GmERF13 mediates salt inhibition of nodulation through interacting with GmLBD16a in soybean

Zhu, Xinfang; Yan, Xifeng; Li, Weijun; Zhang, Mengyue; Leng, Junchen; Yu, Qianqian; Liu, Like; Xue, Dawei; Zhang, Dajian; Ding, Zhaojun

doi:10.1038/s41467-024-55495-1

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Published: 06 January 2025

GmERF13 mediates salt inhibition of nodulation through interacting with GmLBD16a in soybean

Nature Communications volume 16, Article number: 435 (2025) Cite this article

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Abstract

While the genetic regulation of nodule formation has been well explored, the molecular mechanisms by which abiotic stresses, such as salt stress, impede nodule formation remain largely elusive. Here, we identify four APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors, GmERF13s, that are induced by salt stress and play key roles in salt-repressed nodulation. Loss of GmERF13 function increases nodule density, while its overexpression suppresses nodulation. Moreover, salt stress-inhibited nodule formation is greatly attenuated in GmERF13 loss-of-function mutants, whereas it becomes more pronounced when GmERF13 is overexpressed. Furthermore, GmERF13s can interact with Lateral Organ Boundaries Domain 16 (GmLBD16a), which attenuates GmLBD16a’s binding capacity on Expansin17c (GmEXP17c) promoter. Additionally, salt-induced GmERF13s expression relies on abscisic acid signaling, with direct promotion facilitated by GmABI5, illustrating their direct involvement in enhancing GmERF13s expression. Collectively, our study reveals a molecular mechanism by which salt stress impedes nodulation through the GmERF13-GmLBD16a-GmEXP17 module in soybean.

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Introduction

Soybean is one of the most important leguminous plants, providing protein-rich seeds for humans and animals, while also enriching soil nitrogen through biological nitrogen fixation¹. The nodule, resulting from a symbiotic interaction between leguminous plants and soil bacteria like Rhizobium spp. has been finely tuned to provide high efficiency and benefit to agricultural production, especially in nitrogen-deficient environments^2,3. In Lotus japonicus and Medicago truncatula, the symbiosis interaction has been well established. Flavonoids released by roots recruit rhizobia in soil and induce the rhizobia to secret Nod Factors (NFs). The perception of NFs involves a group of receptor-like kinases (RLKs) located on the membrane-like Nod Factor Receptor 1/LysM Domain-containing RLK3 (NFR1 in Lotus japonicus/LYK3 in Medicago truncatula) and Nod Factor Receptor 5/Nod Factor Perception (NFR5 in Lotus japonicus/NFP in Medicago truncatula)⁴. Symbiotic signal transduction also involves the Symbiosis RLK/Does Not Make Infections 2 (SYMRK in Lotus japonicus/DMI2 in Medicago truncatula), which likely forms part of a receptor complex associated with the LysM perception system⁵. This complex activates intracellular signaling pathways by interacting with 3-Hydroxy-3-Methylglutaryl-CoA-Reductase (HMGR), facilitating the production of mevalonate that transmits the NF signal from the plasma membrane to the cell nucleus to activate Ca²⁺ signals in nucleus^6,7. In nucleus, chimeric Ca²⁺/CaM‐dependent protein kinase/Does Not Make Infections 3 (CCaMK in Lotus japonicus/DMI3 in Medicago truncatula) decodes calcium oscillations, and phosphorylates the transcriptional activator CYCLOPS/Interacting Protein of DMI3 (CYCLOPS in Lotus japonicus/IPD3 in Medicago truncatula)^8,9. The activated CYCLOPS/IPD3 interacts with DELLA and Nodulation Signal Pathway 1/2 (NSP1/2) to induce the expression of the core transcription factor encoding genes such as Nodule Inception (NIN), Nuclear Factor-Y (NF-Y), and Ethylene Response Factor Required For Nodulation 1/2 (ERN1/2), leading to rhizobia infection and formation of nodules^{10,11,12,13,14,15}.

Soybean breeding needs to be expedited to fulfil the requirements of a burgeoning global population and to cope with future environmental alterations, which is highly dependent on the discoveries in gene functional studies¹⁶. In recent years, significant advancements in understanding nodulation in soybean have been made. Flavonoids generated by roots and NFs released by rhizobia play central roles in the symbiotic process. Apart from them, the symbiotic relationship between leguminous plants and rhizobia can also be influenced by other signaling molecules. For example, phenoxyacetic acid promotes symbiotic nodulation by regulating the miR172c-Nodule Number Control 1 (NNC1) pathway and GmGA2ox10 expression during the rapid growth stage of soybean¹⁷. Furthermore, the type III effector factor Nodulation Outer Protein L (NopL) promotes symbiotic signalling by interacting with Remorin 1a (GmREM1a) and GmNFR5, thereby mediating the establishment of a symbiotic relationship between rhizobia Sinorhizobium fredii HH103 and soybean¹⁸. The intricate link between plant hormone signaling and rhizobial symbiosis pathways has also been highlighted. For instance, GmRR11d, a B-type Response Regulator induced by rhizobia and cytokinin, functions as an important effector of autoregulation of nodulation (AON) to inhibit the expression of GmNIN1a¹⁹. Similarly, Bri1-EMS-Suppressor 1 (GmBES1), a central transcription factor in the brassinosteroid signaling pathway, inhibits the interaction and DNA-binding activity of GmNSP1 and GmNSP2, thereby modulating nodulation²⁰.

In addition, root nodule nitrogen fixation is also influenced by internal changes in energy levels and carbon sources. For example, light-induced Flowering Locus T (GmFTs) and Soybean TGACG-motif binding Factor 3/4 (GmSTF3/4) move from the shoot to the root, where they are phosphorylated by CCaMK to initiate nodule formation²¹. This finding highlights the critical roles of photosynthetic products and light signals in root nodule development and symbiotic nitrogen fixation in leguminous plants. A newly discovered energy receptor, Nodule AMP Sensor 1/NAS1-Associated Protein 1 (GmNAS1/GmNAP1), regulates nitrogen fixation by modulating carbon distribution within the nodules²². Mutants of the Rhizobia-Induced CLE 1a/2a (ric1a/2a) genes exhibit an increase in both nodule numbers and chlorophyll content, promoting carbon and nitrogen synergies²³. Collectively, these insights into the mechanisms of nodule formation and development in soybeans have significantly advanced our understanding of nitrogen fixation in leguminous plants.

Both the nodule and lateral root serve as lateral tissues of the primary root. Nodule development shares similarities with lateral root development, involving cell proliferation in the cortex layers and pericycle, leading to the initiation of organ primordia. Auxin, a pivotal plant hormone, plays a crucial role in inducing the formation of lateral root primordia (LRP) by activating key regulatory genes such as GATA Transcription Factor 23 (GATA23) and Lateral Organ Boundaries Domain 16/Asymmetric Leaves2 Like 18 (LBD16/ASL18)²⁴. LjLBD16 has been found to promote the initiation of nodules, highlighting a significant overlap between nodule and lateral root development programs^25,26,27. In addition to auxin, other plant hormones and related regulatory factors known to regulate root development are also involved in nodule formation in leguminous plants²⁸. For instance, cytokinin and its signaling components, such as type A and type B regulators, are implicated not only in root development²⁹ but also in regulating root nodulation^19,27.

