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Article

Analysis of GmERF5 Response to the Rhizobial Type III Effector NopAA Underlying the Nodule in Soybeans

by
Lianheng Xia
1,†,‡,
Yunshan Song
1,†,
Tong Yu
1,
Ying Pei
1,
Hongwei Jiang
2,
Qingshan Chen
1 and
Dawei Xin
1,*
1
Key Laboratory of Soybean Biology of the Chinese Ministry of Education, Key Laboratory of Soybean Biology and Breeding, College of Agriculture, Northeast Agricultural University, Harbin 150036, China
2
Jilin Academy of Agricultural Sciences, Soybean Research Institute, Changchun 130033, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
Current address: Department of Peripheral Vascular Diseases, First Affiliated Hospital, Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China.
Nitrogen 2025, 6(2), 38; https://doi.org/10.3390/nitrogen6020038
Submission received: 15 April 2025 / Revised: 13 May 2025 / Accepted: 14 May 2025 / Published: 21 May 2025
(This article belongs to the Special Issue Microbial Interactions with Plants: Advancing Nitrogen Fixation, Uptake, and Utilization)
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Abstract

:
Soybean, an important leguminous crop valued for its high protein and oil content, obtains most of its nitrogen through symbiotic fixation processes. The symbiosis between soybeans and rhizobium can provide sufficient nitrogen for soybean growth. However, the signaling pathways underlying the establishment of the symbiosis are not so clear, especially the rhizobial type III effector-induced host response. In this study, we found that the single mutant HH103 nopAA::kan significantly affected the nodule number in soybeans. To further demonstrate the NopAA-triggered response in soybeans. Initial quantitative real-time PCR (qRT-PCR) tests showed that NopAA affects the expression of the soybean gene GmERF5, which was significantly upregulated upon inoculation with HH103 nopAA::kan, acting as a positive regulator of nodulation. The direct interaction between NopAA and GmERF5 was confirmed through yeast-two hybrid analysis. Furthermore, overexpression of GmERF5 in hairy roots indicated that GmERF5 may underlie the nodule phenotype of soybeans in response to NopAA. These findings provide new insights into the mechanisms by which soybean genes respond to rhizobial type III effectors to regulate symbiosis.

