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

Genetic design of soybean hosts and bradyrhizobial endosymbionts reduces N2O emissions from soybean farming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genetic design of soybean hosts and bradyrhizobial endosymbionts reduces N2O emissions from soybean farming Haruko Imaizumi-Anraku, Hanna Nishida, Manabu Itakura, Khin Win, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5679948/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Soybeans fix atmospheric N 2 through symbiosis with rhizobia, N 2 -fixing bacteria. The relationship between rhizobia and soybeans, particularly those with high nitrous oxide (N 2 O)-reducing activities (Nos ++ ), can be used for reducing N 2 O emissions from agricultural soils. However, inoculating soybeans with Nos ++ rhizobia under field conditions often fails because of the competition from indigenous Nos − (no N 2 O-reducing activities) and Nos + (normal N 2 O-reducing activities) rhizobia. Here, we utilized 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 Nos ++ bradyrhizobia lacking the nopP effector gene, we developed a soybean-bradyrhizobial symbiosis system that Nos ++ rhizobial inoculants predominantly infect. This optimized symbiotic system substantially reduced N 2 O emissions in field and laboratory tests, presenting a promising approach for sustainable agricultural practices. Biological sciences/Plant sciences/Plant symbiosis/Rhizobial symbiosis Biological sciences/Microbiology/Environmental microbiology/Soil microbiology Earth and environmental sciences/Climate sciences/Climate change/Climate-change mitigation Competition GHG Incompatibility N2O NopP effector Predominant infection Rhizobium Soybean Symbiosis Nodule occupancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Food production is expanding to support the growing human population. However, agricultural lands are major sources of anthropogenic nitrous oxide (N 2 O), a greenhouse gas (GHG) with a global warming potential approximately 300 times greater than that of carbon dioxide (CO 2 ) (Tian et al., 2020; Uchida & Akiyama 2013). Meanwhile, chemical nitrogen fertilizers, synthesized from fixed atmospheric N 2 via the Haber–Bosch process using fossil fuels, serve as the primary source of nitrogen for current intensive farming systems (Goyal et al., 2021; Tian et al., 2020; Bourion et al., 2018). Then, nitrogen sources, including fertilizers and biological residues, are converted to inorganic nitrogen by soil microorganisms through the nitrogen cycle (Sanchez & Minamisawa 2019; Uchida & Akiyama 2013). Soil nitrogen, existing as ammonia and nitrate inorganic compounds, transitions between these states through nitrification and denitrification. Among these processes, denitrification involves multiple reduction reactions (NO 3 − → NO 2 − → NO→ N 2 O→ N 2 ). While many soil bacteria lack specific genes that encode reductase for complete denitrification, thereby acting as sources and sinks for N 2 O (Hiis et al., 2024), soil rhizobial bacteria can perform biological N 2 fixation through symbiosis with leguminous plants. For example, Bradyrhizobium , a genus comprising N 2 -fixing bacteria, has symbiotic interactions with legumes, such as soybeans, by infecting the plants, forming nodules in plant roots, and converting N 2 into ammonium in the nodules (Wang et al., 2018; Roy et al., 2020; Nakei et al., 2022). This type of biological N 2 fixation provides N 2 to host plants without the economic effects and GHG emissions associated with chemical N 2 fertilizer production (Goyal et al., 2021; Graham & Vance 2003). However, the nitrogen released from aging and decaying nodules is also a source of N 2 O in the soil (Akiyama et al., 2016; Wasai-Hara et al., 2023; Inaba et al., 2009; Inaba et al., 2012; Toyoda et al.. 2024). Globally, N 2 O emissions from soybean plant residues were estimated to be 19,685 kt CO 2 eq in 2020 (FAO, 2024). Some rhizobacteria in the genus Bradyrhizobium possess N 2 O reductase, an enzyme that reduces N 2 O to N 2 (Samejima-Saito et al., 2006; Itakura et al., 2008; Itakura et al., 2013; Akiyama et al., 2016; Henault et al., 2022; Melissa et al., 2022; Mania et al., 2020). For example, the Bradyrhizobium ottawaense strains isolated in Japan have demonstrated high N 2 O-reducing ability (Nos ++ ) and are classified as Nos ++ rhizobia (Wasai-Hara et al., 2020; Wasai-Hara et al., 2023). In Japan, Andosols—a major soil type covering approximately half of the upland fields—are predominantly occupied by B. japonicum , which lacks nosZ , the gene encoding N 2 O reductase (Nos − rhizobia) (Shiina et al., 2014). Additionally, most paddy fields consist of alluvial soil, primarily colonized by B. diazoefficiens , an N 2 O-reducing (Nos + ) rhizobial phenotype (Shiina et al., 2014). However, the reports on the distribution of Nos ++ rhizobia in Japan remain limited, and only B. ottawaense has been identified in sorghum fields in Fukushima (Wasai-Hara et al., 2020; Wasai-Hara et al., 2023). Since approximately 80% of the soybean fields in Japan are converted from rice paddies, most of the nitrogen derived from soybean residues is likely released as N 2 O without being reduced to N 2 . Inoculating soybeans with Nos ++ bradyrhizobia can reduce post-harvest N 2 O emissions from these fields (Itakura et al., 2013; Akiyama et al., 2016). As the fourth most important crop worldwide, soybeans provide essential vegetable protein and oil for humans and animals (Goyal et al., 2021; Hartman et al., 2011; Rotundo et al., 2024). In 2022, the area for soybean cultivation spanned 133,791,632 hectares, representing approximately 8% of the global total of cropland (1,617,392,600 hectares) (Our World in Data, 2022). Thus, introducing soybean symbiosis with Nos ++ Bradyrhizobium strains can substantially reduce N 2 O emissions from soybean agriculture. However, competition from indigenous soil rhizobia hinders the infection of promising Nos ++ bradyrhizobia after soybeans are inoculated with them. Consequently, the occupancy rate of Nos ++ bradyrhizobia in soybean nodules remains unstable, ranging between 20% and 60% (Itakura et al., 2013). The N 2 O-reducing capacity of Nos ++ bradyrhizobia is likely limited, as most indigenous rhizobia occupying root nodules exhibit little or no N 2 O-reducing capability. Consequently, this “rhizobial competition problem” has been a substantial barrier to the successful introduction of promising rhizobia into agricultural fields (Bourion et al., 2018; Nakei et al., 2022; Triplett & Sadowsky, 1992; Mendiza-Suarez et al., 2020; Mendiza-Suarez et al., 2021). Thus, establishing a soybean symbiotic system that predominantly supports Nos ++ rhizobia infection in nodules is essential for addressing this issue and maximizing the N 2 O-reducing potential of Nos ++ bradyrhizobia. While various factors influence the nodule occupancy rate of rhizobia, the primary determinant is the genotype of the host plant (Triplett & Sadowsky, 1992; Burghardt et al., 2022; Mendiza-Suarez et al., 2021, Grundy et al., 2023). Specifically, soybean incompatibility—a symbiotic inhibition response triggered by rhizobia-released effector proteins via the Type III secretion system—is defined by the soybean genes and corresponding rhizobial effector proteins involved (Teulet et al., 2020; Staehelin & Krishnan, 2015, Grundy et al., 2023). Currently, the genes involved in incompatibility include Rj2 , Rfg1 , Rj4 , and GmNNL1 (Yang et al., 2010; Sugawara et al., 2018; Sugawara et al., 2019; Tang et al., 2016; Ratu et al., 2021; Zhang et al., 2021). Rj2 and GmNNL1 are Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) resistance R genes (Yang et al., 2010; Zhang et al., 2021; Gourion et al., 2015). Rj2 induces incompatibility by recognizing NopP USDA122 , an effector protein derived from B. diazoefficiens USDA122 (Sugawara et al., 2018). In contrast, both NopP USDA6 from B. japonicum USDA6 and NopP USDA110 from B. diazoefficiens USDA110 are recognized by GmNNL1 (Zhang et al., 2021). Most nodulating Bradyrhizobium spp. harbor nopP (Teulet et al.., 2020). In addition, Sugawara et al. (2018) analyzed the nopP sequences from a collection of bradyrhizobial isolates from soil samples across 32 locations in Japan and found that NopP USDA122 , NopP USDA110 , and NopP USDA6 (ST2) were widespread in Japan. Therefore, soybeans with Rj2 and GmNNL1 may suppress the infection of indigenous rhizobia harboring nopP USDA6 , nopP USDA110 , or nopP USDA122 . To test this hypothesis, we crossed soybeans to develop a line with Rj2 and GmNNL1 . Additionally, we generated NopP-deficient Nos ++ bradyrhizobia through induced spontaneous mutations (Sugawara et al., 2018). These NopP-deficient Nos ++ bradyrhizobia bypass symbiotic incompatibility with Rj2 and GmNNL1 , enabling successful infection of the Rj2/GmNNL1 soybean line. The incompatibility-avoiding Nos ++ bradyrhizobia achieved high occupancy rates within the Rj2/GmNNL1 soybean line through competitive infection with B. japonicum USDA6, B. diazoefficiens USDA110, and B. diazoefficiens USDA122, demonstrating substantial N 2 O-reducing capacity. Then, we further examined whether the incompatibility-avoiding Nos ++ bradyrhizobia could outcompete indigenous rhizobia in soybean field soil, thereby reducing N 2 O emissions from the soybean rhizosphere. Results Generation of incompatibility-avoiding bradyrhizobia with high N 2 O-reducing activity It is necessary to delete the effector gene or disable its function to establish bradyrhizbial strains that can overcome incompatibility with NopP effectors. When Rj2 soybeans are inoculated with bradyrhizobia strains carrying nopP USDA122 , huge mature nodules are rarely formed (Sugawara et al., 2018). Where nodules do form, they are often infected by nopP -deficient rhizobia that arise through spontaneous mutation. In addition, none of the eleven strains of B. ottawaense previously isolated for their high N 2 O-reducing activity encode nopP USDA122 (Wasai-Hara et al., 2023). We surveyed the B. ottawaense strains carrying nopP USDA122 strains in Japanese soybean fields (Itakura et al., Unpublished) to generate incompatibility-avoiding rhizobia. As a result, we obtained 2 target strains of B. ottawaense carrying nopP USDA122 (FY2 and GMA461) from Numata (Gunma Prefecture) and 1 strain, identified as a natural nopP mutant (OSA024), from Yao (Osaka Prefecture). The 16S-23S internal transcribed spacer (ITS) sequence of these 3 strains (OSA024, FY2, and GMA461) showed over 99% homology to the high N 2 O-reducing strain SG09 (Wasai-Hara et al., 2023), indicating close phylogenetic relationships (Fig. 1 ). Additionally, these strains demonstrated high N 2 O-reducing activity in free-living cells, comparable to B. ottawaense SG09 and substantially higher than B. diazoefficiens USDA110 (Fig. 1 ). Next, we attempted to isolate mutants with spontaneously disrupted nopP USDA122 by inoculating the Rj2 soybean cultivar Hardee with the FY2 or GMA461 strain carrying nopP USDA122 . When FY2 was inoculated, 3 of the 4 isolates from huge nodules contained an insertion sequence (IS) within nopP USDA122 , and 1 of these isolates was designated as the representative strain, FY2-m1. Among the Rj2 cultivar inoculated with GMA461, 2 of the 4 isolates from giant nodules showed IS insertion in nopP USDA122 , and 1 isolate was designated the representative strain, GMA461-m4 (Figs. 2 a and 2 b). Sequence analysis of nopP in FY2-m1 and GMA461-m4 showed that ISRj2 (Iida T et al., 2015) was inserted in both strains, but the positions of IS insertion differed (Figs. 2 b and 2 c). In the natural nopP mutant OSA024, ISBj11 (Iida T et al., 2015) was inserted in nopP USDA6 (Figs. 2 b and 2 c). Generation of soybean lines accumulating Rj2 and GmNNL1 incompatibility genes and evaluation of rhizobial symbiosis ability of incompatibility-avoiding Nos ++ rhizobia We selected the parent plants for accumulating Rj2 and GmNNL1 through the crossing and initially chose the well-known Japanese cultivar “Bonminori,” carrying the Rj2 genotype (Shiro & Saeki 2022). Additionally, 192 accessions from a soybean mini-core collection with available whole-genome resequencing data (Kajiya-Kanegae, 2021) were analyzed for the Rj2 and GmNNL1 genotypes. We extracted genetic variations in Rj2 and GmNNL1 by detecting single nucleotide polymorphisms (SNPs), small insertions and deletions (InDels), and structural variations (SVs) across the 193 accessions. Since the Rj2/rj2 genotype is determined by an SNP (C/T) in the second exon, which causes a single amino acid substitution (R490 to I490) (Sugawara et al., 2019), we identified 10 accessions with the Rj2 genotype (Supplementary Table 1). Conversely, the null Gmnnl1 genotype results from a 179-bp SINE-like transposon insertion in the second exon (Chen et al., 2016). Only 4 accessions possessed a functional GmNNL1 genotype (Supplementary Table 1). The individual occurrence percentage of Rj2 and NNL1 functional genotypes within the mini-core collection were 5.18% and 2.07%, respectively. Lastly, no accession had a functional genotype of Rj2 and GmNNL1 . Then, we selected the cultivars “Bonminori” and GmWC108 (Karasu-mame), which had similar flowering times, as parent plants for crossing to accumulate Rj2 and GmNNL1 . After crossing, we selected 1 line that was homozygous for Rj2/GmNNL1 and another line that was homozygous for rj2/Gmnnl1 from the F 2 seed population. From the progenies, we obtained the F 3 seeds. The Rj2 and rj2 and the GmNNL1 and Gmnnl1 genotypes of the seeds could be distinguished based on the PCR-amplified fragments of varying sizes that covered an SV near Rj2 and GmNNL1 and were generated using the cotyledon DNA from F 2 seeds (Fig. 3 a). Then, the Rj2/GmNNL1 line was inoculated with FY2 and GMA461, which carried nopP USDA122 , and with FY2-m1 and GMA461-m4, which exhibited the loss of nopP USDA122 function (Figs. 3 b and 3 c). Premature nodule-like structures were observed upon inoculation with FY2 or GMA461. In contrast, inoculation with FY2-m1 and GMA461-m4 resulted in the development of multiple mature nodules. These findings indicate that the loss of nopP USDA122 function due to endogenous transposon insertion enabled FY2-m1 and GMA461-m4 to overcome incompatibility mediated by Rj2 and GmNNL1. Nodule occupancy, NO flux, and symbiotic phenotypes in infection competition between incompatibility-avoiding and other rhizobia We simulated the infection competition between indigenous rhizobia and Nos ++ rhizobia that avoided Rj2/GmNNL1 incompatibility by conducting a simultaneous inoculation experiment using 4 strains: USDA6 (Nos − ), USDA110 (Nos + ), USDA122 (Nos + ), and an incompatibility-avoiding Nos ++ strain. First, we compared infection competition among the 4 strains by assessing nodule occupancy rates based on the differences in the length of restriction enzyme-treated nopP fragments (PCR-RFLP, Supplementary Fig. 1). Also, the nodule occupancy rates of the strains in mature nodules of Rj2/GmNNL1 and rj2/Gmnnl1 soybeans following simultaneous inoculation with strain FY2-m1 and USDA6/USDA110/USDA122 were compared (Fig. 4 a). In the Rj2/GmNNL1 soybean, the FY2-m1 strain occupied 95.3% of the mature nodules, while in