The occurrence of legume nodules is not only regulated by internal developmental signals but also influenced by external environmental signals. For instance, studies have shown that salt stress can impede nodule formation and root hair infection in Vicia faba L. plants^30,31. Subsequent studies revealed that salt stress not only affected crop yield by inhibiting plant growth but also impacted the symbiotic relationship between roots and soil bacteria, including rhizobia, thereby affecting the nitrogen-fixing ability of leguminous plants^32,33,34. Recent research indicates that nearly every aspect of nodulation in leguminous plants is adversely impacted by salt stress^35,36,37. High salt treatment diminishes the root hair infection process in Vicia faba, resulting in fewer nodules being produced³⁰. Additionally, glycogen synthase kinase 3 (GSK3)-like kinase (GSK2-8) directly phosphorylates GmNSP1a to inhibit its transcriptional activity, thus causing reduced nodule numbers under salt stress conditions in soybean³⁸. A key NAC transcription factor GmNAC181, highly induced by salt stress, is able to directly bind to GmNIN1a promoter to activate its expression and promote symbiotic nodulation under salt stress condition³⁹. Abscisic acid (ABA) plays a crucial role in regulating various aspects of plant growth and development under salt stress condition⁴⁰. It inhibits bacterial infection, nodulation, and the expression of nodulin genes in some legumes^41,42. Overexpression of the Abscisic Acid Insensitive 5 (ABI5) gene renders plants more susceptible to salt, drought, and temperature stresses⁴³. In contrast, the ABI5 Like 1 (ABL1) deficiency mutant (abl1) shows reduced ABA responses, modulating plant stress responses by directly regulating a series of genes containing ABA-responsive elements⁴⁴. Notably, the dominant-negative allele abi1 of Abscisic Acid Insensitive 1 is insensitive to ABA and exhibits a hyper-nodulation phenotype⁴⁵. Despite the discovery of salt stress inhibiting root nodulation and nitrogen-fixing efficiency in legumes for ~50 years⁴⁶, the underlying molecular mechanisms remain to be fully elucidated.

In this investigation, we discovered that salt stress induced the expression of GmERF13s, consequently inhibiting root nodulation in soybean. GmERF13s directly bound to the promoter region of GmEXP17c, whose homologous gene EXP17 transcriptionally regulated by LBD16 in Arabidopsis thaliana during lateral root development, leading to the suppression of its transcriptional activity. Additionally, we identified an interaction between GmERF13s and GmLBD16a, an upstream transcriptional activator of GmEXP17c, whereby GmERF13s hindered the binding capacity of GmLBD16a to the promoter of GmEXP17c. Moreover, our research revealed that the upregulation of GmERF13s under salt stress conditions was reliant on GmABI5a and GmABI5b-mediated ABA signalling pathways. This study unveils a groundbreaking molecular mechanism that elucidates how salt stress inhibits nodulation in soybean. Specifically, it uncovers the intricate involvement of the GmABI5-GmERF13s-GmLBD16a-GmEXP17 regulatory module in this process. These findings not only deepen our understanding of the effect of salt stress on nodulation inhibition, but also provide important candidate genes for future molecular design and breeding based on saline soil.

Results

GmERF13 is induced by salt stress and highly expressed in nodules

To elucidate the molecular mechanism of salt stress inhibiting root nodules, we conducted a detailed analysis of the nodulation phenotype in soybean subjected to salt stress treatments. Williams 82 (W82) plants were treated with varying concentrations of NaCl (0 mM, 50 mM, 75 mM, 100 mM, and 150 mM), and nodule numbers were quantified at 28 days post inoculation (dpi) with Bradyrhizobium diazoefficiens strain USDA110 (hereinafter referred to as USDA110). As the NaCl concentration increased, the nodule number in W82 exhibited a corresponding intensified inhibition. At 150 mM NaCl treatment, the plants displayed a significantly reduced ability to produce nodules, dropping to extremely low levels (Supplementary Fig. 1a, b).

Next, we conducted RNA-seq analysis on soybean roots that had been inoculated with USDA110. We extracted RNA from roots with nodules of different sizes, comparing the gene expression profiles between roots with nodules of different sizes treated with 150 mM NaCl for 2 hours and those without NaCl treatment. Several differentially expressed genes (DEGs) were highly induced by NaCl in the roots (Fig. 1a). Notably, among these DEGs, we identified the AP2/ERF transcription factor (Glyma.03G112700), whose homologous gene ERF13 has been previously reported to play a role in the regulation of lateral root development in Arabidopsis thaliana^47,48. There are four GmERF13s identified in soybean, designated as GmERF13a (Glyma.03G112700), GmERF13b (Glyma.07G114000), GmERF13c (Glyma.09G242600), and GmERF13d (Glyma.18G252400), and they are the homolog of Medtr8g006825, Medtr8g006815, Lj3g0017628, Lj1g0020111, and At2G44840 in the phylogenetic tree (Fig. 1b). Furthermore, in order to clearly illustrate the expression pattern of GmERF13s in response to salt stress, we also conducted histochemical assays of ProGmERF13s:GUS transgenic roots and nodules under 150 mM NaCl treatment (Fig. 1c). The results demonstrated that, with the exception of GmERF13a, GmERF13s was primarily expressed in the stele of roots and in the cortical cells of nodules under normal condition. Nevertheless, following 2 hours and 12 hours of salt treatment, GmERF13a expression was markedly induced in both roots and nodules, consistent with the transcriptome data (Fig. 1a). Additionally, the expression of GmERF13b, GmERF13c, and GmERF13d showed significantly upregulated after 12 hours of NaCl treatment (Fig. 1c). Further analysis revealed that both GmERF13b and GmERF13c displayed highly accumulated expression patterns in nodules (Fig. 1c, d). Furthermore, gene expression analysis showed that GmERF13s were significantly induced by rhizobia at the late stage of nodulation in mixed samples of roots and nodules, 7 dpi or even longer for obvious induction to occur (Supplementary Fig. 2a–d). To gain a more detailed understanding of the expression patterns of GmERF13s, we conducted additional ProGmERF13s:GUS histochemical assays in transgenic roots. These assays revealed that, under normal conditions, GmERF13s were mainly expressed in the stele. However, following an inoculation with USDA110 for 21 days, there was a strong induction of GmERF13s, particularly GmERF13b and GmERF13c, in roots (Supplementary Fig. 2e). In summary, our findings suggest that GmERF13 genes are highly expressed in nodules and are significantly activated by both rhizobia and salt stress.

**Fig. 1: *GmERF13s* are induced by salt stress and highly expressed in nodules.**

GmERF13s are negative regulators of nodule formation

To examine the role of GmERF13s on soybean nodulation, we generated GmERF13s^CR mutants with frameshift mutations or large fragmental deletions at all target GmERF13 homologous genes using CRISPR/Cas9 technology (Supplementary Fig. 3a–d). The analysis of nodulation phenotype at 28 dpi, conducted by evaluating the nodule number per gram of root dry weight in GmERF13s^CR mutant roots, demonstrated a significant increase in nodule density as a consequence of loss-of-function mutations in GmERF13s. Among these mutants, notable phenotypic changes were observed in GmERF13b^CR, GmERF13c^CR, and GmERF13d^CR, which exhibited substantial increases in nodule density. However, the GmERF13a^CR mutant did not display a significant nodulation phenotype (Fig. 2a, b). Furthermore, the double mutant GmERF13bc^CR exhibited a greater number of nodules compared to the single mutants GmERF13b^CR or GmERF13c^CR, indicating functional redundancy of GmERF13s (Fig. 2a, b). We also measured the dry weight of root nodules and conducted acetylene reduction experiments to assess the nitrogenase activity of nodules in GmERF13s^CR. The results showed that GmERF13s had little or no impact on nodule weight or size, but they significantly influenced nitrogenase activity in soybean nodules (Fig. 2c, d).

**Fig. 2: GmERF13s function as negative regulators in nodulation and nodule function.**

Next, we analyzed the expression of GmENOD40 and GmENOD11, two key marker genes for nodule formation, in GmERF13s^CR at 7 d and 14 d after inoculated or uninoculated by USDA110 (Fig. 2e–h). The results showed that GmENOD40 and GmENOD11 expression levels were significantly higher in the inoculated GmERF13s^CR roots compared to the inoculated W82 roots.

