Characterization of GmABI3VP1 Associated with Resistance to Soybean Cyst Nematode in Glycine max

Abstract

The ABI3 transcription factor is a key regulator in plant growth and development. Through transcriptome analysis of the resistant soybean cultivar ‘Dongnong L10′ and the susceptible cultivar ‘Heinong 37′ exposed to soybean cyst nematode race 3 (SCN 3) stress, the differentially expressed gene GmABI3VP1 was identified. The GmABI3VP1 gene was then cloned and analyzed through bioinformatics, subcellular localization, and qRT-PCR analysis of resistant and susceptible soybean germplasms, as well as overexpression and gene editing of soybean hairy roots followed by SCN 3 identification analysis. It was found that the protein encoded by GmABI3VP1 is an acidic and hydrophilic protein with transmembrane domains. It has a collinear relationship with Arabidopsis and is widely distributed in plants. Through the analysis of promoter elements, it was shown that this gene contains multiple hormone-responsive promoter elements like ABRE/ABRE3a/ABRE/4a/as-1 and stress-responsive elements such as Myb/MYC/MYc. Transient expression in tobacco indicated that the GmABI3VP1 gene is located in the nucleus. The transcription of GmABI3VP1 responds to the stress of SCN, and its transcriptional level is relatively high in the roots of resistant materials. Genetic transformation mediated by Agrobacterium rhizogenes was used to obtain GmABI3VP1 gene overexpressed and CRISPR-Cas9 gene-edited soybean hairy roots. In comparison to the wild type (WT), the density of nematodes per area was notably lower in hairy roots overexpressing (OX) the gene, whereas the density of SCN per unit area (per cm of lateral root length) significantly increased in gene-edited (KO) soybean hairy roots. Through SCN phenotyping, GmABI3VP1 was identified as a contributor to SCN 3 resistance. This study provides initial insights into the role of the GmABI3VP1 gene in SCN resistance, establishing a robust basis for future research on the mechanisms underlying SCN disease resistance and offering valuable genetic reservoirs for SCN 3 resistance.

1. Introduction

The cultivation of soybeans (Glycine max (L.) Merr.) has been traced back to China, where they have been planted for over 5000 years [1]. It is classified under the genus Glycine and is an annual herbaceous plant. The objectives of modern crop breeders encompass achieving high yields, ensuring excellent quality, maintaining stable production, synchronizing the growth cycle, and adapting to the requirements of modern harvesting [2]. Elevating the per-unit yield and quality of soybeans constitutes the cardinal strategy for safeguarding soybean food security.

Soybean cyst nematode disease (Heterodera glycines Ichinohe, SCN) is widely distributed worldwide. The disease was first discovered in Japan in 1915 [3]. Subsequently, the disease was discovered in the central North American region of the United States. SCN has caused soybean yield losses of up to $1.2 billion [4]. In China, SCN is primarily distributed in the Northeast and Huanghuai regions. The disease exhibits differentiation into multiple physiological races. In the Huanghuai region, a major soybean-producing area in China, physiological races SCN 1 and SCN 4 are dominant. In the Northeast region, another major soybean-producing area, physiological races SCN 3 and SCN 4 are primarily found [5].

J₂ stage SCN causes the most severe damage to soybean root systems. SCN releases cellulase and pectinase through its mouthparts, dissolving the cell walls of plant roots and forming syncytia. Obtaining nutrients from the host soybean [5], leads to a significant reduction in soybean yield. Yields may decrease by 20% to 30%, and, in severe cases, result in complete crop failure. Currently, the primary measures for controlling SCN include crop rotation, SCN chemical agents, and the development of SCN-resistant varieties. In terms of crop rotation, the corn–soybean–corn rotation system is widely adopted. However, it cannot completely eradicate the SCN pathogen. Chemical agents can control the disease to some extent, but they have a significant impact on the surrounding soil ecosystem. Currently, the SCN antigens widely recognized by the scientific community include PI88788, PI54840 (Peaking), and PI437654 [6]. However, with the overuse of antigen resistance, the resistance of antigens gradually weakens, and SCN evolves into more potent physiological populations [7]. The pathogenicity of SCN populations was initially classified as physiological populations based on the responses of four soybean genotypes. Subsequently, seven soybean lines are identified with unique resistance through genotyping methods [8]. As SCN physiological populations continue to evolve, SCN populations have gradually overcome antigens such as PI88788. Therefore, it is crucial to continue screening for new resistant germplasm and genes from soybean germplasm resources, and to expand the genetic background of superior germplasm resources. Rgh1, located on chromosome 18, exhibits incomplete dominance [9], while Rgh4, located on chromosome 8, exhibits complete dominance [10,11]. Rgh1 and Rgh4 have been extensively validated by quantitative trait locus (QTL) studies and are widely recognized as the primary resistance loci for SCN resistance [12,13]. Many studies have focused on developing molecular markers at these loci. However, Rgh1 and Rgh4 can only explain 60% of the genetic variation [14], and the remaining portion still requires further exploration and development.

