). N₂O emissions measured at 1, 2, and 3 weeks after decapitation (WAD) of soybean inoculated with FY2-m1 (d), GMA461-m4 (e), and OSA024 (f). The data are shown as mean ± s.e. Each inoculation combination was performed with n = 4 biological replicates. Asterisks indicate significant differences between Rj2/GmNNL1 and rj2/Gmnnl1 soybean lines on the same WAD (Two-sided Student’s t-test, p < 0.05). Source data are provided as a Source data file.

Fig. 5. Mitigation of N₂O emissions through predominant infection by incompatibility-bypassing bradyrhizobia strains under competition with indigenous soil rhizobia.

a Nodule occupancy. For one experimental plot, 192 nodules were collected and analyzed from 6 plants. b Trends in N₂O emissions following plant decapitation. The data are shown as mean ± s.e. Each inoculation combination was performed with n = 5 biological replicates for mock, FY2-m1, and GMA461-m4, and n = 3 biological replicates for OSA024. c Cumulative N₂O emissions over 1–24 days after plant decapitation. Boxplots indicate medians (centerlines), interquartile ranges (box edges), individual data points (dots), and mean values (crosses). Each inoculation combination was performed with n = 5 biological replicates for mock, FY2-m1, and GMA461-m4, and n = 3 biological replicates for OSA024. Asterisks indicate significant differences between mock and incompatibility-bypassing bradyrhizobia strains on the same day (Multiple comparisons with correction were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test, *p < 0.05; **p < 0.01). Source data are provided as a Source data file.

Fig. 6. Nodule occupancy and N₂O flux from Kashimadai field in Rj2/GmNNL1 soybean plants inoculated with B. ottawaense strains SG09 (nopP_USDA6), GMA461 (nopP_USDA122), and GMA461-m4 (nopP-deficient mutant).

a Nodule occupancy by B. ottawaense-type rhizobia (A total of 186 nodules from 6 plants per plot). Boxplots indicate medians (centerlines), interquartile ranges (boxes), individual data points (dots), and mean values (crosses; n = 6 biological replicates). b N₂O emissions from nodulated field-grown soybean immediately after shoot excision. Boxplots indicate medians (centerlines), interquartile ranges (boxes), individual data points (dots), and mean values (crosses; n = 4 biological replicates). Asterisks indicate statistically significant differences (p < 0.05). Multiple comparisons with correction were performed using one-way analysis of variance (ANOVA), followed by a two-sided Steel test in (a) and Dunnett’s test in (b), with mock inoculation used as the control. Source data are provided as a Source data file.