the rj2/Gmnnl1 soybean, its occupancy was 56.1%. Similarly, the simultaneous inoculation of GMA461-m4 with USDA6/USDA110/USDA122 resulted in a nodule occupancy rate of 92.1% in the Rj2/GmNNL1 line and 54.7% in the rj2/Gmnnl1 line (Fig. 4 b). For OSA024, the simultaneous inoculation with USDA6/USDA110/USDA122 led to 97.4% nodule occupancy in the Rj2/GmNNL1 line and 27.9% in the rj2/Gmnnl1 line (Fig. 4 c). These results indicate that the USDA6/USDA110/USDA122 strains were unable to evade Rj2/GmNNL1 incompatibility, resulting in the predominant infection by Nos ++ incompatibility-avoiding strains in the Rj2/GmNNL1 soybean. N 2 O flux generated from nodulated soybean roots was quantitatively analyzed in inoculated plots, where nodule occupancy was assessed. N 2 O flux was measured 1, 2, and 3 weeks after the senescence and decay induction of nodulated roots. There was no significant difference in N 2 O flux between samples inoculated with FY2-m1 and GMA461-m4 at 1 and 2 weeks after decapitation (WAD); however, a considerable decrease in N 2 O flux was detected at 3 WAD. In the case of inoculating with OSA024, there was a substantial decrease in N 2 O flux at 2 and 3 WAD. Across all 3 Nos ++ incompatibility-avoiding strains, a substantial decrease in N 2 O flux was recorded from the Rj2/GmNNL1 soybean compared with the rj2/Gmnnl1 soybean (Figs. 4 d–f). Additionally, a linear regression analysis was performed to compare the nodule occupancy rates of the incompatibility-avoiding N 2 O-reducing rhizobacteria in rj2/Gmnnl1 and Rj2/GmNNL1 lines at both 2 and 3 WAD. The nodule occupancy of the N 2 O-reducing rhizobacteria was negatively correlated with N 2 O flux (Supplementary Fig. 2). These findings indicate that FY2-m1, GMA461-m4, and OSA024, isolated as Rj2/GmNNL1 incompatibility-avoiding Nos ++ rhizobia, demonstrated remarkably high nodule occupancy in Rj2/GmNNL1 soybeans under competitive inoculation with other rhizobial strains, thereby achieving high N 2 O reduction capacity. We conducted a comparative analysis of soybean growth and symbiotic phenotypes under Rj2 and GmNNL1 expression to address the concern that artificial accumulation of Rj2 and GmNNL1 in soybean lines could trigger defense responses against nopP USDA6 , nopP USDA110 , and nopP USDA122 type rhizobia, potentially negatively affecting soybean growth and symbiotic phenotypes. We inoculated soybean lines Rj2/GmNNL1 and rj2/Gmnnl1 with B. japonicum USDA6, B. diazoefficiens USDA110 and USDA122, and an incompatibility-avoiding N 2 O-reducing bradyrhizobia. Then, we assessed symbiotic phenotypes and soybean growth 5 weeks after inoculation. There was no significant difference in the number of nodules per plant (Supplementary Fig. 3a). However, nodule dry weight per plant and weight per nodule were substantially greater in the Rj2/GmNNL1 line than in the rj2/Gmnnl1 line (Supplementary Fig. 3b, 3c). N 2 -fixing activity was considerably higher in the Rj2/GmNNL1 and FY2-m1 combination (Supplementary Fig. 3d). Furthermore, shoot dry weight (SDW) and root dry weight (RDW) were substantially greater in Rj2/GmNNL1 than in rj2/Gmnnl1 (Supplementary Fig. 3e, 3f). Comparative analysis of nodule occupancy and NO flux between incompatibility-avoiding rhizobia and indigenous soil rhizobia We tested the competition between incompatibility-avoiding Nos ++ rhizobacteria and indigenous rhizobia in the Rj2/GmNNL1 soybean, analyzing their effectiveness in nodule occupancy and N 2 O release using pot culture with field soil at National Agriculture and Food Research Organization (NARO). FY2-m1 and GMA461-m4 exhibited nodule occupancy rate of over 80% occupancy, whereas OSA024 showed less than 60% occupancy (Fig. 5 a). GMA461-m4 had the lowest level of N 2 O release from the rhizosphere, including nodulated roots, following above-ground excision (Fig. 5 b). Cumulative N 2 O emissions of FY2-m1, GMA461-m4 and OSA024 demonstrated a substantial reduction in N 2 O release to that of the mock inoculation (Fig. 5 c). The ability to reduce N 2 O by the high N 2 O-reducing bradyrhizobia strains, FY2-m1 and GMA461-m4, in soybean lines with and without the incompatibility genes was compared to examine the effects of the incompatibility genes. In addition to Rj2/GmNNL1 soybean, the soybean variety “Akuden Shirazu,” which lacks incompatibility genes ( rj2/Gmnnl1 ), was included and examined as a negative control. FY2-m1 inoculation resulted in 69.8% and 25.0% nodule occupancy in the Rj2/GmNNL1 and rj2/Gmnnl1 soybeans, respectively (Supplementary Fig. 4a). N 2 O flux from the respective rhizosphere soils was considerably lower in the Rj2/GmNNL1 soybeans than in the rj2/Gmnnl1 soybeans (Supplementary Fig. 4b). GMA461-m4 inoculation yielded 77.6% and 12.9% nodule occupancy in the Rj2/GmNNL1 rhizosphere soil than in the rj2/Gmnnl1 rhizosphere soil (Supplementary Figs. 4a and 4b). The effect of soybean lines on N 2 O flux was most pronounced 8 to 18 days after decapitation when N 2 O flux values were highest (Supplementary Table 2, Supplementary Fig. 4b). The comparative analysis of cumulative N 2 O release over the measurement period showed that N 2 O release in FY2-m1/ Rj2/GmNNL1 was 40% of that in FY2-m1/ rj2/Gmnnl1 , while N 2 O release in GMA461-m4/ Rj2/GmNNL1 was only 16% of that in GMA461-m4/ rj2/Gmnnl1 (Supplementary Fig. 4c). These results confirm a substantial reduction in N 2 O release associated with increased nodule occupancy in the 2 strains. Nodule occupancy and NO flux in soybean fields The competition between Nos ++ inoculants and indigenous rhizobia in the Rj2/GmNNL1 soybean and their effectiveness in nodule occupancy and N 2 O reduction in a soybean field were analyzed in the Kashimadai soybean field at Tohoku University. The nodule occupancy rates of the treatments were compared (Fig. 6 a). The absence of nosZ of B. ottawaense in the uninoculated control indicated the absence of indigenous Nos ++ B. ottawaense in the field (Fig. 6 a). GMA461-m4, a nopP mutant of B. ottawaense , showed a nodule occupancy of over 60%. In contrast, the parent strain GMA461, which carried nopP USDA122 , could not occupy nodules. Additionally, inoculation with SG09, which carried nopP USDA6 , showed a lower nodule occupancy of approximately 10%. At the maturation stage, 15 weeks after seeding, N 2 O flux from the rhizosphere of each treatment was measured immediately following above-ground excision (Fig. 6 b). The N 2 O fluxes of the SG09 and GMA461 inoculation were not significantly different from that of the uninoculated control, whereas the GMA461-m4 inoculation substantially reduced N 2 O flux compared to SG09 and GMA461. These results indicate that high nodule occupancy of Nos ++ GMA461-m4 contributes to a reduction in N 2 O flux even under field conditions (Fig. 6 a, 6 b). Discussion The discovery of rhizobia capable of reducing N 2 O has paved the way for developing technologies aimed at reducing field-derived N 2 O emissions through rhizobial symbiosis (Sameshima-Saito et al., 2006; Itakura et al., 2013; Akiyama et al., 2016). Since soybean is the world's leading legume crop and forms symbiotic associations with bradyrhizobia species (Hartman et al., 2011), soybean symbiotic systems have been the primary focus for developing N 2 O reduction technologies using Nos + bradyrhizobia. Field inoculation with USDA110, a Nos + rhizobia, and its Nos ++ mutant has successfully reduced N 2 O emissions from soybean fields (Itakura et al., 2013; Akiyama et al., 2016). However, the nodule occupancy of these inoculants was unstable, remaining at 20–60% in farm-scale experiments (Itakura et al., 2013). The instability in nodule occupancy likely limits the inoculants' ability to maximize their N 2 O-reducing potential. In many cases, the introduction of beneficial rhizobium strains into the field is limited by the infection competition from indigenous rhizobia, a phenomenon known as the “rhizobial competition problem” (Triplett & Sadowsky 1992; Mendiza-Suarez et al., 2020; Mendiza-Suarez et al., 2021). The nodule occupancy of inoculated rhizobia is generally approximately 5–40% (McDermott et al., 1989; Hungria et al., 1998), highlighting the need to improve the nodule occupancy rates of inoculated rhizobia. Competition with indigenous rhizobia is influenced by the soil's physical and chemical properties and by biotic factors, such as rhizobial chemotactic response to rhizospheric substances secreted by host plants and rhizobial adhesion to plant roots (Mendiza-Suarez et al., 2021). In addition, the competition between rhizobial strains for carbon resources provided by the host plant plays an essential role in infection competition (Rahman et al., 2023). Establishing a rhizobial symbiotic system that allows beneficial rhizobia to dominate infection is crucial for maximizing the impact of promising rhizobia on host plants (Goyal et al., 2021; Mendiza-Suarez et al., 2021). However, the factors determining rhizobial competitive ability remain largely unknown. Although Cunningham et al. (1991) explored the possibility of chemical control to selectively express nodulation ( nod ) genes in distinct soybean bradyrhizobia lineages, this approach was unlikely to be adopted due to the cost of chemicals. Consequently, there are no reports on artificially controlling the competitive ability of rhizobial inoculants in practice. The soybean symbiotic incompatibility induced by rhizobial NopP, as used in this study, represents a rare interaction mechanism because it involves well-characterized genes on both the rhizobial and host plant sides (Yang et al., 2010; Sugawara et al., 2018; Sugawara et al., 2019; Zhang et al., 2021). This unique mechanism suggests a potential for the functional use of this to control rhizobial infection competition (Shiro et al., 2022). Since the NopP function in rhizobia can be disrupted using Rj2 incompatibility as a selection pressure (Sugawara et al., 2018), we developed 2 rhizobial strains, FY2-m1 and GMA461-m4, that avoided inhibition of infection by incompatibility genes, Rj2 and GmNNL1 . This study represents a novel approach to artificially enhance the host selectivity of promising rhizobia by modifying the host plant and the rhizobia. The inhibition of infection by specific effector-harboring rhizobia by host incompatibility genes is thought to have evolved as a strategy to eliminate rhizobia that are not beneficial to the host plant (Jimenez-Guerrero et al., 2022). However, it remains unclear why most nodulating Bradyrhizobium spp. retain NopPs that induce such incompatibility (Teulet et al., 2020). Since Bradyrhizobium spp. can infect multiple host plants, a NopP protein that induces incompatibility in 1 host may not trigger incompatibility in another (Lopez-Baena et al., 2009; Grundy et al., 2023). Additionally, NopP promotes rhizobial infection in certain host plants (Skorpil et al., 2005), indicating that retaining NopP may enhance the probability of rhizobial survival by enabling movement across symbioses with multiple host plants. Host incompatibility genes generally share homology with R genes, which control infection suppression in response to pathogen attack (Yang et al., 2010; Zhang et al., 2021; Gourion et al., 2015; Grundy et al., 2023). For example, incompatibility induced by B. diazoefficiens USDA122 via Rj2 functions as an effector-triggered immunity with expression of downstream defense response genes (Shine et al., 2019). In addition, Rj2 incompatibility is systemically expressed and triggers foliar resistance to plant pathogens (Shine et al., 2019). Defense response-related genes are also expressed following rhizobial inoculation in response to incompatibility induced by GmNNL1 (Zhang et al., 2021). These findings indicate that incompatibility can be interpreted as a defense response against rhizobia harboring specific effectors. Plants carrying R genes often experience a growth penalty associated with defense responses (Karasov et al., 2017; Ning et al., 2017; He et al., 2022; Gao et al., 2023). Trade-offs between plant growth and immunity have been widely reported across model plants and crop species, underscoring the need to balance productivity with resistance in crop breeding (Ning et al., 2017; Gao et al., 2023). In our study, 2 incompatibility genes were accumulated; thus, it was necessary to verify whether the trade-offs between defense response and plant growth from R genes were evident. We evaluated the effect of incompatibility gene accumulation by comparing the Rj2/GmNNL 1 and rj2/Gmnnl1 lines from the same breeding backgrounds. The Rj2/GmNNL1 lines, where incompatibility genes are functionally expressed through co-inoculation with USDA6, USDA110, and USDA122, and incompatibility genes were not expressed in the rj2/Gmnnl1 lines. The similarity in nodule numbers indicates that the accumulation of incompatibility genes does not affect nodule number control mechanisms. However, nodule dry weight per plant, nodule dry weight per nodule, SDW, and RDW in Rj2/GmNNL1 were substantially higher than in rj2/Gmnnl1 . We observed no negative effects on symbiotic phenotypes or plant growth due to incompatibility gene accumulation within our system. Therefore, these phenotypes appear more influenced by the combination of host soybean and rhizobial strains than by the expression of incompatibility genes (Yuan et al., 2020). According to the geographical distribution of rhizobia in Japanese soil, the major rhizobial flora in the Honshu-Kyushu area can be classified into Nos − USDA6 ( B. japonicum ) and Nos + USDA110 types ( B. diazoefficiens ) (Shiina et al., 2014; Saeki et al., 2013). Although bacterial classification based on ITS and NopP sequences does not always align, NopP sequence analysis using the same bacterial library (Shiina et al., 2014) indicates the widespread presence of NopP USDA110 , NopP USDA122 , and NopP USDA6 types of bradyrhizobia in Japan (Sugawara et al., 2018). This finding suggests that GmNNL1 and Rj2 can effectively inhibit infection competition by indigenous rhizobia. Here, this hypothesis was tested through competitive inoculation experiments with strains USDA6, USDA110, USDA122, and the incompatibility-avoiding N 2 O-reducing rhizobacteria FY2-m1, GMA461-m4, and OSA024. Each incompatibility-avoiding strain had a significantly higher nodule occupancy than USDA6, USDA110, and USDA122. However, in experiments using field soil, the occupancy rate of OSA024 was lower than that of FY2-m1 and GMA461-m4, and the N 2 O release from the OSA024-inoculated soybean rhizospheres trended higher. These data indicate that OSA024 was less effective than the other strains in the field soil used. In our field experiment, the nopP -deficient GMA461-m4 showed higher nodule occupancy than GMA461 with nopP USDA122 and SG09 with nopP USDA6 . The lack of nodulation with GMA461 may be due to the strong incompatibility of Rj2 against the effector NopP USDA122 (Sugawara et al., 2018). In contrast, GmNNL1 incompatibility against rhizobia with nopP USDA6 or nopP USDA110 blocks infection via infection threads but still allows infection through crack entry (Zhang et al., 2021). Thus, the nodule occupancy of SG09 in Rj2/GmNNL1 soybeans is likely higher than that of GMA461. Nodule occupancy of GMA461-m4, at