1. Introduction

Soybean, a member of the Fabaceae family, is the most important legume oilseed crop for many people around the world [1]. Integrating grain legumes into cropping systems improves soil fertility and increases household incomes [2]. In the quest for higher soybean yields, excessive amounts of chemical nitrogen fertilizer have been applied to fields. However, the continuous increase in chemical nitrogen application rates and the inappropriate use of fertilizers have resulted in reduced fertilizer use efficiency over time, causing significant environmental problems such as the eutrophication of water bodies and groundwater pollution. There is an urgent need to address the deterioration of the agro-ecological environment [3]. Therefore, it is crucial to find a sustainable and environmentally friendly method of nitrogen fertilization. Biological nitrogen fixation (BNF) is the main source of nitrogen (N) for soybean growth in intensive agriculture, and nitrogen fertilizer, of which 40−80% is derived from BNF, will directly affect the yield and nutritional quality of soybeans [4,5]. BNF is also considered an effective approach to minimizing carbon emissions from agriculture by reducing the use of energy-intensive nitrogen fertilizers [6]. Soybean, as a typical legume, forms nodules through interaction with compatible rhizobia. The nitrogen-fixing rhizobia in these nodules help legumes thrive under nitrogen limitation, reducing farmers’ reliance on nitrogen fertilizers for legume crops [4,7,8].
Signals from rhizobia and host cultivars regulate all stages of the legume–rhizobium symbiosis [9]. Secreted chemicals play a crucial role in early communication. Flavonoids from plant roots induce the production of rhizobial nodulation factors, which serve as signaling molecules recognized by the plant host [10,11,12,13]. Upon recognition, root hairs curl to capture the bacteria, and localized hydrolysis of the root hair cell wall allows the bacteria to enter plant root cells via an infection thread [14,15]. The formation of the infection thread stimulates the expression of genes involved in symbiotic signaling between the rhizobia and the host, resulting in nodule formation. During the establishment of some legume–rhizobium symbioses, the type III secretion system (T3SS) of the rhizobium plays a critical role by directly delivering type III effectors into eukaryotic host cells, thereby influencing the plant immune recognition process and, consequently, the nodulation ability of plants. The study of type III effectors is therefore a major focus in symbiosis. Host-specific nodulation may also rely on nodulation outer proteins (Nops) secreted by the rhizobial T3SS [16]. NopAA, identified as a glycoside hydrolase belonging to the GH12 family, is one such effector [17]. As a type III effector, NopAA can be induced by flavonoids, and its expression is regulated by TtsI. It can further influence nodule numbers by shaping the number of infection threads, differentially impacting the nodulation of soybean cultivars with distinct genetic characteristics [18]. However, the gene response to NopAA in legume hosts is poorly understood.
The phytohormone ethylene, found in all higher plants, plays a crucial role in modulating normal plant growth and development, including seed dormancy, fruit ripening, flower and leaf senescence, and plant responses to environmental cues [19,20,21]. In addition to regulating various aspects of plant growth, ethylene is involved in defense and symbiotic programs induced by bacteria [22,23], and is known to inhibit the rhizobia-induced nodulation process [24]. In recent years, compelling experimental evidence has demonstrated that ethylene is a master regulator of nodulation that affects multiple hormonal signaling pathways to regulate every step of the process, including rhizobial infection, nodule organogenesis, and nodule senescence. Despite great progress in understanding the involvement of ethylene in nodulation, genetic evidence for the role of ethylene signaling comes mainly from functional analyses of loss-of-function mutants of Arabidopsis EIN2 orthologs. However, the roles of ethylene in rhizobia–soybean interactions and nodule development in soybeans are unclear [19].
Ethylene response factors (ERFs) are encoded by one of the largest families of plant transcription factors. Acting at the end of the ethylene signaling pathway, they are probably located in the most suitable position to regulate the diversity and specificity of different aspects of the ethylene response [25]. To date, ERF family transcription factors have been shown to coordinate with each other and regulate the developmental, physiological, and biochemical responses of plants to a variety of environmental stress conditions, including those that occur in combination with other abiotic and biotic stresses [26], which have revealed critical roles for transcription factors during rhizobial infection and/or nodule organogenesis in legumes [27]. In Medicago truncatula, EFD (for ethylene response factor required for nodule differentiation) belonging to ERF group V plays a key role in the processes of plant and bacteroid differentiation taking place beneath the nodule meristem [28]. In addition, MtEFD and MtEFD2 were also found to be key regulators of nodule numbers. However, the function of ERF in soybeans has not yet been uncovered clearly. In particular, the interaction between ERF and type III effectors was elucidated poorly. As there are no type III effectors that trigger the signaling pathway in Medicago truncatula, this interaction exists between soybeans and rhizobium interactions. Therefore, the ERF appears to play essential roles in promoting various processes necessary for the initiation of nitrogen-fixing symbiosis. To further understand the functions of ERF in the activation of the diverse processes associated with BNF and nodule functions, we thoroughly investigated the expression pattern of the transcription factors-related and the hormone-related genes in response to rhizobium and its derived T3SS mutant. We determined that the transcription factors and hormone-related genes responded to NopAA at a specific stage.
In this study, we found that the expression of GmERF5 was affected by NopAA and the nodule number can be induced by NopAA and GmERF5. NopAA can interact with GmERF5 directly. We therefore propose that the symbiotic functions of GmERF5 may be mediated by NopAA through a modulation of the ethylene pathway.

2. Materials and Methods

2.1. Function Domain Prediction of Candidate Gene GmERF5

An analysis of the candidate gene GmERF5 (Glyma.11G036400) sequence was conducted with the Phytozome database (https://phytozome-next.jgi.doe.gov/search-results/f2d88ddc-cf52-4be7-a266-42ca5c476718, accessed on 16 February 2024). A prediction of the GmERF5 protein structure was performed using NCBI database.

2.2. Construction of Rhizobial nopAA Mutants

Sinorhizobium fredii HH103 (hereafter HH103), Escherichia coli DH5α, and their mutants were employed in this study. First, nopAA containing an inserted Kanamycin antibiotic (Km) resistance gene was cloned and ligated into the vector pJQ200sk to generate pJQ200sk-nopAA-Km, which was transformed into E. coli DH5α at 16 °C for 16 h after ligation. Further, E. coli-carrying pJQ200sk-nopAA-Km, helper strain, and HH103 were mixed in a 1:1:3 ratio following a previously described tri-parental mating protocol [29]. Gene replacement was performed through selection for antibiotic resistance at a concentration of 50 ng/L and growth on 5% (w/v) sucrose [30]. The obtained mutant, HH103 nopAA::kan, was confirmed by PCR analysis (Figure 1) [18].