To test whether GmERF13s could complement all the GmERF13s^CR mutant nodulation phenotypes, we introduced constructs containing ProGmERF13s:GmERF13s into the GmERF13s^CR mutants using Agrobacterium rhizogenes K599-mediated hairy root transformation. The nodule number on mutant roots expressing the complementation constructs was observed to be inhibited compared to GmERF13s^CR mutants expressing the empty vector (pTF102) (Supplementary Fig. 4a, b). This indicates that the increased nodulation phenotypes observed in GmERF13s^CR mutants are attributable to mutations in GmERF13s.

We also generated hairy roots overexpressing GmERF13s (GmERF13a^OX, GmERF13b^OX, GmERF13c^OX, and GmERF13d^OX) and evaluated the nodule number per hairy root at 28 dpi. Consistent with increased nodules in the stable GmERF13s^CR mutants, GmERF13s^OX (GmERF13b^OX, GmERF13c^OX, and GmERF13d^OX) hairy roots exhibited significantly fewer nodules compared to control roots expressing the empty vector (pS1300) (Supplementary Fig. 5a, b). RT-qPCR analysis confirmed that all four GmERF13s were upregulated in GmERF13s^OX hairy roots (Supplementary Fig. 5c), indicating a correlation between the increased nodulation phenotype and the upregulated expression of GmERF13s. Consistently, the expression levels of GmENOD40 and GmENOD11 showed a visible decrease in GmERF13b^OX and GmERF13c^OX hairy roots compared to hairy roots transformed with the empty vector (pS1300; Supplementary Fig. 5d). In summary, these results indicate that GmERF13s play negative roles in nodulation and nodule function.

GmERF13s regulate nodule formation through their interplay and antagonistic interaction with GmLBD16a

To elucidate the molecular mechanisms of GmERF13-mediated regulation of nodule formation, we performed a point-by-point validation by introducing the bait protein GmERF13s, along with key factors known to regulate root nodulation in legumes, into the yeast two-hybrid (Y2H) Gold strain. Notably, we discovered an interaction between GmERF13s and LOB-DOMAIN PROTEIN 16 (GmLBD16a; Glyma.03G161400; Supplementary Table 1). Given that LBD16 has been identified as a downstream target of NIN and a key regulator of root nodulation in Medicago truncatula²⁷, we focused on the regulatory relationship between GmERF13s and GmLBD16a to further investigate their roles in root nodulation. Genome-wide analysis identified three copies of GmLBD16 genes (Glyma.03G161400, Glyma.19G162900, and Glyma.13G121300) in soybean, which were denoted as GmLBD16a, GmLBD16b, and GmLBD16c based on their coding sequences homology to LBD16 in Arabidopsis thaliana (Supplementary Fig. 7a).

To further confirm the interaction between GmERF13s and GmLBD16a, we performed Y2H, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (Co-IP) assays. The interactions between GmERF13s and GmLBD16a were detected in both yeast cells and the nucleus of tobacco leaf cells (Fig. 3a–c). Our results showed that GmERF13a, GmERF13c, and GmERF13d could interact with GmLBD16a, while GmERF13b could not (Fig. 3a–c). We also demonstrated that GmERF13a, GmERF13c, and GmERF13d could interact with each other and GmERF13c was able to form homologous dimers in both yeast cells and the nucleus of tobacco leaf cells (Supplementary Fig. 6a, c, d). In addition, we also confirmed that GmERF13b could interact with GmERF13c in yeast cells (Supplementary Fig. 6b).

**Fig. 3: GmERF13s interact with GmLBD16a.**

We generated hairy roots overexpressing GmLBD16 (GmLBD16a^OX and GmLBD16b^OX) and investigated the impact of GmLBD16 on nodule formation by evaluating the nodule number per hairy root at 28 dpi. The results indicated that GmLBD16a^OX and GmLBD16b^OX roots produced many more nodules compared to the empty vector (pS1300; Supplementary Fig. 7b–d). These results suggest that both GmLBD16a and GmLBD16b play positive roles in regulating nodulation in soybean.

To illustrate the genetic relationship of GmERF13s and GmLBD16a in nodulation, we performed nodulation analysis by either overexpressing GmERF13b/GmERF13c in the stable GmLBD16a overexpression line or overexpressing GmLBD16a in the stable GmERF13bc^CR mutant background. In the stable GmLBD16a overexpression background, the repressive effect of GmERF13b^OX or GmERF13c^OX on nodulation was significantly alleviated (Fig. 4a, b). Conversely, in the GmERF13bc^CR mutant background, overexpression of GmLBD16a led to an even higher number of root nodules compared to the empty vector control (Fig. 4a, b). RT-qPCR experiment confirmed that GmERF13b, GmERF13c, and GmLBD16a were upregulated in the stable GmLBD16a^OX and GmERF13bc^CR mutant backgrounds (Fig. 4c). These results indicate that GmERF13 and GmLBD16a have antagonistic roles in the regulation of nodule formation.

**Fig. 4: GmERF13s antagonize GmLBD16a in regulating root nodulation.**

GmEXP17s function as positive regulators of nodulation

LBD16/18-Expansin14/17 (EXP14/17) module has been well known to regulate lateral root development⁴⁹. We thus suggested that the GmLBD16-GmEXP17 module might play a role in nodulation. Four copies of GmEXP17 genes (Glyma. 20G089100, Glyma. 10G140200, Glyma. 03G225900, and Glyma. 19G222900) were identified in soybean and recognized as GmEXP17a, GmEXP17b, GmEXP17c, and GmEXP17d according to coding sequences homology to EXP17 in Arabidopsis thaliana (Supplementary Fig. 8a). As observed in Arabidopsis thaliana, where LBDs positively regulated the transcriptional expression of EXP17, we observed upregulation of GmEXP17b and GmEXP17c by GmLBD16a (Supplementary Fig. 8c, d). Furthermore, we also detected analogous regulation of EXP17s, which were upregulated in GmERF13s^CR mutant plants (Supplementary Fig. 8b–e). These results showed that GmEXP17s were downstream of GmERF13s and GmLBD16a while GmERF13s and GmLBD16a played antagonistic roles in GmEXP17 expression.

Subsequently, we explored the influence of GmEXP17s on nodule formation through hairy roots. When GmEXP17s were overexpressed, confirmed by the upregulated expression of GmEXP17a, GmEXP17b, GmEXP17c, and GmEXP17d through RT-qPCR analysis (Fig. 5c), the nodule numbers per hairy root after 28 dpi were significantly increased in GmEXP17a^OX, GmEXP17b^OX, and GmEXP17c^OX roots compare to the empty vector (pS1300; Fig. 5a, b). However, in the amiR-GmEXP17 transgenic hairy roots, where the expression of all four GmEXP17s was downregulated using artificial microRNA (amiR) interference technology (Fig. 5d), the amiR-GmEXP17 knockdown roots after 28 dpi displayed much fewer nodules than the empty vector (pS1300; Fig. 5a, b). These results suggest that GmEXP17s function as positive regulators of nodulation in soybean.