over 60%, may substantially mitigate N 2 O emissions from the rhizosphere of Rj2/GmNNL1 soybeans. Therefore, this strain is thought to both outcompete indigenous rhizobia in Rj2/GmNNL1 symbiosis and exhibit N 2 O reduction potential under field soil conditions. Collectively, our findings demonstrate that optimizing the combination of incompatibility genes and effectors-deficient N 2 O-reducing rhizobia can lead to soybean cultivation systems that effectively reduce N 2 O emissions. Globally, soil types vary substantially. In this study, based on the characteristics of Japanese soils, Rj2 and GmNNL1 were used as symbiotic incompatibility genes to prevent infection by the predominant indigenous rhizobia. Since the dominant indigenous rhizobia may differ depending on the soils’ physical, chemical, and biological properties, applying this method broadly will require selecting incompatibility genes specific to the dominant rhizobia in a soil type (Li et al., 2023; Mendiza-Suarez et al., 2021). Agricultural land is a major anthropogenic source of N 2 O emissions (Tian et al., 2020). Under current global warming conditions, biological N 2 fixation through rhizobial symbiosis holds substantial promise as an alternative for agricultural production systems reliant on synthetic nitrogen fertilizers (Bourion et al., 2018; Guilpart et al., 2022; Grundy et al., 2023; Rotundo et al., 2024). In addition to nitrogen fertilizer applied to fields, nitrogen sources released from soybean plant residues contribute to N 2 O emissions (Inaba et al., 2009; Inaba et al., 2012; Toyoda et al., 2024). A rhizobial symbiotic system with N 2 O-reducing rhizobia can help reduce N 2 O emissions from soybean fields by lowering the need for artificial nitrogen fertilizer, a primary source of N 2 O emission, and by reducing N 2 O derived from soybean residues through the rhizobia’s N 2 O-reducing capability. Materials and Methods Isolation of Nos ++ B. ottawaense strains and incompatibility-avoiding strains Mature nodules were collected from soybean roots grown in (1) fields in Gunma Prefecture in 2021 and 2022; and (2) soils collected from a soybean field in Osaka Prefecture in 2022. Collected nodules were surface-sterilized with 0.5% NaOCl, placed in a 96-well microplate, and crushed with a sterile toothpick in 150 µL of sterile water. Then, 75 µL of cell lysate was prepared from the nodule crushing solution (Shiina Y et al., 2014). Next, 25 µL of 50% (v/v) glycerol solution was added to the remaining 75 µL of crushed nodule solution, and the samples were stored at − 80°C until bradyrhizobia isolation. PCR was performed with B. ottawaense nosZ -specific primers using cell lysates as templates to select bradyrhizobia strains possessing B. ottawaense nosZ (Hara et al., 2024). The cell lysates were also used as templates for PCR amplification of the 16S-23S ITS region and nopP (Shiina Y et al., 2014; Sugawara M et al., 2018). DNA sequences of the 16S-23S ITS region and nopP were determined by Sanger sequencing at GENEWIZ (Azenta Life Sciences). Based on ITS and nopP sequencing results, strains with over 99% ITS homology to B. ottawaense SG09 and nopP USDA122 or IS-inserted nopP were selected. Strains FY2, GMA461, and OSA024 were isolated from the nodule crushing solution stored at − 80°C using HM agar medium (Cole et al., 1973). Measurement of NO-reducing activity in cultured rhizobia The N 2 O-reducing activity of B. ottawaense strains was measured (Wasai-Hara et al., 2023). N 2 O-reducing activity was determined by culturing the bacteria under anaerobic conditions with 1% N 2 O supplied as the sole electron acceptor. N 2 O concentrations were measured using a gas chromatograph (GC2014; Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector and a Porapak Q column (GL Sciences, Tokyo, Japan). Bacterial strains were first aerobically cultured for over 6 hours in a 75-mL test tube with an air-permeable plug containing 10 mL of HM liquid medium (Cole et al., 1973) supplemented with 0.1% (w/v) arabinose and 0.025% (w/v) yeast extract, at 28°C with shaking at 200 rpm. Then, an appropriate volume of bacterial culture was transferred to new tubes containing 10 mL of HM medium to reach an optical density (OD) at 660 nm (OD 660 ) of 0.05, measured in a 25-mm diameter test tube (TEST25NP; AGC Techno Glass Co., Ltd., Shizuoka, Japan). After the initial culture, the test tube was sealed with a butyl rubber cap, and the gas phase was replaced with a mixture of 4.98% N 2 O + 95.02% N 2 gas for 12 to 14 hours to induce N 2 O reduction metabolism. Afterward, the gas phase was replaced with 100% N 2 gas, and 100% N 2 O was added to adjust to a final concentration of 1%. The test tube was incubated at 28ºC with shaking at 200 rpm, and 100 µL samples of the gas phase were withdrawn every 1 to 3 hours for analysis by gas chromatography. Selection of incompatibility-avoiding Nos ++ strains Strains FY2 and GMA461 harboring nopP 122 were inoculated into soybean ( Glycine max (L.) Merr. cv. Hardee) carrying the Rj2 incompatibility gene. Soybean seeds were sterilized using 0.5% sodium hypochlorite, sown in Leonardo Jar pots (five seeds per pot) containing sterilized vermiculite, and inoculated with FY2 and GMA461 at 1 × 10 9 cells per seed (Sugawara et al., 2018). Six pots were prepared for each inoculated strain. Soybeans were grown in a growth chamber at 25°C with a 16-hour light and 8-hour dark cycle for 3 weeks. Nodules were collected and surface-sterilized with 0.5% NaOCl and sliced with a sterile razor blade, and the internal bacteroids were spread on HM agar medium to isolate the bradyrhizobia strains. Single-colony isolates were subsequently inoculated onto Hardee soybeans. After 3 weeks of cultivation, the bradyrhizobia strains were re-isolated from nodules on soybean roots with green leaves. Lastly, nopP PCR was performed on the bradyrhizobial isolates to determine the presence of ISs in nopP (Sugawara et al., 2018). Genotypic analysis of Rj2 and GmNNL1 genes in soybean germplasm For whole-genome resequencing of the soybean variety "Bonminori," total DNA was extracted from leaves using the DNeasy Plant Mini Kit (Qiagen). The DNA library was subjected to 150-bp paired-end sequencing on an Illumina NovaSeq instrument (Illumina Co., Ltd.) to achieve 20 × genome coverage. The reads from "Bonminori" and 192 accessions of the mini-core collection were mapped to the G. max Williams 82 genome assembly (v4.0) using BWA-MEM (Li & Durbin 2009), and the duplicates were removed using Picard MarkDuplicates ( http://broadinstitute.github.io/picard/ ). Variants calling followed GATK best practices for germline SNP/Indel discovery (Auwera et al., 2013), using GATK version 4.0.11.0. Variants were initially called individually for each sample with GATK HaplotypeCaller, followed by joint genotyping with GenotypeGVCFs to consolidate variants (Poplin et al., 2017). Variants were first filtered using GATK with the parameters: "QD 50.0 || SOR > 3.0 || MQ < 50.0 || MQRankSum < -2.5 || ReadPosRankSum 3.5" Further filtering was conducted using the bcftools view (Li, 2011) with parameters: -m2 -M2 -g hom --output-type z --exclude-uncalled -e "MAF 0.25." All variants were annotated for potential impact using SnpEff version 4.3 (Cingolani et al., 2012). The genotype of Rj2 was determined based on an SNP (C/T) at Gm16: 37281186 bp, which causes a single amino acid substitute (R490 to I490) (Sugawara et al., 2019). Since the genotype of GmNNL1 is defined by an SV, SV analysis was performed using Manta (Chen et al., 2016), with SV files subsequently merged using SURVIVOR (Jeffares et al., 2017). Breeding soybean with accumulated incompatibility genes The soybean varieties “Bonminori”, harboring Rj2 , and GmWMC108 Karasu-mame, harboring GmNNL1 , were grown simultaneously during flowering and crossbred. To facilitate DNA marker analysis of the Rj2 genotypes, an indel marker was developed based on an SV near Rj2 (see Supplementary Table 3). An indel marker for GmNNL1 genotype analysis was developed using genomic sequences around the position of the SINE-like transposon. For genotyping, genomic DNA was extracted from thin slices of the resulting seed cotyledon (Kamiya and Kiguchi 2003). The Rj2 and GmNNL1 markers were amplified using PCR to select the F 1 seeds heterozygous for Rj2 and GmNNL1 based on migration patterns in agarose gel electrophoresis. Then, the F 1 seeds were planted and grown. After harvesting the F 2 seeds, the lines homozygous for Rj2 and GmNNL1 were selected. Then, F 3 seeds were harvested from the resulting F 2 plants to establish Rj2/GmNNL1 soybean lines. Method of rhizobial inoculation for soybean A rhizobial inoculum was prepared by suspending rhizobial strains in Broughton and Dilworth (B&D) solution (Broughton and Dilworth 1971) at 6.7 × 10 2 cells/mL. Leonard jar pots filled with sterile vermiculite were pre-inoculated with 150 mL of the rhizobial inoculum, and 2 chlorine-gas-treated soybean seeds were sown per pot. Cultivation was conducted in an artificial climatic chamber set to 25°C with a 16-hour light and 8-hour dark photoperiod. On the fourth day after sowing, seedlings were thinned to leave 1 well-germinated plant per pot and then cultivated for 3 to 5 weeks. Pots were periodically supplied with a nitrogen-free B&D solution. Analysis of root nodule occupancy using competitive inoculation tests B. diazoefficiens strains USDA110 and USDA122 and B. japonicum strain USDA6 were selected as competitor strains. Incompatibility-avoiding strains and competitor strains were each suspended in B༆D solution. One of the incompatibility-avoiding strains was mixed with all 3 competitor strains at equal ratios, and a rhizobial inoculum mixture with a total bacterial concentration of 6.7 × 10 2 cells/mL was prepared. In Leonard jar pots filled with sterile vermiculite, 150 mL of the rhizobial inoculum mixture was pre-inoculated, and 2 chlorine-gas-treated Rj2/GmNNL1 or rj2/Gmnnl1 soybeans were sown per pot following the standard soybean inoculation method. At five weeks after inoculation, the number of mature nodules on the roots was counted. Then, mature nodules were collected and surface-sterilized by immersion in 10% NaOCl for 3 min. The nopP region was amplified using PCR with nopP -specific primers. Next, PCR-amplified fragments were digested with the restriction enzymes Alu I and Pst I and analyzed by agarose gel electrophoresis. The strains occupying mature nodules were identified based on the patterns of the restriction enzyme-digested PCR-amplified fragments. N 2 O flux from nodulated soybean roots in Leonard jar pot experiments N 2 O flux was measured (Wasai-Hara et al., 2023). After competitive inoculation, the soybean root system was gently immersed in water to remove excess vermiculite. Then, the roots were transferred to a 100-mL glass vial containing 30 mL of soil obtained from the Kashimadai experimental field (38°27′36.0″N, 141°05′24.0″E) with permission from Tohoku University, Japan. Kashimadai soils had been sieved through a 2-mm mesh to remove large aggregates and stones. Additionally, 5 ml of sterile distilled water was added to each vial. The vials containing roots, soil, and water were incubated aerobically at 25°C for 20 days to induce nodule degradation. The vials were covered with a soft cloth to maintain aeration during incubation. Each week during the incubation, vials were sealed with butyl-rubber caps and kept under atmospheric conditions for 240 to 360 minutes to determine N 2 O flux. N 2 O concentrations in the vial gas phase were measured using a gas chromatograph (GC2014; Shimadzu) equipped with a 63 Ni electron capture detector and tandem Porapak Q columns (GL Sciences; 80/100 mesh; 3.0 mm × 1.0 m and 3.0 mm × 2.0 m). Analysis of root nodule occupancy and N 2 O release in field simulation cultivation with field soil Four seeds were sown in a Wagener pot filled with 3 L of NARO field soil (Andosol) and inoculated with 1 ml of incompatibility-avoiding rhizobial solution at 1 × 10 9 cells/ml per seed. Eight pots were prepared for each test plot. After germination, 2 seedlings were retained per pot. After 42 days of cultivation, soybeans were harvested from 3 pots, and the number of nodules formed on the roots was recorded. Nodule occupancy was determined using PCR to detect the B. ottawaense -type nosZ gene (Hara et al., 2024). For the remaining 5 pots, the above-ground portion of the soybeans was excised, and 30-ml gas samples were collected at 0, 20, and 40 minutes after covering the pots with acrylic plates. Sampling was conducted every 2 to 3 days, and N 2 O concentrations in the gas samples were quantitatively analyzed using gas chromatography (GC-2014, Shimazu, Kyoto, Japan; Sudo, 2021). Analysis of nodule occupancy and N 2 O flux through field cultivation Rj2/GmNNL1 soybeans were inoculated with the Kashimadai field at Tohoku University with peat moss materials containing SG09, GMA461, and GMA461-m4 strains at 1 × 10 10 CFU per seed. Approximately 1 month later, the nodule occupancy of the inoculum in mature nodules was quantified by PCR to detect the B. ottawaense -type nosZ (Hara et al., 2024). About 15 weeks after inoculation and during soybean ripening, N 2 O concentrations over time were monitored with a mobile mid-infrared N 2 O sensor (MIRA Ultra; Aeris Technologies, Heyward, CA, USA). Data analysis Statistical analysis was conducted using JMP16.2.0. The statistical analysis methods applied are as follows. Figure 1 ; Tukey’s HSD test. Figure 3 ; Wilcoxon rank-sum test. Figure 4 ; Student’s t-test. Figure 5 ; Dunnett test using mock inoculation as control. Figure 6 A; Steel test using mock inoculation as control, 6B; Dunnett test using mock inoculation as control. SplFig3; Tukey’s HSD test. SplFig4C; Student’s t-test. Declarations Data availability Upon this paper's acceptance, the NGS data for “Bonminori” will be available in the DDBJ Sequence Read Archive under accession number DRR610375. Authors and Affiliations Hanna Nishida, Khin Thuzar Win, Yukiko Fujisawa, Yoshikazu Shimoda, Haruko Imaizumi-Anraku Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan Manabu Itakura, Kaori Kakizaki, Atsuo Suzuki, Satoshi Ohkubo, Kiwamu Minamisawa Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Feng Li, Koji Takahashi Institute of Crop Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan Masayuki Sugawara Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan Sachiko Masuda, Arisa Shibata, Ken Shirasu Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan Misa Tsubokura, Hiroko Akiyama Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan Competing interests The authors declare having no conflicts of interest. Corresponding authors Haruko IMAIZUMI-ANRAKU Kiwamu MINAMISAWA Author contributions K.M. and H.I-A oversaw the project and designed the experiments. H.N., M.I., K.T.W., F. L., K.K., A.S., S.O., M.S., K.T., S.M., A.S., K.S., Y.F., M.T., H.A., Y.S., K.M. and H.I-A performed experiments. M.I., K.K., A.S., S.O. and K.M. isolated Nos ++ B. ottawaense . H.N., M.I., K.K., M.S., Y.F., Y.S., and H.I-A. analyzed nodule occupancy of Nos ++ bradyrhizobia. M.I., K.T.W., S.O., M.T., and H.A. analyzed N 2 O flux from the rhizosphere. F.L. analyzed the genotypes of incompatibility genes in the NARO soybean core collection and selected the Rj2/GmNNL1 -accumulating soybean lines. K.T. crossbred soybean cultivars possessing incompatibility genes. S.M., A. S., and K.S. sequenced the whole genome of Nos ++ bradyrhizobia. H.I-A., K.M., H.N., M.I., and S.O. analyzed the data and wrote the manuscript. Acknowledgments We thank Shusei Sato (Tohoku University) for this manuscript's critical reading and useful comments. We also acknowledge Dr. Matthew Shenton (NARO) for supplying a pipeline for whole genome resequencing analysis of soybeans. This research was supported by a JPNP18016 project commissioned by the New Energy and Industrial Technology Development Organization (NEDO). References Tian H. et al. A comprehensive quantification of global nitrous oxide sources and sinks Nature 586, 248–256 (2020). Uchida Y. & Akiyama H. Mitigation of postharvest nitrous oxide emissions from soybean ecosystems: a review. Soil Science and Plant Nutrition 59, 477-487 (2013) Goyal R. K., Mattoo A. K., Schmidt M. A. Rhizobial–Host Interactions and Symbiotic Nitrogen Fixation in Legume Crops Toward Agriculture Sustainability. Front Microbiol. 