2.3. qRT-PCR Analysis of GmERF5 Expression

The soybean cultivar Suinong 14 (SN14) was used for nodulation assays following Cl2 sterilization and growth in sterile vermiculite under controlled conditions (12 h photoperiod, 28 °C) with B&D nitrogen-free nutrient solution [31]. Mid-log phase cultures (OD600 = 0.4) of wild-type S. fredii HH103 and its isogenic nopAA mutant were used to inoculate Vc-stage seedlings. Quantification of nodules at 25 dpi was performed in three biological replicates (n = 20 plants per replicate), with statistical analysis by one-way ANOVA. Gene expression profiling was performed by (TB Green™ Fast Mix, Takara Bio, Nanjing, China) using GmUKN1 (Glyma.12G020500) as the reference gene, with technical triplicates and negative controls [32].

2.4. Yeast Two-Hybrid Analysis

The yeast two-hybrid assay was performed to determine the interactions between the GmERF5 protein and the type III effector NopAA protein. Expression vectors pGADT7-GmERF5 and pGBKT7-GmERF5, as well as pGADT7-GmERF5 and pGBKT7-GmERF5 were constructed from the entry vectors pGWC-NopAA and pGWC-GmERF5 using the Gateway™ LR (Invitrogen, Carlsbad, CA, USA), reaction. The plasmids were extracted, and their construction was verified through PCR amplification and sequencing. The experimental group plasmid pGBKT7-NopAA, positive control plasmid pGBKT7-53, and the negative control plasmid pGBKT7-GmERF5 were transformed into yeast AH109 using the transformation method described above. Then, they were plated on SD/-Trp plates. At the same time, mating the plasmid pGADT7-GmERF5, the positive control plasmid pGADT7-Rect and the negative control plasmid pGADT7-NopAA were transformed into yeast Y187 and plated on SD/-Leu plates. The plates were incubated inversely at 30 °C for 3–5 days for the growth of the transformed yeast cells. Monoclones greater than 2 mm in diameter were harvested and placed in 0.5 mL of Yeast Peptone Dextrose (YPD) liquid medium. After complete resuspension and mixing, they were paired overnight and shaken at 200 r/min at 30 °C. The next day, 100 µL of mating products was applied to culture plates. After open-air drying, the transformed yeast cells were cultured at 30 °C for 3–5 days for the observation of their growth [33].

2.5. Hairy Root Transformation and Detection of Positively Transformed Hairy Roots

The Fu28 entry vector was recombined with the green fluorescent protein (GFP)-tagged pSOY1 expression vector through the Gateway™ LR reaction to produce the pSOY1-GmERF5 plasmid [34]. After sequencing verification, the extracted plasmid was transferred into Agrobacterium tumefaciens K599 via heat shock transformation or electroporation [35]. The Agrobacterium cultures were then used to infect the hypocotyls of cultivar SN14 by wounding them with a syringe needle dipped in the bacterial cultures. The nodule phenotype was assessed 25 days post-inoculation with rhizobia and the derived nopAA mutant separately [36].

3. Results

3.1. Predictive Analysis of GmERF5 Protein Structural Domains

The bioinformatic analysis demonstrated that GmERF5 is an intronless single-copy gene spanning 995 bp on chromosome 11, containing an 831 bp open reading frame that encodes a 276-residue protein (predicted MW: 69.23 kDa). Protein structure prediction identified a canonical AP2/ERF domain (amino acids 138–196), consistent with its putative function in mediating ethylene response signaling through DNA-binding activity (Figure 2).

3.2. Nodule Capacity Identification of Mutant HH103 nopAA::kan

The wild-type S. fredii HH103 and the derived single mutant HH103 nopAA::kan were inoculated into SN14 to assess nodule capacity. After 25 days of inoculation, the number of nodules, nodule dry weight, fresh weight, and nodule size were measured. The single mutant HH103 nopAA::kan exhibited a decrease in nodule number and showed significant differences compared to HH103 in terms of nodule number, fresh weight, and the number of small nodules. The differences in nodule dry weight and the number of large nodules were also significant. These results indicate that NopAA positively affects the nodulation phenotype in SN14. (Figure 3).