**Fig. 5: GmEXP17s are positive regulators of nodulation in hairy root.**

GmERF13s and GmLBD16a directly target GmEXP17s promoters and regulate their expression

The discovery highlights a noteworthy interaction between GmERF13s and GmLBD16a, both demonstrating antagonistic effects on the expression of GmEXP17b and GmEXP17c (Supplementary Fig. 8c, d). This finding impelled us continuously to explore the underlying molecular mechanism. Consequently, we proceeded by introducing more than 2000 bp promoters of GmEXP17s into the pHIS2.1 vector to investigate whether GmLBD16a could directly target the promoter of GmEXP17s through yeast one-hybrid (Y1H) assays in yeast cells. Surprisingly, GmLBD16a could only bind to the promoter of GmEXP17c, not GmEXP17a, GmEXP17b, or GmEXP17d (Fig. 6a). To further validate this observation, we conducted chromatin immunoprecipitation (ChIP) assays using hairy roots at 10 dpi. Remarkably, the ChIP assay confirmed a substantial enrichment of GmLBD16a across nearly all regions of the GmEXP17c promoter containing GCC-Like motif (Fig. 6b). Furthermore, we investigated whether GmERF13s could directly target the promoters of GmEXP17s through Y1H assays. The results showed that GmERF13s could bind to the promoters of GmEXP17a, GmEXP17b, and GmEXP17c, but not GmEXP17d (Supplementary Fig. 9). Subsequent ChIP-qPCR assays provided additional evidence, demonstrating that GmERF13a, GmERF13b, GmERF13c, and GmERF13d exhibited varying binding patterns within the promoter regions of GmEXP17c (Fig. 6b). These findings indicate that both GmLBD16a and GmERF13s directly regulate GmEXP17c expression.

**Fig. 6: GmERF13s inhibit the binding ability of GmLBD16a to *GmEXP17c* promoter.**

To show the expression levels of GmLBD16a and GmEXP17c genes during nodulation, RT-qPCR analysis was conducted, and the results showed that GmLBD16a was also significantly induced at 10 dpi and remained elevated expression levels in mixed samples of roots and nodules. However, GmEXP17c shows only mild induction at the early stages of nodulation, with no significantly induced expression in later stages (Supplementary Fig. 10a, b). Given that GmERF13s were upregulated during the late stages of rhizobia treatment (Supplementary Fig. 2a–d), we hypothesized that GmERF13s might suppress GmEXP17 expression at later nodulation stages, thereby inhibiting root nodulation. Additionally, we performed histochemical assays using ProGmLBD16a:GUS and ProGmEXP17c:GUS transgenic roots to further assess their expression after rhizobia treatment. The results showed that similar to GmERF13s, GmLBD16a and GmEXP17c were predominantly expressed in the stele cells under normal conditions. After inoculation with USDA110, GmLBD16a was induced in 21 dpi roots, while GmEXP17c exhibited only a minor induction in roots treated with rhizobia for 3 hours (Supplementary Fig. 10c, d).

To further illustrate the underlying significance of interaction between GmERF13s and GmLBD16a, we transiently co-expressed ProGmEXP17c-LUC with different combinations with empty vector (pBI221), 35S:GmERF13s, and 35S:GmLBD16a in soybean protoplast. The LUC assays showed that the expression level of GmEXP17c was markedly decreased when ProGmEXP17c-LUC was co-expressed with 35S:GmERF13s. Conversely, a notable increase in the expression level of GmEXP17c was observed when ProGmEXP17c-LUC was co-expressed with 35S:GmLBD16a (Fig. 6c). Moreover, the function of GmLBD16a on GmEXP17c expression was apparently reduced by 35S:GmERF13s when ProGmEXP17c-LUC was co-expressed with 35S:GmERF13s and 35S:GmLBD16a (Fig. 6c). During the early stages of nodulation, GmEXP17c shows a minimal induction due to the lower transcriptional levels of GmERF13s. Conversely, at the late stages of nodulation, when GmERF13 expression is elevated, GmEXP17c expression remains relatively low (Supplementary Fig. 2a–d; Supplementary Fig. 10b). The interaction between GmERF13s and GmLBD16a likely disrupts the transcriptional activity of GmLBD16a.

In order to verify the effect of GmERF13s on the DNA-binding ability of GmLBD16a, we conducted ChIP-qPCR assays in hairy roots after 10 dpi. Our findings revealed a significant enrichment of GmLBD16a across multiple regions of the GmEXP17c promoter (Fig. 6b, d). Interestingly, the ChIP assay also demonstrated that the enrichment of GmLBD16a at the GmEXP17c promoter was notably enhanced in specific regions (B, C, F, and G) in the GmERF13bc^CR mutant plants (Fig. 6d). These results suggested the negative role of GmERF13s on the enrichment of GmLBD16a at the GmEXP17c promoter. Therefore, we conclude that the interaction between GmERF13s and GmLBD16a primarily centres on disrupting the binding ability of GmLBD16a to the promoter of GmEXP17c.

GmEXP17c acts downstream of GmERF13s and GmLBD16a in root nodulation

To genetically investigate the regulation of GmLBD16a on GmEXP17c, the composite roots overexpressing GmLBD16a (GmLBD16a^OX) and silencing GmEXP17 (amiR-GmEXP17) using an artificial microRNA interference technology were generated. The GmLBD16a^OX/amiR-GmEXP17 composite roots showed a similar nodule number phenotype as amiR-GmEXP17 (Fig. 7a, b). Due to the similar function of GmERF13a, GmERF13b, GmERF13c, and GmERF13d on GmEXP17c, we took GmERF13b and GmERF13c as examples to investigate the genetic relationship between GmERF13s and GmEXP17c. The results showed that the composite roots GmERF13b^OX/GmEXP17c^OX and GmERF13c^OX/GmEXP17c^OX had similar increased nodule number phenotype as GmEXP17c^OX (Fig. 7c, d). Taken together, these results suggest that GmEXP17c functions downstream of both GmERF13s and GmLBD16a in the regulation of root nodulation in soybean.

**Fig. 7: GmEXP17s function downstream of GmERF13 and GmLBD16a in nodulation.**

Salt stress-induced expression of GmERF13s is dependent on ABA

According to the data from RNA-seq, the expression of GmERF13a is induced by 150 mM NaCl after 2 hours of treatment (Fig. 1a). Salt stress can induce a rapid and massive accumulation of ABA in plant tissues⁵⁰. In order to determine whether the induction of GmERF13s by salt stress depended on ABA, we subjected seven-day-old seedlings to treatment with 150 mM NaCl and 100 µM Fluridone, an ABA biosynthesis inhibitor. Analysis through RT-qPCR revealed that GmERF13s were highly expressed in roots after 150 mM NaCl treatment and the expression of GmERF13s induced by 150 mM NaCl was significantly reduced by ABA biosynthesis inhibitor (Fig. 8a–d). Subsequently, to further elucidate this relationship, we examined the expression of GmERF13s under treatment with 10 µM ABA, which demonstrated a significant induction of GmERF13s expression (Fig. 8e). These findings unequivocally indicate the dependency of GmERF13s induction by salt stress on ABA signaling.

**Fig. 8: Salt stress-induced expression of *GmERF13s* is ABA-dependent.**

GmABI5a and GmABI5b directly promote the transcription of GmERF13s

Given the induction of GmERF13s expression by ABA (Fig. 8e), it is easy to have visions of GmABI3/4/5 activating the expression of GmERF13s probably through ABA signaling. To test this hypothesis, we examined the expression of GmERF13s in GmABIs^OX roots. The data revealed a significant upregulation of GmERF13s expression in GmABI3/4/5^OX (Fig. 8f). Furthermore, we also transiently co-expressed ProGmERF13s-LUC with empty vector (pBI221) and 35S:GmABIs in soybean protoplast. The LUC assays showed that the expression levels of GmERF13s were markedly increased when co-expressed with all six variants of 35S:GmABIs (Fig. 8g).

In order to identify whether GmABIs could bind to the promoters of GmERF13s directly, we carried out ChIP-qPCR assays using GmABI5a^OX and GmABI5b^OX hairy roots after 10 dpi. The data indicated that GmABI5a was significantly enriched in region C, E, and H of GmERF13b promoter, region D of GmERF13c promoter, and region B, D, and F of GmERF13d promoter. On the other hand, GmABI5b exhibited significant enrichment across almost all regions of GmERF13a promoter and GmERF13b promoter, region A, C, and D of GmERF13c promoter, and regions A, B, D, E, and F of GmERF13d promoter (Fig. 8h–k).