12: 669404 (2021) Bourion V. et al. 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Jeffares, D.C., Jolly, C., Hoti, M. et al. (2017). Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Nature Communications 8, 14061 Kamiya M. and Kiguchi T. Rapid DNA Extraction Method from Soybean Seeds. Breeding Science 53: 277-279 (2003) Broughton W.J. and Dilworth M.J. Control of leghaemoglobin synthesis in snake beans. Biochem J 125:1075–1080 (1971) Sudo S. and Yamamoto A. Three-component simultaneous analysis device and three-component simultaneous analysis method. (2021) Patent JP 6843395 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTables.docx Supplemental Tables SupplementalFigures.docx Supplemental Figures 111.pdf Reporting Summary Cite Share Download PDF Status: Published Journal Publication published 04 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5679948","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":397742626,"identity":"d841f1e7-fd96-4b84-80c7-1c9786e7f00f","order_by":0,"name":"Haruko 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(NARO)","correspondingAuthor":false,"prefix":"","firstName":"Yukiko","middleName":"","lastName":"Fujisawa","suffix":""},{"id":397742640,"identity":"c90fd0e9-9293-4ed1-bdb5-752cac9c17f1","order_by":14,"name":"Misa Tsubokura","email":"","orcid":"","institution":"Institute for Agro-Environmental Sciences, National Agriculture and Food Research Organization (NARO)","correspondingAuthor":false,"prefix":"","firstName":"Misa","middleName":"","lastName":"Tsubokura","suffix":""},{"id":397742641,"identity":"e7c1aa5c-e39a-41a8-a228-0672b976dfd7","order_by":15,"name":"Hiroko Akiyama","email":"","orcid":"","institution":"Institute for Agro-Environmental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hiroko","middleName":"","lastName":"Akiyama","suffix":""},{"id":397742642,"identity":"27cf1bba-d6a9-4b68-b1e9-cb1933e1faba","order_by":16,"name":"Yoshikazu Shimoda","email":"","orcid":"","institution":"National Institute of Agrobiological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yoshikazu","middleName":"","lastName":"Shimoda","suffix":""},{"id":397742643,"identity":"4074b4cc-2617-4263-9abe-dc24a27d64ca","order_by":17,"name":"Kiwamu Minamisawa","email":"","orcid":"https://orcid.org/0000-0001-7302-4079","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Kiwamu","middleName":"","lastName":"Minamisawa","suffix":""}],"badges":[],"createdAt":"2024-12-20 01:25:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5679948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5679948/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-63223-6","type":"published","date":"2025-09-04T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73926640,"identity":"123f96b3-1db6-4e81-88fb-73e830d1540c","added_by":"auto","created_at":"2025-01-16 04:58:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191781,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of soybean bradyrhizobial strains based on Internal Transcribed Sequences (ITS) between 16S and 23S ribosomal RNA genes and their N\u003csub\u003e2\u003c/sub\u003eO-reducing activities. The ITS phylogenetic tree was constructed by aligning ITS sequences using ClustalW and applying the neighbor-joining method.\u003cem\u003e *B. ottawaense \u003c/em\u003eisolated in this study. (\u003cem\u003en\u003c/em\u003e=3).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/5aa86157783917f840b7a80e.png"},{"id":73926641,"identity":"6aabd80c-3d38-48cc-b7c8-8d262b264aee","added_by":"auto","created_at":"2025-01-16 04:58:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1159019,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation of incompatibility-avoiding bradyrhizobia strains.\u003c/p\u003e\n\u003cp\u003ea. Giant nodule (arrowhead) formed when soybean cultivar Hardee was inoculated with \u003cem\u003eB. ottawaense \u003c/em\u003eGMA461. b. Positions of endogenous transposon insertions in \u003cem\u003enopP\u003c/em\u003e of FY2-m1, GMA461-m4, and OSA024. White arrowheads, the positions of the 4 amino acid residues that define the NopP type; amino acid residues and their positions are noted above each arrowhead in the NopP sequence. c. Amplified fragments of the \u003cem\u003enopP\u003c/em\u003eregion using \u003cem\u003enopP\u003c/em\u003e-specificprimers. Red arrowheads, bands that shifted toward a larger molecular size due to transposon insertion.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/192f99663f8effd9cbe7d11d.png"},{"id":73926644,"identity":"21b6a7dc-e83c-4c9f-bf43-ab89c51daf9d","added_by":"auto","created_at":"2025-01-16 04:58:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1202207,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean and its nodulation phenotypes.\u003c/p\u003e\n\u003cp\u003ea. Electrophoresis images of \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e markers in soybean. b-c. Number of mature nodules on the roots of \u003cem\u003eRj2/GmNNL1\u003c/em\u003egene-accumulated soybean inoculated with FY2 or FY2-m1 (b), and GMA461 or GMA461-m4 (c). Photographs of each nodulated root were presented below the graphs.*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; \u003cem\u003en\u003c/em\u003e=4.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/01d08590ab1e2c89e3100b4b.png"},{"id":73926650,"identity":"ff9e64d8-d0ec-4ae6-9025-eef56a0ca8e9","added_by":"auto","created_at":"2025-01-16 04:58:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210108,"visible":true,"origin":"","legend":"\u003cp\u003eMitigation of N\u003csub\u003e2\u003c/sub\u003eO emission through predominant infection by incompatibility-avoiding Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia strains.\u003c/p\u003e\n\u003cp\u003ea–c. Nodule occupancy of USDA6, USDA110, USDA122, and incompatibility-avoiding rhizobia in \u003cem\u003eRj2/GmNNL1\u003c/em\u003e and \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybean lines FY2-m1 (a), GMA461-m4 (b), and OSA024 (c). d–f. N\u003csub\u003e2\u003c/sub\u003eO emission was measured 1, 2, and 3 weeks after decapitation (WAD) in FY2-m1 (d), GMA461-m4 (e), and OSA024 (f). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; \u003cem\u003en\u003c/em\u003e=4.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/77a0769bf397348698ff3253.png"},{"id":73926647,"identity":"91826644-87d2-4adf-8048-4c8e9fcd405b","added_by":"auto","created_at":"2025-01-16 04:58:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":274123,"visible":true,"origin":"","legend":"\u003cp\u003eMitigation of N\u003csub\u003e2\u003c/sub\u003eO emissions through predominant infection by incompatibility-avoiding Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia strains under competition with indigenous soil rhizobia.\u003c/p\u003e\n\u003cp\u003ea. Nodule occupancy. b. Trends in N\u003csub\u003e2\u003c/sub\u003eO emissions over time after plant decapitation. c. Cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003e p\u003c/em\u003e\u0026lt;0.01; n=3 (OSA024); n=5 (FY2-m1, GMA461-m4).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/d4141329e3ae29f037d25848.png"},{"id":73926655,"identity":"bd32fb22-4aa3-4507-9cf7-e7b5f1739daf","added_by":"auto","created_at":"2025-01-16 04:58:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":108771,"visible":true,"origin":"","legend":"\u003cp\u003eNodule occupancy and N\u003csub\u003e2\u003c/sub\u003eO flux from Kashimadai field in \u003cem\u003eGmNNL1/Rj2\u003c/em\u003e soybean plants inoculated with \u003cem\u003eB. ottawaense \u003c/em\u003estrains SG09 (\u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA6\u003c/sub\u003e), GMA461 (\u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e), and GMA461-m4 (\u003cem\u003enopP\u003c/em\u003e deficient mutant).\u003c/p\u003e\n\u003cp\u003ea. Nodule occupancy of \u003cem\u003eB. ottawaense\u003c/em\u003e-type rhizobia. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; \u003cem\u003en\u003c/em\u003e=6. b. N\u003csub\u003e2\u003c/sub\u003eO release from the field immediately after the excision of above-ground parts of nodulated soybean. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; \u003cem\u003en\u003c/em\u003e=4.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/76d936dc24ea82768b5df16e.png"},{"id":90642695,"identity":"7322bd67-5c3d-4953-b8d5-d2beb42aee92","added_by":"auto","created_at":"2025-09-05 07:05:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4962146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/1127c2ad-48b5-481e-9cd3-b373affe3d44.pdf"},{"id":73926970,"identity":"58d34c95-bc45-42a1-99fd-ee4d778d3ce2","added_by":"auto","created_at":"2025-01-16 05:06:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":45388,"visible":true,"origin":"","legend":"Supplemental Tables","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/2922e6202df96b5121440686.docx"},{"id":73926656,"identity":"eec9e441-b19c-4905-86dc-7b529f04ec43","added_by":"auto","created_at":"2025-01-16 04:58:40","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1728203,"visible":true,"origin":"","legend":"Supplemental Figures","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/5c065c3945d1f71e353c4472.docx"},{"id":73926646,"identity":"2f10f8e3-71cf-4745-9c77-dafe3193957a","added_by":"auto","created_at":"2025-01-16 04:58:39","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2098272,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"111.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5679948/v1/05c19dc8071f2f7a678519cd.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Genetic design of soybean hosts and bradyrhizobial endosymbionts reduces N2O emissions from soybean farming","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFood production is expanding to support the growing human population. However, agricultural lands are major sources of anthropogenic nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO), a greenhouse gas (GHG) with a global warming potential approximately 300 times greater than that of carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) (Tian et al., 2020; Uchida \u0026amp; Akiyama 2013). Meanwhile, chemical nitrogen fertilizers, synthesized from fixed atmospheric N\u003csub\u003e2\u003c/sub\u003e via the Haber\u0026ndash;Bosch process using fossil fuels, serve as the primary source of nitrogen for current intensive farming systems (Goyal et al., 2021; Tian et al., 2020; Bourion et al., 2018). Then, nitrogen sources, including fertilizers and biological residues, are converted to inorganic nitrogen by soil microorganisms through the nitrogen cycle (Sanchez \u0026amp; Minamisawa 2019; Uchida \u0026amp; Akiyama 2013). Soil nitrogen, existing as ammonia and nitrate inorganic compounds, transitions between these states through nitrification and denitrification. Among these processes, denitrification involves multiple reduction reactions (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026rarr; NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026rarr; NO\u0026rarr; N\u003csub\u003e2\u003c/sub\u003eO\u0026rarr; N\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eWhile many soil bacteria lack specific genes that encode reductase for complete denitrification, thereby acting as sources and sinks for N\u003csub\u003e2\u003c/sub\u003eO (Hiis et al., 2024), soil rhizobial bacteria can perform biological N\u003csub\u003e2\u003c/sub\u003e fixation through symbiosis with leguminous plants. For example, \u003cem\u003eBradyrhizobium\u003c/em\u003e, a genus comprising N\u003csub\u003e2\u003c/sub\u003e-fixing bacteria, has symbiotic interactions with legumes, such as soybeans, by infecting the plants, forming nodules in plant roots, and converting N\u003csub\u003e2\u003c/sub\u003e into ammonium in the nodules (Wang et al., 2018; Roy et al., 2020; Nakei et al., 2022). This type of biological N\u003csub\u003e2\u003c/sub\u003e fixation provides N\u003csub\u003e2\u003c/sub\u003e to host plants without the economic effects and GHG emissions associated with chemical N\u003csub\u003e2\u003c/sub\u003e fertilizer production (Goyal et al., 2021; Graham \u0026amp; Vance 2003). However, the nitrogen released from aging and decaying nodules is also a source of N\u003csub\u003e2\u003c/sub\u003eO in the soil (Akiyama et al., 2016; Wasai-Hara et al., 2023; Inaba et al., 2009; Inaba et al., 2012; Toyoda et al.. 2024). Globally, N\u003csub\u003e2\u003c/sub\u003eO emissions from soybean plant residues were estimated to be 19,685 kt CO\u003csub\u003e2\u003c/sub\u003e eq in 2020 (FAO, 2024).\u003c/p\u003e \u003cp\u003eSome rhizobacteria in the genus \u003cem\u003eBradyrhizobium\u003c/em\u003e possess N\u003csub\u003e2\u003c/sub\u003eO reductase, an enzyme that reduces N\u003csub\u003e2\u003c/sub\u003eO to N\u003csub\u003e2\u003c/sub\u003e (Samejima-Saito et al., 2006; Itakura et al., 2008; Itakura et al., 2013; Akiyama et al., 2016; Henault et al., 2022; Melissa et al., 2022; Mania et al., 2020). For example, the \u003cem\u003eBradyrhizobium ottawaense\u003c/em\u003e strains isolated in Japan have demonstrated high N\u003csub\u003e2\u003c/sub\u003eO-reducing ability (Nos\u003csup\u003e++\u003c/sup\u003e) and are classified as Nos\u003csup\u003e++\u003c/sup\u003e rhizobia (Wasai-Hara et al., 2020; Wasai-Hara et al., 2023). In Japan, Andosols\u0026mdash;a major soil type covering approximately half of the upland fields\u0026mdash;are predominantly occupied by \u003cem\u003eB. japonicum\u003c/em\u003e, which lacks \u003cem\u003enosZ\u003c/em\u003e, the gene encoding N\u003csub\u003e2\u003c/sub\u003eO reductase (Nos\u003csup\u003e\u0026minus;\u003c/sup\u003e rhizobia) (Shiina et al., 2014). Additionally, most paddy fields consist of alluvial soil, primarily colonized by \u003cem\u003eB. diazoefficiens\u003c/em\u003e, an N\u003csub\u003e2\u003c/sub\u003eO-reducing (Nos\u003csup\u003e+\u003c/sup\u003e) rhizobial phenotype (Shiina et al., 2014). However, the reports on the distribution of Nos\u003csup\u003e++\u003c/sup\u003e rhizobia in Japan remain limited, and only \u003cem\u003eB. ottawaense\u003c/em\u003e has been identified in sorghum fields in Fukushima (Wasai-Hara et al., 2020; Wasai-Hara et al., 2023).