3.3. GmERF5 Responses to NopAA

Ethylene response factor 5 (ERF5) is involved in the chitin-induced innate immune response and plays a critical regulatory role in plant responses to both biotic and abiotic stresses. A novel ERF transcription factor, GmERF5, has been identified in soybeans. We confirmed the interaction between the type-III effector NopAA and the protein encoded by the GmERF5 gene using a yeast two-hybrid assay.
To investigate the regulatory influence of NopAA on GmERF5 expression, qRT-PCR was conducted using GmUKN1 as the reference gene. The expression analysis revealed distinct temporal regulation patterns of GmERF5 in response to different rhizobial strains. At 12 h post-inoculation (hpi), GmERF5 transcript levels were higher in plants inoculated with HH103 nopAA::kan compared to wild-type HH103. This expression pattern was reversed at 24 hpi, when wild-type HH103-inoculated plants showed significantly elevated GmERF5 levels relative to the mutant strain. The wild-type HH103 induced maximal GmERF5 expression at 24 hpi, demonstrating strain-specific temporal regulation of this transcription factor during symbiotic interactions (Figure 4a). These results provide further evidence for the ability of the NopAA effector to promote the expression of the GmERF5 gene.
To determine whether there is a direct interaction between NopAA and GmERF5, a self-activation assay was performed using the vectors pGBKT7-NopAA and pGBKT7-GmERF5. It was found that pGBKT7-GmERF5 was self-activating. Therefore, pGADT7-GmERF5 and pGBKT7-NopAA were used as the experimental group, with pGADT7-RecT and pGBKT7-53 serving as the positive control. The results showed that the two negative controls did not grow to clones at a dilution of 10−3, while both the experimental and positive controls grew to clones, demonstrating that GmERF5 interacts with NopAA in the yeast system (Figure 4b).

3.4. GmERF5 Effect on Nodule of Soybeans

To investigate the effect of the GmERF5 gene on soybean nodulation, soybean hairy roots with over-expressed GmERF5 were used to inoculate with S.fredii HH103 and derived mutant HH103 nopAA::kan. Significant differences in nodule number, nodule fresh weight, and nodule dry weight were observed when comparing HH103 nopAA::kan and HH103 (Figure 5). Hairy roots showed significantly less nodule formation after inoculation with HH103 nopAA::kan compared to the control plants SN14, while nodule number was decreased significantly when inoculating with HH103 nopAA::kan compared to inoculation with HH103. When GmERF5 was over expressed, the nodule number was increased compared to the wild type soybean SN14 (Figure 5b). nopAA mutant cannot induce more nodules than wild type HH103, even in wild type soybeans and GmERF5 over-expressed lines. The nodule number showed an increased phenotype in GmERF5 over-expressed lines by inoculating with HH103 nopAA::kan compared to wild type soybeans. These results support the role of GmERF5 in regulating the effect of NopAA on soybean nodulation, and GmERF5 acts downstream of the signaling pathway involved in NopAA.