We next examined the effect of GmABI3/4/5 on nodulation by creating GmABIs^OX (GmABI3a^OX, GmABI3b^OX, GmABI4^OX, GmABI5a^OX, GmABI5b^OX, and GmABI5c^OX) hairy roots and assessing nodule numbers of GmABIs^OX per hairy root at 28 dpi. The results showed that all the GmABIs^OX hairy roots produced obviously fewer nodules than empty vector (pS1300; Supplementary Fig. 11a, c, e). Subsequently, we employed CRISPR/Cas9 technology to create knockout mutants for GmABI3a and GmABI3b (GmABI3^CR), GmABI4 (GmABI4^CR), GmABI5a and GmABI5b (GmABI5ab^CR), and GmABI5c (GmABI5c^CR) in hairy roots. Remarkably, all four GmABIs^CR mutants exhibited significantly higher nodule formation compared to the empty vector control (pGES401; Supplementary Fig. 11b, d). In addition, we also generated the double mutant GmABI5bc^CR with base deletion or large fragmental deletions at GmABI5 homologous genes using CRISPR/Cas9 technology (Supplementary Fig. 3e, f) to analyze the impact on root nodulation. The results showed that the GmABI5bc^CR mutant exhibited a significant increase in nodule density compared to the W82 control (Supplementary Fig. 14a, b).

In summary, these data strongly support the conclusion that GmABI3/4/5 act as negative regulators of nodulation in soybean by directly activating the transcription of GmERF13s.

GmERF13s mediate salt stress inhibition of nodulation

Based on our findings, GmERF13s had been identified as salt-induced genes and played negative roles during nodulation. We next further investigated the impact of GmERF13s on the inhibition of nodulation under salt stress conditions. Seven-day-old W82 and GmERF13s^CR seedlings were treated with or without 75 mM NaCl, and nodule density was analyzed at 28 dpi with USDA110. Surprisingly, the nodule density in W82 decreased by 39.73% after being treated with 75 mM NaCl, while the reduction rates in the GmERF13s^CR mutants were significantly lower than that of W82 except in GmERF13d^CR. Specifically, the reduction rates for GmERF13a^CR, GmERF13b^CR, GmERF13c^CR, GmERF13d^CR, and GmERF13bc^CR after treated with 75 mM NaCl were 21.68%, 19.43%, 25.27%, 46.75%, and 16.48%, respectively. Notably, the reduction rate of the GmERF13bc^CR double mutant was significantly lower compared to that of GmERF13b^CR and GmERF13c^CR single mutants (Fig. 9a, b), which also indicates functional redundancy among GmERF13s. We also evaluated the nodule number of GmERF13s^OX transgenic hairy roots confirmed by RT-qPCR experiments (Fig. 9e) with or without 75 mM NaCl treatment at 28 dpi. Significantly, the nodule numbers of GmERF13s^OX were reduced by 52.67%, 66.14%, 40.13%, and 40.14%, respectively, which were significantly higher than the 31.62% reduction observed in the empty vector (pS1300; Fig. 9c, d). These findings indicate that GmERF13s play substantial roles in the salt stress-mediated inhibition of nodulation in soybean.

To assess the function of GmERF13s in mature nodules under salt stress condition, we conducted the nitrogenase activity assay to analyze the effects of GmERF13s on salt tolerance of mature nodules (Supplementary Fig. 12). The results showed that after treatment with 75 mM NaCl, the nitrogenase activity in wild-type W82 nodules decreased by 36.40%. However, the reduction of nitrogenase activity in GmERF13^CR mutants was significantly lower than in W82, with the double mutant GmERF13bc^CR showing a notably lower reduction rate compared to the single mutants GmERF13b^CR and GmERF13c^CR. These findings suggest that GmERF13s not only mediate the inhibition of nodule density under salt stress condition but also contribute to the suppression of nitrogenase activity.

GmLBD16a, GmABI5, and GmEXP17c mediate the inhibitory effects of salt stress on nodulation

To gain deeper insights into the role of GmLBD16s, GmEXP17s, and GmABIs in regulating root nodulation under salt stress condition, we analyzed their transcriptional profiles using RNA-seq data. Our analysis revealed that the expression levels of all these genes were altered in response to salt stress (Supplementary Fig. 13a). These findings were further validated by RT-qPCR, which showed a significant upregulation of GmABI3, GmABI4, and GmABI5 upon NaCl treatment (Supplementary Fig. 13b, c). Conversely, the expression of GmLBD16a and GmEXP17s was downregulated under the same condition (Supplementary Fig. 13a, d). The transcriptional data were also validated by histochemical assays of transgenic roots and nodules. Under normal condition, GmLBD16a was mostly expressed in the cortical cells of nodules, but GmABI5a and GmABI5b were only weakly expressed in roots and nodules. Under salt stress condition, the expression of GmABI5a and GmABI5b was significantly upregulated, while GmEXP17c expression was notably suppressed in both roots and nodules (Supplementary Fig. 13e).

GmERF13s had been proven to be involved in the salt stress inhibition of nodulation in soybean from the phenotype data (Fig. 9). To confirm the roles of GmABIs, GmLBD16a, and GmEXP17 after salt treatment, GmLBD16a overexpression lines (GmLBD16a^OX-1 and GmLBD16a^OX-5) and GmABI5bc^CR stable mutants were used to carry out the phenotypic experiments under salt stress condition. The phenotypic analysis of root nodules showed that both GmLBD16a overexpression lines (GmLBD16a^OX-1 and GmLBD16a^OX-5) and the GmABI5bc^CR stable double mutant exhibited increased root nodule density. Moreover, salt stress, which typically inhibited root nodulation, was alleviated in both GmLBD16a overexpression lines (GmLBD16a^OX-1 and GmLBD16a^OX-5) and the GmABI5bc^CR stable double mutant compared to the W82 control (Supplementary Fig. 14a, b). In addition, GmEXP17c^OX hairy roots were also been used to conduct phenotypic data under salt stress condition. The results showed that the nodule number of GmEXP17c^OX was reduced by 26.57%, which was significantly lower than the 36.91% reduction observed in the empty vector (pS1300; Supplementary Fig. 14c, d). The expression level of GmEXP17c has been validated in GmEXP17c^OX (Supplementary Fig. 14e). These findings suggest that the GmABI5-GmERF13-GmLBD16a-GmEXP17c regulatory module plays a key role in the inhibition of nodulation by salt stress in soybean.

Discussion

The AP2/ERF family of transcription factors plays a vital role in plant development and environmental adaptation by regulating key physiological processes through the modulation of gene expression. In this study, we highlight the pivotal role of GmERF13, a transcription factor within the AP2/ERF family, and its involvement in inhibiting nodulation under salt stress condition in soybean. Mutant analysis revealed that GmERF13b^CR, GmERF13c^CR, and GmERF13d^CR all exhibited significantly increased nodule numbers, while there was no notable phenotypic change observed in GmERF13a^CR (Fig. 2a, b). Conversely, GmERF13b^OX, GmERF13c^OX, and GmERF13d^OX all displayed fewer nodule numbers, while GmERF13a^OX did not exhibit a noticeable nodulation phenotype (Supplementary Fig. 5a, b). Although the most significant induction of GmERF13a under salt stress condition (Fig. 1a), these inconsistent nodulation phenotypes observed in both mutant and overexpression roots of GmERF13a suggest complex regulatory mechanisms among these four homologous genes in nodulation. Further investigation, including the generation of various double mutants, is necessary to fully elucidate the mechanisms underlying GmERF13-mediated nodulation inhibition under salt stress condition.