\u003c/p\u003e \u003cp\u003eSince approximately 80% of the soybean fields in Japan are converted from rice paddies, most of the nitrogen derived from soybean residues is likely released as N\u003csub\u003e2\u003c/sub\u003eO without being reduced to N\u003csub\u003e2\u003c/sub\u003e. Inoculating soybeans with Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia can reduce post-harvest N\u003csub\u003e2\u003c/sub\u003eO emissions from these fields (Itakura et al., 2013; Akiyama et al., 2016). As the fourth most important crop worldwide, soybeans provide essential vegetable protein and oil for humans and animals (Goyal et al., 2021; Hartman et al., 2011; Rotundo et al., 2024). In 2022, the area for soybean cultivation spanned 133,791,632 hectares, representing approximately 8% of the global total of cropland (1,617,392,600 hectares) (Our World in Data, 2022). Thus, introducing soybean symbiosis with Nos\u003csup\u003e++\u003c/sup\u003e \u003cem\u003eBradyrhizobium\u003c/em\u003e strains can substantially reduce N\u003csub\u003e2\u003c/sub\u003eO emissions from soybean agriculture.\u003c/p\u003e \u003cp\u003eHowever, competition from indigenous soil rhizobia hinders the infection of promising Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia after soybeans are inoculated with them. Consequently, the occupancy rate of Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia in soybean nodules remains unstable, ranging between 20% and 60% (Itakura et al., 2013). The N\u003csub\u003e2\u003c/sub\u003eO-reducing capacity of Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia is likely limited, as most indigenous rhizobia occupying root nodules exhibit little or no N\u003csub\u003e2\u003c/sub\u003eO-reducing capability. Consequently, this \u0026ldquo;rhizobial competition problem\u0026rdquo; has been a substantial barrier to the successful introduction of promising rhizobia into agricultural fields (Bourion et al., 2018; Nakei et al., 2022; Triplett \u0026amp; Sadowsky, 1992; Mendiza-Suarez et al., 2020; Mendiza-Suarez et al., 2021). Thus, establishing a soybean symbiotic system that predominantly supports Nos\u003csup\u003e\u003cem\u003e++\u003c/em\u003e\u003c/sup\u003e rhizobia infection in nodules is essential for addressing this issue and maximizing the N\u003csub\u003e2\u003c/sub\u003eO-reducing potential of Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia.\u003c/p\u003e \u003cp\u003eWhile various factors influence the nodule occupancy rate of rhizobia, the primary determinant is the genotype of the host plant (Triplett \u0026amp; Sadowsky, 1992; Burghardt et al., 2022; Mendiza-Suarez et al., 2021, Grundy et al., 2023). Specifically, soybean incompatibility\u0026mdash;a symbiotic inhibition response triggered by rhizobia-released effector proteins via the Type III secretion system\u0026mdash;is defined by the soybean genes and corresponding rhizobial effector proteins involved (Teulet et al., 2020; Staehelin \u0026amp; Krishnan, 2015, Grundy et al., 2023). Currently, the genes involved in incompatibility include \u003cem\u003eRj2\u003c/em\u003e, \u003cem\u003eRfg1\u003c/em\u003e, \u003cem\u003eRj4\u003c/em\u003e, and \u003cem\u003eGmNNL1\u003c/em\u003e (Yang et al., 2010; Sugawara et al., 2018; Sugawara et al., 2019; Tang et al., 2016; Ratu et al., 2021; Zhang et al., 2021). \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e are Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) resistance R genes (Yang et al., 2010; Zhang et al., 2021; Gourion et al., 2015). Rj2 induces incompatibility by recognizing NopP\u003csub\u003eUSDA122\u003c/sub\u003e, an effector protein derived from \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA122 (Sugawara et al., 2018). In contrast, both NopP\u003csub\u003eUSDA6\u003c/sub\u003e from \u003cem\u003eB. japonicum\u003c/em\u003e USDA6 and NopP\u003csub\u003eUSDA110\u003c/sub\u003e from \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA110 are recognized by GmNNL1 (Zhang et al., 2021). Most nodulating Bradyrhizobium spp. harbor \u003cem\u003enopP\u003c/em\u003e (Teulet et al.., 2020). In addition, Sugawara et al. (2018) analyzed the \u003cem\u003enopP\u003c/em\u003e sequences from a collection of bradyrhizobial isolates from soil samples across 32 locations in Japan and found that NopP\u003csub\u003eUSDA122\u003c/sub\u003e, NopP\u003csub\u003eUSDA110\u003c/sub\u003e, and NopP\u003csub\u003eUSDA6 (ST2)\u003c/sub\u003e were widespread in Japan. Therefore, soybeans with \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e may suppress the infection of indigenous rhizobia harboring \u003cem\u003enopP\u003c/em\u003e\u003csub\u003e\u003cem\u003eUSDA6\u003c/em\u003e\u003c/sub\u003e, \u003cem\u003enopP\u003c/em\u003e\u003csub\u003e\u003cem\u003eUSDA110\u003c/em\u003e\u003c/sub\u003e, or \u003cem\u003enopP\u003c/em\u003e\u003csub\u003e\u003cem\u003eUSDA122\u003c/em\u003e\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we crossed soybeans to develop a line with \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e. Additionally, we generated NopP-deficient Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia through induced spontaneous mutations (Sugawara et al., 2018). These NopP-deficient Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia bypass symbiotic incompatibility with \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e, enabling successful infection of the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean line. The incompatibility-avoiding Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia achieved high occupancy rates within the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean line through competitive infection with \u003cem\u003eB. japonicum\u003c/em\u003e USDA6, \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA110, and \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA122, demonstrating substantial N\u003csub\u003e2\u003c/sub\u003eO-reducing capacity. Then, we further examined whether the incompatibility-avoiding Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia could outcompete indigenous rhizobia in soybean field soil, thereby reducing N\u003csub\u003e2\u003c/sub\u003eO emissions from the soybean rhizosphere.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneration of incompatibility-avoiding bradyrhizobia with high N\u003csub\u003e2\u003c/sub\u003eO-reducing activity\u003c/h2\u003e\n \u003cp\u003eIt is necessary to delete the effector gene or disable its function to establish bradyrhizbial strains that can overcome incompatibility with NopP effectors. When \u003cem\u003eRj2\u003c/em\u003e soybeans are inoculated with bradyrhizobia strains carrying \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e, huge mature nodules are rarely formed (Sugawara et al., 2018). Where nodules do form, they are often infected by \u003cem\u003enopP\u003c/em\u003e-deficient rhizobia that arise through spontaneous mutation. In addition, none of the eleven strains of \u003cem\u003eB. ottawaense\u003c/em\u003e previously isolated for their high N\u003csub\u003e2\u003c/sub\u003eO-reducing activity encode \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e (Wasai-Hara et al., 2023).\u003c/p\u003e\n \u003cp\u003eWe surveyed the \u003cem\u003eB. ottawaense\u003c/em\u003e strains carrying \u003cem\u003enopP\u003c/em\u003e\u003csub\u003e\u003cem\u003eUSDA122\u003c/em\u003e\u003c/sub\u003e strains in Japanese soybean fields (Itakura et al., Unpublished) to generate incompatibility-avoiding rhizobia. As a result, we obtained 2 target strains of \u003cem\u003eB. ottawaense\u003c/em\u003e carrying \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e (FY2 and GMA461) from Numata (Gunma Prefecture) and 1 strain, identified as a natural \u003cem\u003enopP\u003c/em\u003e mutant (OSA024), from Yao (Osaka Prefecture).\u003c/p\u003e\n \u003cp\u003eThe 16S-23S internal transcribed spacer (ITS) sequence of these 3 strains (OSA024, FY2, and GMA461) showed over 99% homology to the high N\u003csub\u003e2\u003c/sub\u003eO-reducing strain SG09 (Wasai-Hara et al., 2023), indicating close phylogenetic relationships (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, these strains demonstrated high N\u003csub\u003e2\u003c/sub\u003eO-reducing activity in free-living cells, comparable to \u003cem\u003eB. ottawaense\u003c/em\u003e SG09 and substantially higher than \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA110 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eNext, we attempted to isolate mutants with spontaneously disrupted \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e by inoculating the \u003cem\u003eRj2\u003c/em\u003e soybean cultivar Hardee with the FY2 or GMA461 strain carrying \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e. When FY2 was inoculated, 3 of the 4 isolates from huge nodules contained an insertion sequence (IS) within \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e, and 1 of these isolates was designated as the representative strain, FY2-m1. Among the \u003cem\u003eRj2\u003c/em\u003e cultivar inoculated with GMA461, 2 of the 4 isolates from giant nodules showed IS insertion in \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e, and 1 isolate was designated the representative strain, GMA461-m4 (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Sequence analysis of \u003cem\u003enopP\u003c/em\u003e in FY2-m1 and GMA461-m4 showed that \u003cem\u003eISRj2\u003c/em\u003e (Iida T et al., 2015) was inserted in both strains, but the positions of IS insertion differed (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). In the natural \u003cem\u003enopP\u003c/em\u003e mutant OSA024, \u003cem\u003eISBj11\u003c/em\u003e (Iida T et al., 2015) was inserted in \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA6\u003c/sub\u003e (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGeneration of soybean lines accumulating\u003c/strong\u003e \u003cstrong\u003eRj2\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eGmNNL1\u003c/strong\u003e \u003cstrong\u003eincompatibility genes and evaluation of rhizobial symbiosis ability of incompatibility-avoiding Nos\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e++\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003erhizobia\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWe selected the parent plants for accumulating \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e through the crossing and initially chose the well-known Japanese cultivar \u0026ldquo;Bonminori,\u0026rdquo; carrying the \u003cem\u003eRj2\u003c/em\u003e genotype (Shiro \u0026amp; Saeki 2022). Additionally, 192 accessions from a soybean mini-core collection with available whole-genome resequencing data (Kajiya-Kanegae, 2021) were analyzed for the \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e genotypes. We extracted genetic variations in \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e by detecting single nucleotide polymorphisms (SNPs), small insertions and deletions (InDels), and structural variations (SVs) across the 193 accessions. Since the \u003cem\u003eRj2/rj2\u003c/em\u003e genotype is determined by an SNP (C/T) in the second exon, which causes a single amino acid substitution (R490 to I490) (Sugawara et al., 2019), we identified 10 accessions with the \u003cem\u003eRj2\u003c/em\u003e genotype (Supplementary Table 1). Conversely, the null \u003cem\u003eGmnnl1\u003c/em\u003e genotype results from a 179-bp SINE-like transposon insertion in the second exon (Chen et al., 2016). Only 4 accessions possessed a functional \u003cem\u003eGmNNL1\u003c/em\u003e genotype (Supplementary Table 1). The individual occurrence percentage of \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eNNL1\u003c/em\u003e functional genotypes within the mini-core collection were 5.18% and 2.07%, respectively. Lastly, no accession had a functional genotype of \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eThen, we selected the cultivars \u0026ldquo;Bonminori\u0026rdquo; and GmWC108 (Karasu-mame), which had similar flowering times, as parent plants for crossing to accumulate \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e. After crossing, we selected 1 line that was homozygous for \u003cem\u003eRj2/GmNNL1\u003c/em\u003e and another line that was homozygous for \u003cem\u003erj2/Gmnnl1\u003c/em\u003e from the F\u003csub\u003e2\u003c/sub\u003e seed population. From the progenies, we obtained the F\u003csub\u003e3\u003c/sub\u003e seeds. The \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003erj2\u003c/em\u003e and the \u003cem\u003eGmNNL1\u003c/em\u003e and \u003cem\u003eGmnnl1\u003c/em\u003e genotypes of the seeds could be distinguished based on the PCR-amplified fragments of varying sizes that covered an SV near \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e and were generated using the cotyledon DNA from F\u003csub\u003e2\u003c/sub\u003e seeds (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\n \u003cp\u003eThen, the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e line was inoculated with FY2 and GMA461, which carried \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e, and with FY2-m1 and GMA461-m4, which exhibited the loss of \u003cem\u003enopP\u003c/em\u003e \u003csub\u003eUSDA122\u003c/sub\u003e function (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec). Premature nodule-like structures were observed upon inoculation with FY2 or GMA461. In contrast, inoculation with FY2-m1 and GMA461-m4 resulted in the development of multiple mature nodules. These findings indicate that the loss of \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e function due to endogenous transposon insertion enabled FY2-m1 and GMA461-m4 to overcome incompatibility mediated by Rj2 and GmNNL1.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eNodule occupancy, NO flux, and symbiotic phenotypes in infection competition between incompatibility-avoiding and other rhizobia\u003c/h3\u003e\n\u003cp\u003eWe simulated the infection competition between indigenous rhizobia and Nos\u003csup\u003e\u003cem\u003e++\u003c/em\u003e\u003c/sup\u003e rhizobia that avoided \u003cem\u003eRj2/GmNNL1\u003c/em\u003e incompatibility by conducting a simultaneous inoculation experiment using 4 strains: USDA6 (Nos\u003csup\u003e\u0026minus;\u003c/sup\u003e), USDA110 (Nos\u003csup\u003e+\u003c/sup\u003e), USDA122 (Nos\u003csup\u003e+\u003c/sup\u003e), and an incompatibility-avoiding Nos\u003csup\u003e\u003cem\u003e++\u003c/em\u003e\u003c/sup\u003e strain. First, we compared infection competition among the 4 strains by assessing nodule occupancy rates based on the differences in the length of restriction enzyme-treated \u003cem\u003enopP\u003c/em\u003e fragments (PCR-RFLP, Supplementary Fig. 1). Also, the nodule occupancy rates of the strains in mature nodules of \u003cem\u003eRj2/GmNNL1\u003c/em\u003e and \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybeans following simultaneous inoculation with strain FY2-m1 and USDA6/USDA110/USDA122 were compared (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean, the FY2-m1 strain occupied 95.3% of the mature nodules, while in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybean, its occupancy was 56.1%. Similarly, the simultaneous inoculation of GMA461-m4 with USDA6/USDA110/USDA122 resulted in a nodule occupancy rate of 92.1% in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e line and 54.7% in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e line (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). For OSA024, the simultaneous inoculation with USDA6/USDA110/USDA122 led to 97.4% nodule occupancy in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e line and 27.9% in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e line (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results indicate that the USDA6/USDA110/USDA122 strains were unable to evade \u003cem\u003eRj2/GmNNL1\u003c/em\u003e incompatibility, resulting in the predominant infection by Nos\u003csup\u003e++\u003c/sup\u003e incompatibility-avoiding strains in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean.\u003c/p\u003e\n\u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO flux generated from nodulated soybean roots was quantitatively analyzed in inoculated plots, where nodule occupancy was assessed. N\u003csub\u003e2\u003c/sub\u003eO flux was measured 1, 2, and 3 weeks after the senescence and decay induction of nodulated roots. There was no significant difference in N\u003csub\u003e2\u003c/sub\u003eO flux between samples inoculated with FY2-m1 and GMA461-m4 at 1 and 2 weeks after decapitation (WAD); however, a considerable decrease in N\u003csub\u003e2\u003c/sub\u003eO flux was detected at 3 WAD. In the case of inoculating with OSA024, there was a substantial decrease in N\u003csub\u003e2\u003c/sub\u003eO flux at 2 and 3 WAD. Across all 3 Nos\u003csup\u003e++\u003c/sup\u003e incompatibility-avoiding strains, a substantial decrease in N\u003csub\u003e2\u003c/sub\u003eO flux was recorded from the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean compared with the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybean (Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed\u0026ndash;f).\u003c/p\u003e\n\u003cp\u003eAdditionally, a linear regression analysis was performed to compare the nodule occupancy rates of the incompatibility-avoiding N\u003csub\u003e2\u003c/sub\u003eO-reducing rhizobacteria in \u003cem\u003erj2/Gmnnl1\u003c/em\u003e and \u003cem\u003eRj2/GmNNL1\u003c/em\u003e lines at both 2 and 3 WAD. The nodule occupancy of the N\u003csub\u003e2\u003c/sub\u003eO-reducing rhizobacteria was negatively correlated with N\u003csub\u003e2\u003c/sub\u003eO flux (Supplementary Fig. 2). These findings indicate that FY2-m1, GMA461-m4, and OSA024, isolated as \u003cem\u003eRj2/GmNNL1\u003c/em\u003e incompatibility-avoiding Nos\u003csup\u003e\u003cem\u003e++\u003c/em\u003e\u003c/sup\u003e rhizobia, demonstrated remarkably high nodule occupancy in \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybeans under competitive inoculation with other rhizobial strains, thereby achieving high N\u003csub\u003e2\u003c/sub\u003eO reduction capacity.\u003c/p\u003e\n\u003cp\u003eWe conducted a comparative analysis of soybean growth and symbiotic phenotypes under \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e expression to address the concern that artificial accumulation of \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e in soybean lines could trigger defense responses against \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA6\u003c/sub\u003e, \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA110\u003c/sub\u003e, and \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e type rhizobia, potentially negatively affecting soybean growth and symbiotic phenotypes. We inoculated soybean lines \u003cem\u003eRj2/GmNNL1\u003c/em\u003e and \u003cem\u003erj2/Gmnnl1\u003c/em\u003e with \u003cem\u003eB. japonicum\u003c/em\u003e USDA6, \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA110 and USDA122, and an incompatibility-avoiding N\u003csub\u003e2\u003c/sub\u003eO-reducing bradyrhizobia.\u003c/p\u003e\n\u003cp\u003eThen, we assessed symbiotic phenotypes and soybean growth 5 weeks after inoculation. There was no significant difference in the number of nodules per plant (Supplementary Fig. 3a). However, nodule dry weight per plant and weight per nodule were substantially greater in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e line than in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e line (Supplementary Fig. 3b, 3c). N\u003csub\u003e2\u003c/sub\u003e-fixing activity was considerably higher in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e and FY2-m1 combination (Supplementary Fig. 3d). Furthermore, shoot dry weight (SDW) and root dry weight (RDW) were substantially greater in \u003cem\u003eRj2/GmNNL1\u003c/em\u003e than in \u003cem\u003erj2/Gmnnl1\u003c/em\u003e (Supplementary Fig. 3e, 3f).\u003c/p\u003e\n\u003ch3\u003eComparative analysis of nodule occupancy and NO flux between incompatibility-avoiding rhizobia and indigenous soil rhizobia\u003c/h3\u003e\n\u003cp\u003eWe tested the competition between incompatibility-avoiding Nos\u003csup\u003e++\u003c/sup\u003e rhizobacteria and indigenous rhizobia in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean, analyzing their effectiveness in nodule occupancy and N\u003csub\u003e2\u003c/sub\u003eO release using pot culture with field soil at National Agriculture and Food Research Organization (NARO). FY2-m1 and GMA461-m4 exhibited nodule occupancy rate of over 80% occupancy, whereas OSA024 showed less than 60% occupancy (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). GMA461-m4 had the lowest level of N\u003csub\u003e2\u003c/sub\u003eO release from the rhizosphere, including nodulated roots, following above-ground excision (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions of FY2-m1, GMA461-m4 and OSA024 demonstrated a substantial reduction in N\u003csub\u003e2\u003c/sub\u003eO release to that of the mock inoculation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eThe ability to reduce N\u003csub\u003e2\u003c/sub\u003eO by the high N\u003csub\u003e2\u003c/sub\u003eO-reducing bradyrhizobia strains, FY2-m1 and GMA461-m4, in soybean lines with and without the incompatibility genes was compared to examine the effects of the incompatibility genes. In addition to \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean, the soybean variety \u0026ldquo;Akuden Shirazu,\u0026rdquo; which lacks incompatibility genes (\u003cem\u003erj2/Gmnnl1\u003c/em\u003e), was included and examined as a negative control. FY2-m1 inoculation resulted in 69.8% and 25.0% nodule occupancy in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e and \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybeans, respectively (Supplementary Fig. 4a). N\u003csub\u003e2\u003c/sub\u003eO flux from the respective rhizosphere soils was considerably lower in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybeans than in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybeans (Supplementary Fig. 4b). GMA461-m4 inoculation yielded 77.6% and 12.9% nodule occupancy in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e rhizosphere soil than in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e rhizosphere soil (Supplementary Figs. 4a and 4b). The effect of soybean lines on N\u003csub\u003e2\u003c/sub\u003eO flux was most pronounced 8 to 18 days after decapitation when N\u003csub\u003e2\u003c/sub\u003eO flux values were highest (Supplementary Table\u0026nbsp;2, Supplementary Fig.\u0026nbsp;4b). The comparative analysis of cumulative N\u003csub\u003e2\u003c/sub\u003eO release over the measurement period showed that N\u003csub\u003e2\u003c/sub\u003eO release in FY2-m1/\u003cem\u003eRj2/GmNNL1\u003c/em\u003e was 40% of that in FY2-m1/\u003cem\u003erj2/Gmnnl1\u003c/em\u003e, while N\u003csub\u003e2\u003c/sub\u003eO release in GMA461-m4/\u003cem\u003eRj2/GmNNL1\u003c/em\u003e was only 16% of that in GMA461-m4/\u003cem\u003erj2/Gmnnl1\u003c/em\u003e (Supplementary Fig. 4c). These results confirm a substantial reduction in N\u003csub\u003e2\u003c/sub\u003eO release associated with increased nodule occupancy in the 2 strains.\u003c/p\u003e\n\u003ch3\u003eNodule occupancy and NO flux in soybean fields\u003c/h3\u003e\n\u003cp\u003eThe competition between Nos\u003csup\u003e++\u003c/sup\u003e inoculants and indigenous rhizobia in the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean and their effectiveness in nodule occupancy and N\u003csub\u003e2\u003c/sub\u003eO reduction in a soybean field were analyzed in the Kashimadai soybean field at Tohoku University. The nodule occupancy rates of the treatments were compared (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). The absence of \u003cem\u003enosZ\u003c/em\u003e of \u003cem\u003eB. ottawaense\u003c/em\u003e in the uninoculated control indicated the absence of indigenous Nos\u003csup\u003e++\u003c/sup\u003e \u003cem\u003eB. ottawaense\u003c/em\u003e in the field (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). GMA461-m4, a \u003cem\u003enopP\u003c/em\u003e mutant of \u003cem\u003eB. ottawaense\u003c/em\u003e, showed a nodule occupancy of over 60%. In contrast, the parent strain GMA461, which carried \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e, could not occupy nodules. Additionally, inoculation with SG09, which carried \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA6\u003c/sub\u003e, showed a lower nodule occupancy of approximately 10%.\u003c/p\u003e\n\u003cp\u003eAt the maturation stage, 15 weeks after seeding, N\u003csub\u003e2\u003c/sub\u003eO flux from the rhizosphere of each treatment was measured immediately following above-ground excision (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). The N\u003csub\u003e2\u003c/sub\u003eO fluxes of the SG09 and GMA461 inoculation were not significantly different from that of the uninoculated control, whereas the GMA461-m4 inoculation substantially reduced N\u003csub\u003e2\u003c/sub\u003eO flux compared to SG09 and GMA461. These results indicate that high nodule occupancy of Nos\u003csup\u003e++\u003c/sup\u003e GMA461-m4 contributes to a reduction in N\u003csub\u003e2\u003c/sub\u003eO flux even under field conditions (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe discovery of rhizobia capable of reducing N\u003csub\u003e2\u003c/sub\u003eO has paved the way for developing technologies aimed at reducing field-derived N\u003csub\u003e2\u003c/sub\u003eO emissions through rhizobial symbiosis (Sameshima-Saito et al., 2006; Itakura et al., 2013; Akiyama et al., 2016). Since soybean is the world's leading legume crop and forms symbiotic associations with bradyrhizobia species (Hartman et al., 2011), soybean symbiotic systems have been the primary focus for developing N\u003csub\u003e2\u003c/sub\u003eO reduction technologies using Nos\u003csup\u003e+\u003c/sup\u003e bradyrhizobia. Field inoculation with USDA110, a Nos\u003csup\u003e+\u003c/sup\u003e rhizobia, and its Nos\u003csup\u003e++\u003c/sup\u003e mutant has successfully reduced N\u003csub\u003e2\u003c/sub\u003eO emissions from soybean fields (Itakura et al., 2013; Akiyama et al., 2016). However, the nodule occupancy of these inoculants was unstable, remaining at 20\u0026ndash;60% in farm-scale experiments (Itakura et al., 2013). The instability in nodule occupancy likely limits the inoculants' ability to maximize their N\u003csub\u003e2\u003c/sub\u003eO-reducing potential.\u003c/p\u003e \u003cp\u003eIn many cases, the introduction of beneficial rhizobium strains into the field is limited by the infection competition from indigenous rhizobia, a phenomenon known as the \u0026ldquo;rhizobial competition problem\u0026rdquo; (Triplett \u0026amp; Sadowsky 1992; Mendiza-Suarez et al., 2020; Mendiza-Suarez et al., 2021). The nodule occupancy of inoculated rhizobia is generally approximately 5\u0026ndash;40% (McDermott et al., 1989; Hungria et al., 1998), highlighting the need to improve the nodule occupancy rates of inoculated rhizobia. Competition with indigenous rhizobia is influenced by the soil's physical and chemical properties and by biotic factors, such as rhizobial chemotactic response to rhizospheric substances secreted by host plants and rhizobial adhesion to plant roots (Mendiza-Suarez et al., 2021). In addition, the competition between rhizobial strains for carbon resources provided by the host plant plays an essential role in infection competition (Rahman et al., 2023). Establishing a rhizobial symbiotic system that allows beneficial rhizobia to dominate infection is crucial for maximizing the impact of promising rhizobia on host plants (Goyal et al., 2021; Mendiza-Suarez et al., 2021). However, the factors determining rhizobial competitive ability remain largely unknown. Although Cunningham et al. (1991) explored the possibility of chemical control to selectively express nodulation (\u003cem\u003enod\u003c/em\u003e) genes in distinct soybean bradyrhizobia lineages, this approach was unlikely to be adopted due to the cost of chemicals. Consequently, there are no reports on artificially controlling the competitive ability of rhizobial inoculants in practice.\u003c/p\u003e \u003cp\u003eThe soybean symbiotic incompatibility induced by rhizobial NopP, as used in this study, represents a rare interaction mechanism because it involves well-characterized genes on both the rhizobial and host plant sides (Yang et al., 2010; Sugawara et al., 2018; Sugawara et al., 2019; Zhang et al., 2021). This unique mechanism suggests a potential for the functional use of this to control rhizobial infection competition (Shiro et al., 2022). Since the NopP function in rhizobia can be disrupted using \u003cem\u003eRj2\u003c/em\u003e incompatibility as a selection pressure (Sugawara et al., 2018), we developed 2 rhizobial strains, FY2-m1 and GMA461-m4, that avoided inhibition of infection by incompatibility genes, \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e. This study represents a novel approach to artificially enhance the host selectivity of promising rhizobia by modifying the host plant and the rhizobia.\u003c/p\u003e \u003cp\u003eThe inhibition of infection by specific effector-harboring rhizobia by host incompatibility genes is thought to have evolved as a strategy to eliminate rhizobia that are not beneficial to the host plant (Jimenez-Guerrero et al., 2022). However, it remains unclear why most nodulating \u003cem\u003eBradyrhizobium\u003c/em\u003e spp. retain NopPs that induce such incompatibility (Teulet et al., 2020). Since \u003cem\u003eBradyrhizobium\u003c/em\u003e spp. can infect multiple host plants, a NopP protein that induces incompatibility in 1 host may not trigger incompatibility in another (Lopez-Baena et al., 2009; Grundy et al., 2023). Additionally, NopP promotes rhizobial infection in certain host plants (Skorpil et al., 2005), indicating that retaining NopP may enhance the probability of rhizobial survival by enabling movement across symbioses with multiple host plants.