4. Discussion

Host-specific nodulation in legumes, such as soybeans, is heavily influenced by Nops, which are secreted by the rhizobial T3SS. By constructing T3SS knockout mutants (such as NopA, NopB, or the regulatory gene ttsI) and performing nodulation assays, it was observed that most legumes exhibited either improved or reduced nodulation with these T3SS knockouts, indicating the affected competitiveness of strains in co-inoculation experiments [37]. In this study, the nodulation assay showed that the nodule number in soybeans inoculated with HH103 nopAA::kan was decreased, suggesting that NopAA functions as a positive regulator of nodulation. qRT-PCR analyses of SN14 and GmERF5 over-expressed plants were performed to identify the regulatory effect of NopAA on GmERF5. The upregulation of GmERF5 expression in SN14 indicated that NopAA promotes GmERF5 expression. Inoculation with the nopAA mutant led to a significant decrease in nodulation levels in both SN14 and GmERF5 over-expressed plants, possibly due to the over-expression of GmERF5 leading to different nodulation effects. Compared to SN14, GmERF5 over-expressed hairy roots had more nodules, demonstrating that GmERF5 promotes nodulation. This result is consistent with the function that MtEFD positively regulates the nodule number of Medicago truncatula [28]. However, the precise molecular mechanism through which NopAA influences soybean nodulation via GmERF5 interaction requires further experimental investigation.
An analysis of the interaction between effectors and GmERF5 revealed feedback regulation of GmERF5 expression by NopAA. NopAA was identified as a positive effector that promotes nodulation by increasing GmERF5 expression. NopAA primarily impacted the expression of regulatory genes like GmERF5, suggesting that it may influence defense response activity during the establishment of symbiosis. These findings provide new insights into how soybean symbiotic N fixation works, regulated by rhizobial type III effectors interacting with soybean proteins. To test these hypotheses, however, further experiments are essential. The construction of GmERF5 knockout soybean plants will allow experiments to directly confirm the functional role of GmERF5 in symbiosis, and screening for relevant genetic variations in different soybean populations could help breed soybean varieties with improved nitrogen-fixation efficiency. Meanwhile, the expression of GmERF family genes was induced by drought, salt, methyl jasmonate (MeJA), ethylene (ETH) and abscisic acid (ABA), and heat treatments. Field conditions should then be taken into account when breeding varieties [38,39].
In addition, NopAA belongs to the Xyloglucan hydrolase protein family, and contains the domain for these original structural properties, which, combined with the specificity of NopAA for xyloglucan, is a key component of the root cell wall which is also secreted by roots in the soil. This may put NopAA in a strategic position to participate in recognition between bacteria and plant roots and intervene in the nodulation process [40]. In this study, it was identified that NopAA can interact with GmERF5 in yeast. These results showed that NopAA poses multiple functions similar to other type III effectors of rhizobium [41,42]. Given GmERF5’s involvement in ethylene signaling, the functional consequences of NopAA–GmERF5 interactions on this pathway warrant further investigation. Based on our findings, we propose a model wherein NopAA is secreted into host cells via the T3SS and modulate nodulation through interaction with GmERF5 to regulate ethylene signaling (Figure 6). This mechanism provides a plausible explanation for the observed nodulation phenotypes.

5. Conclusions

We identified the defense-related gene GmERF5, which may respond to NopAA, an effector that plays a critical role in determining nodulation capacity and rhizobial interactions. GmERF5 can interact with NopAA in yeast and the over-expressed GmERF5 can affect the NopAA effect on soybean nodule formation. These results suggested that GmERF5 functions downstream of the NopAA signaling pathway.

Author Contributions

Conceptualization, D.X. and Q.C.; methodology, L.X.; software, Y.S.; validation, H.J.; formal analysis, Y.S.; investigation, T.Y.; resources, L.X.; data curation, Y.S.; writing—original draft preparation, Y.S.; writing—review and editing, D.X.; visualization, Y.S.; supervision, D.X. and Q.C.; project administration, Y.P.; funding acquisition, D.X. and Q.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (Grant numbers: 32070274, 31771882, 32072014, U20A2027, 31801389), the National Key Research and Development Program of China (2021YFF1001206), and High Oil and Yield Germplasm Development and Breeding Utilization (CZKYF2021-2-C009).

Data Availability Statement

Data are available upon request.

Acknowledgments

The authors are grateful for the funding received from the Opening Project from The Heilongjiang Province Postdoctoral Science Foundation (LBHZ24090), the National Natural Science Foundation of China (Grant numbers: 32070274, 31771882, 32072014, U20A2027, 31801389).