During root nodule development, GmERF13s and GmLBD16a are induced by rhizobia at the late stages of nodulation, while GmEXP17c is slightly induced at the early stages, with no discernible upregulation at later stages (Supplementary Fig. 2a–d; Supplementary Fig. 10a, b). Given that GmERF13s interact with GmLBD16a in both in vivo and in vitro assays (Fig. 3), we propose that GmERF13s may suppress GmEXP17 expression by inhibiting the transcriptional activity of GmLBD16a at later stages of nodulation, thereby inhibiting root nodulation.

ERF13 has been identified as a negative regulator of lateral root development in Arabidopsis thaliana. MPK14-mediated auxin signaling phosphorylates and degrades ERF13, thereby promoting lateral root formation^47,48. In this study, we have found that GmERF13s are induced by salt stress via ABA signaling and impede nodulation by interacting with and interfering GmLBD16a functions (Figs. 3, 6 and 8). This highlights a shared mechanism underlying both root and nodule development regulated by the GmERF13-GmLBD16 module. This discovery not only improves our understanding about the molecular mechanism of underlying nodulation in response to salt stress, but also further underscores the potential close evolutionary relationship between nodule formation in leguminous plants and the regulation of plant root development. Recent studies have highlighted the dual involvement of several regulatory factors in both root development and the formation of nodules in leguminous plants⁵¹. For instance, in species like Medicago truncatula and Glycine max, the SHR-SCR module, renowned for its pivotal role in root development, is recruited by symbiotic signals to govern nodule organogenesis through activating cortical cell division^52,53,54. Moreover, in Medicago truncatula, PLETHORA genes are expressed in nodule primordia and later at the apex of mature nodules, playing a regulatory role in nodule formation⁵⁵. Additionally, research in Medicago truncatula and Lotus japonicus has shown that the LBD16/ASL18 gene is activated by NIN and forms a complex with the NF-Y transcription factor, thereby promoting nodule development²⁵. All these studies suggest that the process of nodule formation in leguminous plants and the regulation of plant root development may be closely related to evolution.

The cell wall is essential in plant cells, shaping their structure, and supporting important functions like organ growth, nutrient transport, and protection from the environment. Additionally, it also plays a critical role in various plant growth and development through controlling cell differentiation and intercellular communication⁵⁶. For example, both EXP14 and EXP17 are transcriptionally upregulated by LBD18 and cause cell wall-loosening during auxin-promoted lateral root emergence^57,58. The IDA-HAE/HSL2 signaling module facilitates the passage of LRP through overlaying root tissues during lateral root emergence by regulating the expression of cell wall remodeling genes⁵⁹. Lateral root initiation also entails the selective distribution of pectin species⁶⁰ and asymmetric cell divisions in the pericycle mediated by EXPANSIN A1 (EXPA1)⁶¹. In leguminous plants, the process of nodulation involves infection threads passing through several cell layers and the subsequent colonization of differentiating cells in nodule primordia. Much like in lateral root development, these processes are intricately connected to changes in the composition and properties of the cell wall, although our understanding in this area is still somewhat limited⁶². For instance, a β-expansin gene, GmEXPB2, has been shown to regulate nodule formation and development by influencing cell wall modification and extension in soybean⁶³. Additionally, a recent study demonstrated that symbiosis-specific pectin methyl esterases (SyPME1) and nodulation pectate lyase (NPL) coordinated to govern the intracellular progression of infection threads through the entire root cortical tissue⁶⁴. Here, we demonstrate that GmEXP17s, which act downstream of GmERF13s and GmLBD16a, promote nodule formation (Fig. 7). This discovery not only reaffirms the crucial role of cell wall-related regulatory factors in the process of nodule formation in leguminous plants but also underscores the conservation of regulatory mechanisms shared between plant root development and the occurrence of nodules in leguminous plants. Moreover, our understanding of plant root development gained from the model plant Arabidopsis thaliana will have significant implications for gaining deeper insights into the regulatory mechanisms governing nodule formation in leguminous plants.

In summary, we propose a model to explain how abiotic stress inhibits nodulation between legumes and rhizobia. Under normal conditions, the symbiotic signaling pathway is activated by rhizobia in response to root flavonoids, leading to the release of NFs and subsequent nodule development. In this process, the downstream factor GmLBD16a binds directly to the promoter of the symbiotic gene GmEXP17c, activating its expression in response to the upstream symbiotic signal. During this phase, GmERF13s are expressed at relatively low levels. However, under abiotic stress conditions, such as salt stress, GmERF13s are strongly induced via ABI5-mediated ABA signaling. The reduced nodule numbers are a consequence of the interaction between upregulated GmERF13s and GmLBD16a, which reduces GmLBD16a’s binding affinity to the GmEXP17c promoter, thereby inhibiting nodule formation (Fig. 10). In soybean, we identified four GmERF13 genes, while in Arabidopsis, only one ERF13 gene is present. Future studies should investigate the specific roles of these four GmERF13 genes in nitrogen fixation and legume-rhizobia interactions.

**Fig. 10: Proposed model for GmERF13-mediated salt stress inhibition of nodulation.**

Methods

Plant materials and growth conditions

All materials used in this study were in soybean cultivar Williams 82 (W82) background. Seedlings were grown on sterilized vermiculite under 16 h/8 h light/dark conditions in a culturing room at 26 °C. For the nodulation phenotype assay, seedlings were germinated in sterilized vermiculite, and after seven days, they were inoculated with Bradyrhizobium diaefficiens strain USDA110 (OD₆₀₀ = 0.05). Phenotypic assessments were conducted at 28 days post inoculation (dpi) with USDA110. To generate GmERF13 mutants and GmABI5bc mutants by CRISPR/Cas9-mediated gene editing technology, we designed three sgRNAs specifically targeting the single GmERF13 coding region, four sgRNAs targeting both the GmERF13b and GmERF13c coding regions, and three sgRNAs targeting both the GmABI5b and GmABI5c coding regions. These sgRNAs were then incorporated into the pGES401 vector. To produce GmLBD16a overexpression lines, we constructed the coding sequence of GmLBD16a into an overexpression vector pROKII. Subsequently, the plasmids containing the sgRNAs and overexpressed GmLBD16a were introduced into W82 through cotyledon node transformation method. Different transgenic lines were obtained by Sanger sequencing. The primers were listed in Supplementary Data 1.

Salt treatment dosage assay

For the nodulation phenotype assay of W82 under salt stress condition, seven-day-old seedlings germinated in sterilized vermiculite were treated with 50 mL of NaCl solutions at concentrations of 50 mM, 75 mM, 100 mM, and 150 mM per plant bowl. Approximately four hours after salt treatment, the seedlings were inoculated with B. diaefficiens strain USDA110 (OD₆₀₀ = 0.05), which was diluted in a low nitrogen nutrient solution containing 50 μM nitrogen (following the nutrient solution formulation described in ref. ³⁸). The plants were subjected to the NaCl treatments and USDA110 inoculation weekly. Control plants (0 mM NaCl) received an equal volume of low nitrogen nutrient solution without NaCl. After 28 days of treatment with USDA110, the number of nodules formed on the roots was quantified.

RNA sequencing

For RNA sequencing analysis, seedlings were grown on vermiculite. Following a growth period of 28 days, plants were treated with 150 mM NaCl for 2 h. The control plants were treated with equal volume low nitrogen nutrient solution. At the designated sampling time point, three biological replicates were collected and quickly immersed in liquid N₂ before total RNA extraction. The whole transcriptome sequencing was performed by OE Biotech Co., Ltd. (Shanghai, China). Differential expression analysis was performed using the DESeq2 R package. P < 0.05 and log₂FC (fold change) >5 were set as the threshold for significantly differential expression.