\u003c/p\u003e \u003cp\u003eHost incompatibility genes generally share homology with R genes, which control infection suppression in response to pathogen attack (Yang et al., 2010; Zhang et al., 2021; Gourion et al., 2015; Grundy et al., 2023). For example, incompatibility induced by \u003cem\u003eB. diazoefficiens\u003c/em\u003e USDA122 via \u003cem\u003eRj2\u003c/em\u003e functions as an effector-triggered immunity with expression of downstream defense response genes (Shine et al., 2019). In addition, \u003cem\u003eRj2\u003c/em\u003e incompatibility is systemically expressed and triggers foliar resistance to plant pathogens (Shine et al., 2019). Defense response-related genes are also expressed following rhizobial inoculation in response to incompatibility induced by \u003cem\u003eGmNNL1\u003c/em\u003e (Zhang et al., 2021). These findings indicate that incompatibility can be interpreted as a defense response against rhizobia harboring specific effectors. Plants carrying R genes often experience a growth penalty associated with defense responses (Karasov et al., 2017; Ning et al., 2017; He et al., 2022; Gao et al., 2023). Trade-offs between plant growth and immunity have been widely reported across model plants and crop species, underscoring the need to balance productivity with resistance in crop breeding (Ning et al., 2017; Gao et al., 2023). In our study, 2 incompatibility genes were accumulated; thus, it was necessary to verify whether the trade-offs between defense response and plant growth from R genes were evident. We evaluated the effect of incompatibility gene accumulation by comparing the \u003cem\u003eRj2/GmNNL\u003c/em\u003e1 and \u003cem\u003erj2/Gmnnl1\u003c/em\u003e lines from the same breeding backgrounds. The \u003cem\u003eRj2/GmNNL1\u003c/em\u003e lines, where incompatibility genes are functionally expressed through co-inoculation with USDA6, USDA110, and USDA122, and incompatibility genes were not expressed in the \u003cem\u003erj2/Gmnnl1\u003c/em\u003e lines. The similarity in nodule numbers indicates that the accumulation of incompatibility genes does not affect nodule number control mechanisms. However, nodule dry weight per plant, nodule dry weight per nodule, SDW, and RDW in \u003cem\u003eRj2/GmNNL1\u003c/em\u003e were substantially higher than in \u003cem\u003erj2/Gmnnl1\u003c/em\u003e. We observed no negative effects on symbiotic phenotypes or plant growth due to incompatibility gene accumulation within our system. Therefore, these phenotypes appear more influenced by the combination of host soybean and rhizobial strains than by the expression of incompatibility genes (Yuan et al., 2020).\u003c/p\u003e \u003cp\u003eAccording to the geographical distribution of rhizobia in Japanese soil, the major rhizobial flora in the Honshu-Kyushu area can be classified into Nos\u003csup\u003e\u0026minus;\u003c/sup\u003e USDA6 (\u003cem\u003eB. japonicum\u003c/em\u003e) and Nos\u003csup\u003e+\u003c/sup\u003e USDA110 types (\u003cem\u003eB. diazoefficiens\u003c/em\u003e) (Shiina et al., 2014; Saeki et al., 2013). Although bacterial classification based on ITS and NopP sequences does not always align, NopP sequence analysis using the same bacterial library (Shiina et al., 2014) indicates the widespread presence of NopP\u003csub\u003eUSDA110\u003c/sub\u003e, NopP\u003csub\u003eUSDA122\u003c/sub\u003e, and NopP\u003csub\u003eUSDA6\u003c/sub\u003e types of bradyrhizobia in Japan (Sugawara et al., 2018). This finding suggests that \u003cem\u003eGmNNL1\u003c/em\u003e and \u003cem\u003eRj2\u003c/em\u003e can effectively inhibit infection competition by indigenous rhizobia. Here, this hypothesis was tested through competitive inoculation experiments with strains USDA6, USDA110, USDA122, and the incompatibility-avoiding N\u003csub\u003e2\u003c/sub\u003eO-reducing rhizobacteria FY2-m1, GMA461-m4, and OSA024. Each incompatibility-avoiding strain had a significantly higher nodule occupancy than USDA6, USDA110, and USDA122. However, in experiments using field soil, the occupancy rate of OSA024 was lower than that of FY2-m1 and GMA461-m4, and the N\u003csub\u003e2\u003c/sub\u003eO release from the OSA024-inoculated soybean rhizospheres trended higher. These data indicate that OSA024 was less effective than the other strains in the field soil used.\u003c/p\u003e \u003cp\u003eIn our field experiment, the \u003cem\u003enopP\u003c/em\u003e-deficient GMA461-m4 showed higher nodule occupancy than GMA461 with \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e and SG09 with \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA6\u003c/sub\u003e. The lack of nodulation with GMA461 may be due to the strong incompatibility of \u003cem\u003eRj2\u003c/em\u003e against the effector NopP\u003csub\u003eUSDA122\u003c/sub\u003e (Sugawara et al., 2018). In contrast, \u003cem\u003eGmNNL1\u003c/em\u003e incompatibility against rhizobia with \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA6\u003c/sub\u003e or \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA110\u003c/sub\u003e blocks infection via infection threads but still allows infection through crack entry (Zhang et al., 2021). Thus, the nodule occupancy of SG09 in \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybeans is likely higher than that of GMA461. Nodule occupancy of GMA461-m4, at over 60%, may substantially mitigate N\u003csub\u003e2\u003c/sub\u003eO emissions from the rhizosphere of \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybeans. Therefore, this strain is thought to both outcompete indigenous rhizobia in \u003cem\u003eRj2/GmNNL1\u003c/em\u003e symbiosis and exhibit N\u003csub\u003e2\u003c/sub\u003eO reduction potential under field soil conditions. Collectively, our findings demonstrate that optimizing the combination of incompatibility genes and effectors-deficient N\u003csub\u003e2\u003c/sub\u003eO-reducing rhizobia can lead to soybean cultivation systems that effectively reduce N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e \u003cp\u003eGlobally, soil types vary substantially. In this study, based on the characteristics of Japanese soils, \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e were used as symbiotic incompatibility genes to prevent infection by the predominant indigenous rhizobia. Since the dominant indigenous rhizobia may differ depending on the soils\u0026rsquo; physical, chemical, and biological properties, applying this method broadly will require selecting incompatibility genes specific to the dominant rhizobia in a soil type (Li et al., 2023; Mendiza-Suarez et al., 2021).\u003c/p\u003e \u003cp\u003eAgricultural land is a major anthropogenic source of N\u003csub\u003e2\u003c/sub\u003eO emissions (Tian et al., 2020). Under current global warming conditions, biological N\u003csub\u003e2\u003c/sub\u003e fixation through rhizobial symbiosis holds substantial promise as an alternative for agricultural production systems reliant on synthetic nitrogen fertilizers (Bourion et al., 2018; Guilpart et al., 2022; Grundy et al., 2023; Rotundo et al., 2024). In addition to nitrogen fertilizer applied to fields, nitrogen sources released from soybean plant residues contribute to N\u003csub\u003e2\u003c/sub\u003eO emissions (Inaba et al., 2009; Inaba et al., 2012; Toyoda et al., 2024). A rhizobial symbiotic system with N\u003csub\u003e2\u003c/sub\u003eO-reducing rhizobia can help reduce N\u003csub\u003e2\u003c/sub\u003eO emissions from soybean fields by lowering the need for artificial nitrogen fertilizer, a primary source of N\u003csub\u003e2\u003c/sub\u003eO emission, and by reducing N\u003csub\u003e2\u003c/sub\u003eO derived from soybean residues through the rhizobia\u0026rsquo;s N\u003csub\u003e2\u003c/sub\u003eO-reducing capability.\u003c/p\u003e "},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eIsolation of Nos\u003c/b\u003e \u003csup\u003e \u003cb\u003e++\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eB. ottawaense\u003c/b\u003e \u003cb\u003estrains and incompatibility-avoiding strains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMature nodules were collected from soybean roots grown in (1) fields in Gunma Prefecture in 2021 and 2022; and (2) soils collected from a soybean field in Osaka Prefecture in 2022. Collected nodules were surface-sterilized with 0.5% NaOCl, placed in a 96-well microplate, and crushed with a sterile toothpick in 150 \u0026micro;L of sterile water. Then, 75 \u0026micro;L of cell lysate was prepared from the nodule crushing solution (Shiina Y et al., 2014). Next, 25 \u0026micro;L of 50% (v/v) glycerol solution was added to the remaining 75 \u0026micro;L of crushed nodule solution, and the samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until bradyrhizobia isolation. PCR was performed with \u003cem\u003eB. ottawaense nosZ\u003c/em\u003e-specific primers using cell lysates as templates to select bradyrhizobia strains possessing \u003cem\u003eB. ottawaense nosZ\u003c/em\u003e (Hara et al., 2024). The cell lysates were also used as templates for PCR amplification of the 16S-23S ITS region and \u003cem\u003enopP\u003c/em\u003e (Shiina Y et al., 2014; Sugawara M et al., 2018). DNA sequences of the 16S-23S ITS region and \u003cem\u003enopP\u003c/em\u003e were determined by Sanger sequencing at GENEWIZ (Azenta Life Sciences). Based on ITS and \u003cem\u003enopP\u003c/em\u003e sequencing results, strains with over 99% ITS homology to \u003cem\u003eB. ottawaense\u003c/em\u003e SG09 and \u003cem\u003enopP\u003c/em\u003e\u003csub\u003eUSDA122\u003c/sub\u003e or IS-inserted \u003cem\u003enopP\u003c/em\u003e were selected. Strains FY2, GMA461, and OSA024 were isolated from the nodule crushing solution stored at \u0026minus;\u0026thinsp;80\u0026deg;C using HM agar medium (Cole et al., 1973).\u003c/p\u003e\n\u003ch3\u003eMeasurement of NO-reducing activity in cultured rhizobia\u003c/h3\u003e\n\u003cp\u003eThe N\u003csub\u003e2\u003c/sub\u003eO-reducing activity of \u003cem\u003eB. ottawaense\u003c/em\u003e strains was measured (Wasai-Hara et al., 2023). N\u003csub\u003e2\u003c/sub\u003eO-reducing activity was determined by culturing the bacteria under anaerobic conditions with 1% N\u003csub\u003e2\u003c/sub\u003eO supplied as the sole electron acceptor. N\u003csub\u003e2\u003c/sub\u003eO concentrations were measured using a gas chromatograph (GC2014; Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector and a Porapak Q column (GL Sciences, Tokyo, Japan). Bacterial strains were first aerobically cultured for over 6 hours in a 75-mL test tube with an air-permeable plug containing 10 mL of HM liquid medium (Cole et al., 1973) supplemented with 0.1% (w/v) arabinose and 0.025% (w/v) yeast extract, at 28\u0026deg;C with shaking at 200 rpm. Then, an appropriate volume of bacterial culture was transferred to new tubes containing 10 mL of HM medium to reach an optical density (OD) at 660 nm (OD\u003csub\u003e660\u003c/sub\u003e) of 0.05, measured in a 25-mm diameter test tube (TEST25NP; AGC Techno Glass Co., Ltd., Shizuoka, Japan). After the initial culture, the test tube was sealed with a butyl rubber cap, and the gas phase was replaced with a mixture of 4.98% N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;95.02% N\u003csub\u003e2\u003c/sub\u003e gas for 12 to 14 hours to induce N\u003csub\u003e2\u003c/sub\u003eO reduction metabolism. Afterward, the gas phase was replaced with 100% N\u003csub\u003e2\u003c/sub\u003e gas, and 100% N\u003csub\u003e2\u003c/sub\u003eO was added to adjust to a final concentration of 1%. The test tube was incubated at 28\u0026ordm;C with shaking at 200 rpm, and 100 \u0026micro;L samples of the gas phase were withdrawn every 1 to 3 hours for analysis by gas chromatography.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelection of incompatibility-avoiding Nos\u003c/b\u003e \u003csup\u003e \u003cb\u003e++\u003c/b\u003e \u003c/sup\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eStrains FY2 and GMA461 harboring \u003cem\u003enopP\u003c/em\u003e\u003csub\u003e122\u003c/sub\u003e were inoculated into soybean (\u003cem\u003eGlycine max\u003c/em\u003e (L.) Merr. cv. Hardee) carrying the \u003cem\u003eRj2\u003c/em\u003e incompatibility gene. Soybean seeds were sterilized using 0.5% sodium hypochlorite, sown in Leonardo Jar pots (five seeds per pot) containing sterilized vermiculite, and inoculated with FY2 and GMA461 at 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e cells per seed (Sugawara et al., 2018). Six pots were prepared for each inoculated strain. Soybeans were grown in a growth chamber at 25\u0026deg;C with a 16-hour light and 8-hour dark cycle for 3 weeks. Nodules were collected and surface-sterilized with 0.5% NaOCl and sliced with a sterile razor blade, and the internal bacteroids were spread on HM agar medium to isolate the bradyrhizobia strains. Single-colony isolates were subsequently inoculated onto Hardee soybeans. After 3 weeks of cultivation, the bradyrhizobia strains were re-isolated from nodules on soybean roots with green leaves. Lastly, \u003cem\u003enopP\u003c/em\u003e PCR was performed on the bradyrhizobial isolates to determine the presence of ISs in \u003cem\u003enopP\u003c/em\u003e (Sugawara et al., 2018).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenotypic analysis of\u003c/b\u003e \u003cb\u003eRj2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eGmNNL1\u003c/b\u003e \u003cb\u003egenes in soybean germplasm\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor whole-genome resequencing of the soybean variety \"Bonminori,\" total DNA was extracted from leaves using the DNeasy Plant Mini Kit (Qiagen). The DNA library was subjected to 150-bp paired-end sequencing on an Illumina NovaSeq instrument (Illumina Co., Ltd.) to achieve 20 \u0026times; genome coverage.\u003c/p\u003e \u003cp\u003eThe reads from \"Bonminori\" and 192 accessions of the mini-core collection were mapped to the \u003cem\u003eG. max\u003c/em\u003e Williams 82 genome assembly (v4.0) using BWA-MEM (Li \u0026amp; Durbin 2009), and the duplicates were removed using Picard MarkDuplicates (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://broadinstitute.github.io/picard/\u003c/span\u003e\u003cspan address=\"http://broadinstitute.github.io/picard/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Variants calling followed GATK best practices for germline SNP/Indel discovery (Auwera et al., 2013), using GATK version 4.0.11.0. Variants were initially called individually for each sample with GATK HaplotypeCaller, followed by joint genotyping with GenotypeGVCFs to consolidate variants (Poplin et al., 2017). Variants were first filtered using GATK with the parameters: \"QD\u0026thinsp;\u0026lt;\u0026thinsp;5.0 || FS\u0026thinsp;\u0026gt;\u0026thinsp;50.0 || SOR\u0026thinsp;\u0026gt;\u0026thinsp;3.0 || MQ\u0026thinsp;\u0026lt;\u0026thinsp;50.0 || MQRankSum \u0026lt; -2.5 || ReadPosRankSum \u0026lt; -1.0 || ReadPosRankSum\u0026thinsp;\u0026gt;\u0026thinsp;3.5\" Further filtering was conducted using the bcftools view (Li, 2011) with parameters: -m2 -M2 -g hom --output-type z --exclude-uncalled -e \"MAF\u0026thinsp;\u0026lt;\u0026thinsp;0.05 || F_MISSING\u0026thinsp;\u0026gt;\u0026thinsp;0.25.\" All variants were annotated for potential impact using SnpEff version 4.3 (Cingolani et al., 2012). The genotype of \u003cem\u003eRj2\u003c/em\u003e was determined based on an SNP (C/T) at Gm16: 37281186 bp, which causes a single amino acid substitute (R490 to I490) (Sugawara et al., 2019). Since the genotype of \u003cem\u003eGmNNL1\u003c/em\u003e is defined by an SV, SV analysis was performed using Manta (Chen et al., 2016), with SV files subsequently merged using SURVIVOR (Jeffares et al., 2017).