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. PCR identification analysis of HH103 nopAA::kan by primer Km-F and NopAA-R (a); PCR identification analysis of HH103 nopAA::kan by primer NopAA-F and NopAA-R (b). M for Trans 2K Plus DNA marker, lane 1–3 for HH103 nopAA::kan, lane 4–6 for the wild strain HH103.
Figure 1. PCR identification analysis of HH103 nopAA::kan by primer Km-F and NopAA-R (a); PCR identification analysis of HH103 nopAA::kan by primer NopAA-F and NopAA-R (b). M for Trans 2K Plus DNA marker, lane 1–3 for HH103 nopAA::kan, lane 4–6 for the wild strain HH103.
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Figure 2. The function domain prediction of candidate gene GmERF5.
Figure 2. The function domain prediction of candidate gene GmERF5.
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Figure 3. Nodule identification of different strains inoculated with SN14 on nodule number (a), nodule fresh weight (b), nodule dry weight (c), numbers of nodules > 2 mm in diameter and ≤2 mm in diameter (d,e), respectively. Significance was determined using ANOVA. ** indicates significant difference at p < 0.01; *** indicates significant difference at p < 0.001. Error bars represent mean ± SE for triplicates.
Figure 3. Nodule identification of different strains inoculated with SN14 on nodule number (a), nodule fresh weight (b), nodule dry weight (c), numbers of nodules > 2 mm in diameter and ≤2 mm in diameter (d,e), respectively. Significance was determined using ANOVA. ** indicates significant difference at p < 0.01; *** indicates significant difference at p < 0.001. Error bars represent mean ± SE for triplicates.
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Figure 4. qRT-PCR analysis of GmERF5 gene expression under different treatments. Expression levels were normalized to the reference gene GmUKN1 (Glyma.12G020500). Significance was determined using ANOVA. * Indicates a significant difference at p < 0.05; ** indicates a significant difference at p < 0.01. Error bars represent mean ± SE for triplicates (a). Yeast two-hybrid assay results shows the interaction between NopAA and GmERF5 (b).
Figure 4. qRT-PCR analysis of GmERF5 gene expression under different treatments. Expression levels were normalized to the reference gene GmUKN1 (Glyma.12G020500). Significance was determined using ANOVA. * Indicates a significant difference at p < 0.05; ** indicates a significant difference at p < 0.01. Error bars represent mean ± SE for triplicates (a). Yeast two-hybrid assay results shows the interaction between NopAA and GmERF5 (b).
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Figure 5. The GmERF5 gene hairy root transformation and nodule identification. Positive soybean hairy roots with GFP-tag under bright field and GFP fluorescence (a). Quantitative analyses corresponding to nodule number (b); nodule fresh weight (c); nodule dry weight (d); numbers of nodules > 2 mm in diameter and ≤2 mm in diameter (e,f), respectively. Significance was determined using ANOVA. ** indicates a significant difference at p < 0.01; *** indicates a significant difference at p < 0.001. Error bars represent mean ± SE for triplicates.
Figure 5. The GmERF5 gene hairy root transformation and nodule identification. Positive soybean hairy roots with GFP-tag under bright field and GFP fluorescence (a). Quantitative analyses corresponding to nodule number (b); nodule fresh weight (c); nodule dry weight (d); numbers of nodules > 2 mm in diameter and ≤2 mm in diameter (e,f), respectively. Significance was determined using ANOVA. ** indicates a significant difference at p < 0.01; *** indicates a significant difference at p < 0.001. Error bars represent mean ± SE for triplicates.
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Figure 6. Schematic diagram illustrating the interaction between effectors and plant candidate genes.
Figure 6. Schematic diagram illustrating the interaction between effectors and plant candidate genes.
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MDPI and ACS Style

Xia, L.; Song, Y.; Yu, T.; Pei, Y.; Jiang, H.; Chen, Q.; Xin, D. Analysis of GmERF5 Response to the Rhizobial Type III Effector NopAA Underlying the Nodule in Soybeans. Nitrogen 2025, 6, 38. https://doi.org/10.3390/nitrogen6020038

AMA Style

Xia L, Song Y, Yu T, Pei Y, Jiang H, Chen Q, Xin D. Analysis of GmERF5 Response to the Rhizobial Type III Effector NopAA Underlying the Nodule in Soybeans. Nitrogen. 2025; 6(2):38. https://doi.org/10.3390/nitrogen6020038

Chicago/Turabian Style

Xia, Lianheng, Yunshan Song, Tong Yu, Ying Pei, Hongwei Jiang, Qingshan Chen, and Dawei Xin. 2025. "Analysis of GmERF5 Response to the Rhizobial Type III Effector NopAA Underlying the Nodule in Soybeans" Nitrogen 6, no. 2: 38. https://doi.org/10.3390/nitrogen6020038

APA Style

Xia, L., Song, Y., Yu, T., Pei, Y., Jiang, H., Chen, Q., & Xin, D. (2025). Analysis of GmERF5 Response to the Rhizobial Type III Effector NopAA Underlying the Nodule in Soybeans. Nitrogen, 6(2), 38. https://doi.org/10.3390/nitrogen6020038

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