Average dry weight of root nodules statistical assays

The fresh nodules were collected after rhizobia treatment for 28 days in absorbent paper. After the surface moisture was absorbed, the nodules were dried in a 60 °C oven until the weight no longer decreases. The total dry weight of nodules for W82 and GmERF13 CRISPR/Cas9 mutants was weighed, and the number of nodules for each plant was counted. The total dry weight of nodules/number of nodules was calculated to represent average dry weight of root nodule.

Nitrogenase activity

Fresh nodules, harvested after 28 days of rhizobia treatment, were placed in sealed anaerobic tubes. A volume of 2 mL of air was extracted from each tube using a syringe, followed by the addition of 2 mL of acetylene. The tubes were then incubated at 30 °C for 6 hours, after which the reaction was terminated by immersing the tubes in ice water.

Relative ethylene production, indicating nitrogenase activity, was measured using a gas chromatograph (GC; Agilent 7890, USA) equipped with a flame ionization detector (FID) and a PLOT/Q capillary column (30 m × 0.53 mm × 40 μm). Nitrogen was used as the carrier gas at a flow rate of 2.0 mL/min. The FID was maintained at 250 °C, with hydrogen and air supplied at flow rates of 40 mL/min and 450 mL/min, respectively. The method for calculating ethylene production and nitrogenase activity follows a previously reported protocol⁶⁵. For each sample, 0.2 g of fresh nodules were collected for analysis, with 10 parallel samples used per treatment in each experimental repeat. To assess nitrogenase activity under salt stress conditions, fresh nodules from plants treated with or without 75 mM NaCl for 28 days were analyzed using the same measurement protocol.

Salt stress phenotype analysis

For the nodulation phenotype assay under salt stress conditions, seven-day-old seedlings or hairy roots cultured in sterilized vermiculite were treated with 75 mM NaCl per plant bowl. Approximately four hours later, plant materials were inoculated with B. diaefficiens strain USDA110 (OD₆₀₀ = 0.05) diluted with low nitrogen nutrient solution containing 50 μM N. The plant materials were treated with 75 mM NaCl and USDA110 (OD₆₀₀ = 0.05) once a week. The control of W82, transgenic material plants, and hairy roots (0 mM NaCl) were treated with equal volume low nitrogen nutrient solution. The phenotypes were evaluated at 28 dpi. To detect the expression of GmERF13s in NaCl, ABA, and NaCl with ABA inhibitor, seven-day-old seedlings were treated with low nitrogen nutrient solution containing 150 mM NaCl, 10 µM ABA, and 150 mM NaCl with 100 µM ABA inhibitor (Fluridone) for 0 h, 6 h, 12 h, and 24 h. RNA extraction and RT-qPCR were followed.

RNA extraction and RT-qPCR

Inoculated roots, uninoculated roots, NaCl treated roots, ABA treated roots, NaCl and ABA inhibitor treated roots and nodules all are obtained from plants for gene expression analysis. Total RNAs of the plant materials were extracted by total RNA Extraction Kit (TIANGEN). cDNAs were reversed by 4× gDNA Wiper Mix (Vazyme) and 5× Hiscript® qRT SuperMix II (Vazyme). RT-qPCR analysis of three biological replicates was carried out by ABclonal 2× Universal SYBR Green Fast qPCR Mix and Bio-Rad CFX96 system. GmActin expression was used as an internal reference.

Vector construction

For overexpression constructs, the coding sequences of GmERF13s, GmLBD16a/b, GmEXP17s, and GmABI3/4/5 were amplified and cloned into pSuper 1300 (pS1300) vector using ApaI and SpeI. The pGES401 vector was used as template to construct four sgRNAs vector of GmERF13bc^CR double mutant and three sgRNAs vector of GmERF13s^CR single mutants, GmABI3^CR, GmABI4^CR, GmABI5ab^CR, GmABI5bc^CR, and GmABI5c^CR by Golden Gate reaction using BsaI. For complementation assay in hairy roots, the promoters (2000 bp upstream of ATG) and coding sequences of GmERF13s were amplified and cloned into pTF102 using BamHI and EcoRI. RS300 (miR319a pBSK) was used for artificial microRNA construction. The microRNA fragments were amplified and cloned into pS1300 vector using ApaI and SpeI. For genetic relationship analysis, the two fragments were amplified and cloned into pS1300-transformed vector by a single restriction enzyme digestion site. The two enzyme digestion sites were HindIII before MYC label and SalI before FLAG label, respectively.

Hairy root transformation

Plump seeds were sowed in vermiculite for germination. Four days later, the hypocotyls were cut off with scissors and applied with Agrobacterium rhizogenes K599 containing the indicated constructs in the wounds. Subsequently, the seedlings were transferred to plates immersed in water and left to cultivate overnight in darkness. On the following day, the seedlings were moved to holes filled with moist vermiculite and pruned the adventitious roots on the hypocotyl.

GUS staining analysis

Hairy roots and nodules were used for promoter GUS staining assays. For GUS staining experiments of salt treatment, roots and nodules treated with B. diaefficiens strain USDA110 (OD₆₀₀ = 0.05) for 21 days were collected after 150 mM NaCl treatment. For GUS staining experiments of root treatment with rhizobia, roots were collected after USDA110 (OD₆₀₀ = 0.05) treatment for a specific time. The clean roots and nodules were fixed with 4% polyformaldehyde (PFA) for 15 min. Samples were incubated in 5-bromo-4-chloro-3-indoyl-β-D-glucuronic acid (X-Gluc) solution (50 mM NaH₂PO₄•2H₂O, 50 mM Na₂HPO₄•12H₂O, 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄[Fe(CN)₆]•3H₂O, 10 mM Na₂EDTA•2H₂O, 0.1% Triton X-100, and 2 mM X-Gluc) for 6-8 h at 37°C. After staining, the roots and root segments containing nodules were washed by 1× phosphate buffer saline (PBS) buffer (Coolaber, Catalog number SL61102, Beijing, China). The roots were observed by a microscope (Olympus BX53, Tokyo, Japan) immediately while root segments containing nodules were successively incubated in 10% sucrose solution with 1% PFA, 20% sucrose solution with 1% PFA, and 30% sucrose solution with 1% PFA for 20 min at 4 °C. The materials were subsequently embedded in low melting agarose (Coolaber, Catalog number CA1351, Beijing, China) and 50 µm sections were prepared using Leica VT1000S microtome before microscope observation.

Phylogenetic analysis

The coding sequences of Glycine max, Medicago truncatula, Lotus japonicus, and Arabidopsis thaliana were obtained from Phytozome (https://phytozome-next.jgi.doe.gov/), SoyBase (https://www.soybase.org/sgn/), and Tair (https://www.arabidopsis.org/). MEGAX software was used to construct evolutionary tree by Neighbor Joining Method.

DNA extraction and identification of transgenic materials

Firstly, the plant materials were ground in liquid N₂ to powder states. CTAB extraction buffer (100 mM, Tris-HCl [pH 8.0], 4 M NaCl, and 20 mM EDTA) was added into the powder and incubated at 65 °C for 30 min. Chloroform: isopentanol (24:1) was used for DNA extraction. After Centrifuge at 13,000 × g for 15 min, anhydrous ethanol was used for precipitation of DNA and 75% ethanol was used for washing precipitation. For identification of CRISPR/Cas9 transgenic plants GmERF13s^CR, GmABI5bc^CR, GmLBD16a overexpression lines, and hairy roots GmABI3/4/5^CR, the fragments containing sgRNAs were amplified and sequenced. For identification of CRISPR/Cas9 transgenic plants, except for fragments containing sgRNAs amplification, bar gene was also amplificated to identify the presence or absence of the vector. The primers were listed in Supplementary Data 1.