\u003c/p\u003e\n\u003ch3\u003eBreeding soybean with accumulated incompatibility genes\u003c/h3\u003e\n\u003cp\u003eThe soybean varieties \u0026ldquo;Bonminori\u0026rdquo;, harboring \u003cem\u003eRj2\u003c/em\u003e, and GmWMC108 Karasu-mame, harboring \u003cem\u003eGmNNL1\u003c/em\u003e, were grown simultaneously during flowering and crossbred. To facilitate DNA marker analysis of the \u003cem\u003eRj2\u003c/em\u003e genotypes, an indel marker was developed based on an SV near \u003cem\u003eRj2\u003c/em\u003e (see Supplementary Table\u0026nbsp;3). An indel marker for \u003cem\u003eGmNNL1\u003c/em\u003e genotype analysis was developed using genomic sequences around the position of the SINE-like transposon. For genotyping, genomic DNA was extracted from thin slices of the resulting seed cotyledon (Kamiya and Kiguchi 2003). The \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e markers were amplified using PCR to select the F\u003csub\u003e1\u003c/sub\u003e seeds heterozygous for \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e based on migration patterns in agarose gel electrophoresis. Then, the F\u003csub\u003e1\u003c/sub\u003e seeds were planted and grown. After harvesting the F\u003csub\u003e2\u003c/sub\u003e seeds, the lines homozygous for \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e were selected. Then, F\u003csub\u003e3\u003c/sub\u003e seeds were harvested from the resulting F\u003csub\u003e2\u003c/sub\u003e plants to establish \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybean lines.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMethod of rhizobial inoculation for soybean\u003c/h2\u003e \u003cp\u003eA rhizobial inoculum was prepared by suspending rhizobial strains in Broughton and Dilworth (B\u0026amp;D) solution (Broughton and Dilworth 1971) at 6.7 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e cells/mL. Leonard jar pots filled with sterile vermiculite were pre-inoculated with 150 mL of the rhizobial inoculum, and 2 chlorine-gas-treated soybean seeds were sown per pot. Cultivation was conducted in an artificial climatic chamber set to 25\u0026deg;C with a 16-hour light and 8-hour dark photoperiod. On the fourth day after sowing, seedlings were thinned to leave 1 well-germinated plant per pot and then cultivated for 3 to 5 weeks. Pots were periodically supplied with a nitrogen-free B\u0026amp;D solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of root nodule occupancy using competitive inoculation tests\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. diazoefficiens\u003c/em\u003e strains USDA110 and USDA122 and \u003cem\u003eB. japonicum\u003c/em\u003e strain USDA6 were selected as competitor strains. Incompatibility-avoiding strains and competitor strains were each suspended in B༆D solution. One of the incompatibility-avoiding strains was mixed with all 3 competitor strains at equal ratios, and a rhizobial inoculum mixture with a total bacterial concentration of 6.7 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e cells/mL was prepared. In Leonard jar pots filled with sterile vermiculite, 150 mL of the rhizobial inoculum mixture was pre-inoculated, and 2 chlorine-gas-treated \u003cem\u003eRj2/GmNNL1\u003c/em\u003e or \u003cem\u003erj2/Gmnnl1\u003c/em\u003e soybeans were sown per pot following the standard soybean inoculation method. At five weeks after inoculation, the number of mature nodules on the roots was counted. Then, mature nodules were collected and surface-sterilized by immersion in 10% NaOCl for 3 min. The \u003cem\u003enopP\u003c/em\u003e region was amplified using PCR with \u003cem\u003enopP\u003c/em\u003e-specific primers. Next, PCR-amplified fragments were digested with the restriction enzymes \u003cem\u003eAlu\u003c/em\u003eI and \u003cem\u003ePst\u003c/em\u003eI and analyzed by agarose gel electrophoresis. The strains occupying mature nodules were identified based on the patterns of the restriction enzyme-digested PCR-amplified fragments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eN\u003csub\u003e2\u003c/sub\u003eO flux from nodulated soybean roots in Leonard jar pot experiments\u003c/h2\u003e \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO flux was measured (Wasai-Hara et al., 2023). After competitive inoculation, the soybean root system was gently immersed in water to remove excess vermiculite. Then, the roots were transferred to a 100-mL glass vial containing 30 mL of soil obtained from the Kashimadai experimental field (38\u0026deg;27\u0026prime;36.0\u0026Prime;N, 141\u0026deg;05\u0026prime;24.0\u0026Prime;E) with permission from Tohoku University, Japan. Kashimadai soils had been sieved through a 2-mm mesh to remove large aggregates and stones. Additionally, 5 ml of sterile distilled water was added to each vial. The vials containing roots, soil, and water were incubated aerobically at 25\u0026deg;C for 20 days to induce nodule degradation. The vials were covered with a soft cloth to maintain aeration during incubation. Each week during the incubation, vials were sealed with butyl-rubber caps and kept under atmospheric conditions for 240 to 360 minutes to determine N\u003csub\u003e2\u003c/sub\u003eO flux. N\u003csub\u003e2\u003c/sub\u003eO concentrations in the vial gas phase were measured using a gas chromatograph (GC2014; Shimadzu) equipped with a \u003csup\u003e63\u003c/sup\u003eNi electron capture detector and tandem Porapak Q columns (GL Sciences; 80/100 mesh; 3.0 mm \u0026times; 1.0 m and 3.0 mm \u0026times; 2.0 m).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of root nodule occupancy and N\u003csub\u003e2\u003c/sub\u003eO release in field simulation cultivation with field soil\u003c/h2\u003e \u003cp\u003eFour seeds were sown in a Wagener pot filled with 3 L of NARO field soil (Andosol) and inoculated with 1 ml of incompatibility-avoiding rhizobial solution at 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e cells/ml per seed. Eight pots were prepared for each test plot. After germination, 2 seedlings were retained per pot. After 42 days of cultivation, soybeans were harvested from 3 pots, and the number of nodules formed on the roots was recorded. Nodule occupancy was determined using PCR to detect the \u003cem\u003eB. ottawaense\u003c/em\u003e-type \u003cem\u003enosZ\u003c/em\u003e gene (Hara et al., 2024).\u003c/p\u003e \u003cp\u003eFor the remaining 5 pots, the above-ground portion of the soybeans was excised, and 30-ml gas samples were collected at 0, 20, and 40 minutes after covering the pots with acrylic plates. Sampling was conducted every 2 to 3 days, and N\u003csub\u003e2\u003c/sub\u003eO concentrations in the gas samples were quantitatively analyzed using gas chromatography (GC-2014, Shimazu, Kyoto, Japan; Sudo, 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of nodule occupancy and N\u003csub\u003e2\u003c/sub\u003eO flux through field cultivation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eRj2/GmNNL1\u003c/em\u003e soybeans were inoculated with the Kashimadai field at Tohoku University with peat moss materials containing SG09, GMA461, and GMA461-m4 strains at 1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e CFU per seed. Approximately 1 month later, the nodule occupancy of the inoculum in mature nodules was quantified by PCR to detect the \u003cem\u003eB. ottawaense\u003c/em\u003e-type \u003cem\u003enosZ\u003c/em\u003e (Hara et al., 2024). About 15 weeks after inoculation and during soybean ripening, N\u003csub\u003e2\u003c/sub\u003eO concentrations over time were monitored with a mobile mid-infrared N\u003csub\u003e2\u003c/sub\u003eO sensor (MIRA Ultra; Aeris Technologies, Heyward, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using JMP16.2.0. The statistical analysis methods applied are as follows. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Tukey\u0026rsquo;s HSD test. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Wilcoxon rank-sum test. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Student\u0026rsquo;s t-test. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Dunnett test using mock inoculation as control. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; Steel test using mock inoculation as control, 6B; Dunnett test using mock inoculation as control. SplFig3; Tukey\u0026rsquo;s HSD test. SplFig4C; Student\u0026rsquo;s t-test.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eUpon this paper's acceptance, the NGS data for \u0026ldquo;Bonminori\u0026rdquo; will be available in the DDBJ Sequence Read Archive under accession number DRR610375.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eAuthors and Affiliations\u003c/h2\u003e\n\u003cp\u003eHanna Nishida, Khin Thuzar Win, Yukiko Fujisawa, Yoshikazu Shimoda, Haruko Imaizumi-Anraku\u003c/p\u003e\n\u003cp\u003eInstitute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan\u003c/p\u003e\n\u003cp\u003eManabu Itakura, Kaori Kakizaki, Atsuo Suzuki, Satoshi Ohkubo, Kiwamu Minamisawa\u003c/p\u003e\n\u003cp\u003eGraduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan\u003c/p\u003e\n\u003cp\u003eFeng Li, Koji Takahashi\u003c/p\u003e\n\u003cp\u003eInstitute of Crop Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan\u003c/p\u003e\n\u003cp\u003eMasayuki Sugawara\u003c/p\u003e\n\u003cp\u003eDepartment of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan\u003c/p\u003e\n\u003cp\u003eSachiko Masuda, Arisa Shibata, Ken Shirasu\u003c/p\u003e\n\u003cp\u003ePlant Immunity Research Group, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan\u003c/p\u003e\n\u003cp\u003eMisa Tsubokura, Hiroko Akiyama\u003c/p\u003e\n\u003cp\u003eInstitute for Agro-Environmental Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare having no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eCorresponding authors\u003c/h2\u003e\n\u003cp\u003eHaruko IMAIZUMI-ANRAKU\u003c/p\u003e\n\u003cp\u003eKiwamu MINAMISAWA\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eK.M. and H.I-A oversaw the project and designed the experiments. H.N., M.I., K.T.W., F. L., K.K., A.S., S.O., M.S., K.T., S.M., A.S., K.S., Y.F., M.T., H.A., Y.S., K.M. and H.I-A performed experiments. M.I., K.K., A.S., S.O. and K.M. isolated Nos\u003csup\u003e++\u003c/sup\u003e \u003cem\u003eB. ottawaense\u003c/em\u003e. H.N., M.I., K.K., M.S., Y.F., Y.S., and H.I-A. analyzed nodule occupancy of Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia. M.I., K.T.W., S.O., M.T., and H.A. analyzed N\u003csub\u003e2\u003c/sub\u003eO flux from the rhizosphere. F.L. analyzed the genotypes of incompatibility genes in the NARO soybean core collection and selected the \u003cem\u003eRj2/GmNNL1\u003c/em\u003e-accumulating soybean lines. K.T. crossbred soybean cultivars possessing incompatibility genes. S.M., A. S., and K.S. sequenced the whole genome of Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia. H.I-A., K.M., H.N., M.I., and S.O. analyzed the data and wrote the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank Shusei Sato (Tohoku University) for this manuscript\u0026apos;s critical reading and useful comments. We also acknowledge Dr. Matthew Shenton (NARO) for supplying a pipeline for whole genome resequencing analysis of soybeans. This research was supported by a JPNP18016 project commissioned by the New Energy and Industrial Technology Development Organization (NEDO).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTian H. et al. A comprehensive quantification of global nitrous oxide sources and sinks Nature 586, 248\u0026ndash;256 (2020).\u003c/li\u003e\n\u003cli\u003eUchida Y. \u0026amp; Akiyama H. Mitigation of postharvest nitrous oxide emissions from soybean ecosystems: a review. Soil Science and Plant Nutrition 59, 477-487 (2013)\u003c/li\u003e\n\u003cli\u003eGoyal R. K., Mattoo A. 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(2021) Patent JP 6843395\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Competition, GHG, Incompatibility, N2O, NopP effector, Predominant infection, Rhizobium, Soybean, Symbiosis, Nodule occupancy","lastPublishedDoi":"10.21203/rs.3.rs-5679948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5679948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoybeans fix atmospheric N\u003csub\u003e2\u003c/sub\u003e through symbiosis with rhizobia, N\u003csub\u003e2\u003c/sub\u003e-fixing bacteria. The relationship between rhizobia and soybeans, particularly those with high nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO)-reducing activities (Nos\u003csup\u003e++\u003c/sup\u003e), can be used for reducing N\u003csub\u003e2\u003c/sub\u003eO emissions from agricultural soils. However, inoculating soybeans with Nos\u003csup\u003e++\u003c/sup\u003e rhizobia under field conditions often fails because of the competition from indigenous Nos\u003csup\u003e−\u003c/sup\u003e (no N\u003csub\u003e2\u003c/sub\u003eO-reducing activities) and Nos\u003csup\u003e+\u003c/sup\u003e (normal N\u003csub\u003e2\u003c/sub\u003eO-reducing activities) rhizobia. Here, we utilized natural incompatibility systems between soybean and rhizobia to address this challenge. Specifically, \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e inhibit certain rhizobial infections in response to NopP, an effector protein. By combining a soybean line with a hybrid accumulation of the \u003cem\u003eRj2\u003c/em\u003e and \u003cem\u003eGmNNL1\u003c/em\u003e genes and Nos\u003csup\u003e++\u003c/sup\u003e bradyrhizobia lacking the \u003cem\u003enopP\u003c/em\u003e effector gene, we developed a soybean-bradyrhizobial symbiosis system that Nos\u003csup\u003e++\u003c/sup\u003e rhizobial inoculants predominantly infect. This optimized symbiotic system substantially reduced N\u003csub\u003e2\u003c/sub\u003eO emissions in field and laboratory tests, presenting a promising approach for sustainable agricultural practices.\u003c/p\u003e","manuscriptTitle":"Genetic design of soybean hosts and bradyrhizobial endosymbionts reduces N2O emissions from soybean farming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-16 04:58:35","doi":"10.21203/rs.3.rs-5679948/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8a4a86fe-b173-4ad3-b427-7f3046924b8b","owner":[],"postedDate":"January 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42371475,"name":"Biological sciences/Plant sciences/Plant symbiosis/Rhizobial symbiosis"},{"id":42371476,"name":"Biological sciences/Microbiology/Environmental microbiology/Soil microbiology"},{"id":42371477,"name":"Earth and environmental sciences/Climate sciences/Climate change/Climate-change mitigation"}],"tags":[],"updatedAt":"2025-09-05T07:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-5679948","link":"https://doi.org/10.1038/s41467-025-63223-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-09-04 04:00:00","publishedOnDateReadable":"September 4th, 2025"},"versionCreatedAt":"2025-01-16 04:58:35","video":"","vorDoi":"10.1038/s41467-025-63223-6","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63223-6","workflowStages":[]},"version":"v1","identity":"rs-5679948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5679948","identity":"rs-5679948","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}