Yeast two-hybrid assay

For Y2H assay, the coding sequences of proteins to be verified for interaction were amplified and cloned into pGBKT7-DEST (BD) and pGADT7-DEST (AD) by NdeI and NcoI, respectively. Y2H assays were carried out according to GAL4 Two-hybrid system (Clontech, Mountain View, CA). Yeast Extract Peptone Dextrose Medium (YPD) culture medium with adenine hemisul fate, SD/-Leu/-Trp (SD-LW) culture medium, TE/LiAc, and PEG4000/LiAc were used for the preparation of yeast receptive state. Bait plasmid, prey plasmid, carrier DNA, and PEG3350 were used for yeast transformation. DMSO was used as a conversion-promoting agent. 3-Amino-1, 2, 4-triazole (3-AT) was used to inhibit self-activation in SD/-Leu/-Trp/-His/-Ade (SD-LWHA) culture medium. Primers used for Y2H assays are listed in Supplementary Data 1.

Yeast one-hybrid assay

For Y1H assay, the coding sequences of proteins were amplified and cloned into pGADT7-DEST (AD) by NdeI and NcoI and the sequences of the target promoters (2000 bp upstream of ATG) were introduced to pHIS2.1 by EcoRI and SmaI, respectively. Yeast transformation method process was basically consistent with Y2H assay except SD/-Leu/-Trp/-His (SD-LWH) culture medium used in interactions. Primers used for Y1H assays are listed in Supplementary Data 1.

BiFC assay

The coding sequences of interaction proteins were amplified and cloned into pUC-SPYNE and pUC-SPYCE by BamHI and SalI, respectively. The constructs were transformed into Agrobacterium tumefaciens GV3101 for the transformation of tobacco leaf cells. Fluorescence signals were obtained laser scanning confocal microscope (Zeiss LSM 880, Germany). 4′, 6-diamidino-2-phenylindole was used as a nuclear dye. Lower epidermis leaves were teared off and stained in dark conditions for 5 min before observation. Primers used for BiFC assay are listed in Supplementary Data 1.

Co-IP

The GmERF13-GFP and GmLBD16a-MYC constructs were co-expressed in tobacoo leaf cells. Total proteins were extracted by protein extraction buffer (50 mM Tris pH = 7.5, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 0.1% TritonX-100, 5 mM DTT, 1 mM PMSF, and 1× protease inhibitor cocktail) and incubated with GFP-Trap® Magnetic Agarose beads (Chromo Tek, Catalog number ytma, 1:100 dilution, Germany)⁶⁶ for 1.5 h–2 h at 4 °C. Protein extraction buffer was used to wash beads five times. The proteins eluted with SDS loading buffer were denatured in boiling water for 8 min before western bloting and analyzed by immunoblotting. Anti-MYC antibody (ABclonal, Catalog number AE010, 1:5,000 dilution, Boston, MA, USA) was used to detect the levels of GmLBD16a-MYC.

ChIP-qPCR assay

For the ChIP-qPCR assays, hairy roots were inoculated with B. diazoefficiens strain USDA110 (OD₆₀₀ = 0.05). After 10 dpi, materials were fixed with 1% formaldehyde for 30 min and treated with 0.125 M glycine under vacuum. The materials were ground thoroughly in liquid N2 to powder states, and the chromatin was sonicated to fragments with various sizes (250 bp–500 bp). Anti-MYC-tag mAb-Magnetic Beads (M047-11; MBL, Nagoya, Japan) and Anti-GFP-tag mAb-Magnetic Beads (D153-11; MBL, Nagoya, Japan) were used for immunoprecipitation. Empty vector (pS1300) and GmLBD16a^OX were used to be controls. GmActin expression was used as an internal reference. The primers used in this experiment were listed in Supplementary Data 1.

LUC assay in soybean protoplasts

Soybean protoplasts isolation and protoplast transformation were consistent with a protocol described previously⁶⁷. Seedlings were grown for two weeks. Fresh leaves were cut into strips and incubated in 1.0% cellulase with 0.4% pectolase solutions for 6 h. 20% PEG4000 and 10 µg plasmid DNA were incubated for 15 min at 25 °C. 12 h–15 h later, the dual-LUC assays were performed using the Dual Luciferase Reporter Assay Kit (DL101-01; Vazyme, Nanjing, China) as instructed. The luciferase activities were detected by microporous plate luminescent instrument. Relative luciferase activity was determined based on the ratio of LUC/REN.

Statistical analysis

RNA-seq data, phenotypic data, and other statistical data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA). Statistical significance was performed by ANOVA with Student’s t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001), the least significant difference, or Duncan test. The statistical test was indicated in the figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data generated in this study have been deposited in the SRA database under accession codes PRJNA1100712 and PRJNA1164464. Source data for Figures and Supplementary Figs. are provided as a Source Data file. Source data are provided with this paper. Sequence data from this article can be found in the TAIR, Phytozome, SoyBase, and NCBI databases under the following accession numbers: AtERF13 (AT2G44840), MtERF13a (Medtr8g006825), MtERF13b (Medtr8g006815), GmERF13a (Glyma.03G112700), GmERF13b (Glyma.07G114000), GmERF13c (Glyma.09G242600), GmERF13d (Glyma.18G252400), AtLBD16 (AT2G42430), GmLBD16a (Glyma.03G161400), GmLBD16b (Glyma.19G162900), GmLBD16c (Glyma.13G121300), GmENOD40 (Glyma.01G028500), GmENOD11 (Glyma.09G092700), AtEXP17 (AT4G01630), GmEXP17a (Glyma.20G089100), GmEXP17b (Glyma.10G140200), GmEXP17c (Glyma.03G225900), GmEXP17d (Glyma.19G222900), GmABI3a (Glyma.08G357600), GmABI3b (Glyma.18G176100), GmABI4 (Glyma.02G264700), GmABI5a (Glyma.19G194500), GmABI5b (Glyma.13G153200), and GmABI5c (Glyma.10G071700).

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Acknowledgements

This work was supported by the National Key Research and Development Program (2022YFD1201702), by the Taishan Scholar Special Project Fund of Shandong Province to Z.D., and by the Intramural Joint Program Fund of State Key Laboratory of Microbial Technology (Project SKLMTIJP-2024-11). The authors thank Haiyan Yu, Xiaomin Zhao, Yuyu Guo, and Sen Wang from Core Facilities for Life and Environmental Sciences of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for assistance in microscopy imaging of Laser scanning confocal microscopy analysis. Thanks to Yiteng Xu from the Core Facility and Service Platform at School of Life Sciences, Shandong University for the assistance provided in laser scanning confocal microscopy imaging.

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The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education; Shandong Key Laboratory of Precision Molecular Crop Design and Breeding; School of Life Sciences, Shandong University, Qingdao, Shandong, China
Xinfang Zhu, Xifeng Yan, Weijun Li, Mengyue Zhang, Junchen Leng & Zhaojun Ding
College of Life Sciences, Liaocheng University, Liaocheng, Shandong, China
Qianqian Yu & Like Liu
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China
Dawei Xue
College of Agriculture, and State Key Laboratory of Crop Biology, Shangdong Agricultural University, Taian, Shandong, China
Dajian Zhang

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Xinfang Zhu
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Xifeng Yan
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Weijun Li
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Junchen Leng
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Z.D., X.Z., and D.Z. planned and designed the experiments. X.Z., X.Y., W.L., M.Z., J.L., Q.Y., L.L., and D.X. performed the major experiments; Z.D. and X.Z. wrote the paper.

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Correspondence to Dajian Zhang or Zhaojun Ding.

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Zhu, X., Yan, X., Li, W. et al. GmERF13 mediates salt inhibition of nodulation through interacting with GmLBD16a in soybean. Nat Commun 16, 435 (2025). https://doi.org/10.1038/s41467-024-55495-1

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Received: 30 May 2024
Accepted: 15 December 2024
Published: 06 January 2025
DOI: https://doi.org/10.1038/s41467-024-55495-1

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