ABI transcription factors are widely distributed in plants such as soybeans, corn, wheat, tomatoes, and Arabidopsis thaliana [15,16]. This type of transcription factor contains four conserved domains, namely A, B₁, B₂, and B₃. Among them, the N-terminal A structure is rich in acidic residues; and, in the B₁ domain, there are essential elements necessary for the interaction with bZIP transcription factors [17]. The B₂ domain encompasses a putative nuclear-localization signal and is considered to play a crucial function in transcriptional regulation [18]. The B₃ domain is a highly conserved DNA-binding domain. It is widely involved in the regulation of seed development, participates in stress responses, and affects the growth and development of plants, among other aspects [19].

ABI3 is widely involved in plant abiotic stress and plays an important regulatory role under conditions such as temperature stress and dehydration stress. ABI3 mediates the dehydration stress response in non-seed plants and seed plants by regulating downstream genes [20]. When AtABI3 is absent in A. thaliana its recovery capacity under dehydration stress is impaired [21]. The development of seed coats in Brassica napus is regulated by BnABI3, which plays a crucial role in seed dehydration tolerance [22]. In response to dehydration stress, ABI3 inhibits the transcription of the RAV1 gene by suppressing the recruitment of RNA polymerase II. Under stress conditions, the downregulation of RAV1 gene expression induces a decrease in the expression of ethylene-insensitive 2 (EIN2) and ethylene receptor 1 (ETR1), thereby generating an effective dehydration stress response [23]. In A. thaliana, ABI3 transgenic plants exhibit enhanced cold tolerance under short-term ABA treatment, suggesting that the ectopic expression of the ABI3 gene may regulate cold-induced cold tolerance and confer higher cold temperature tolerance [24]. The transcription of genes such as SOS1, SOS2, SOS3 sodium/hydrogen ion exchanger, drought stress response 29B, abscisic acid insensitive protein 2, ABI3, MYB15, and δ-1-pyrrolidine-5-carboxylic acid synthase 1 is promoted by salt stress [25]. The ABI3VP1 protein possesses a B₃ DNA-binding domain and is widely involved in regulation during development and under adverse stress conditions [26].

In this study, RNA sequencing (RNA-Seq) was conducted on the root systems of highly resistant and susceptible soybean varieties. Analysis of the transcriptome data revealed the differentially expressed gene GmABI3VP1, which was identified between the resistant and susceptible root samples. Overexpression and gene editing vectors were constructed, and transgenic root systems were developed using Agrobacterium-mediated genetic transformation technology. By analyzing the gene expression patterns in both overexpressed and gene-edited roots, the role of this gene in response to SCN stress was established. Additionally, SCN disease resistance to SCN and measurements of root physiological parameters were conducted. This study aims to explore the function of the GmABI3VP1 gene and provide insights into the mechanisms underlying its role in SCN disease resistance.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The soybean materials used in the experiment included Dongnong 50 (DN50, SCN-susceptible varieties; it is a variety highly susceptible to SCN), Dongnong L10 (SCN-resistant varieties), Suinong14 (SN14,SCN-susceptible varieties), and Heinong 37 (HN 37,SCN-susceptible varieties). Y16 (SCN-resistant varieties; RIL_F15, Recombinant Inbred Line_F15, line 16). All soybean germplasm resources were planted in 2023 in China, Harbin City, Heilongjiang Province (45°45′16.2″ N, 126°54′39.6″ E) in Xiangyang Farm of Northeast Agricultural University. The field experiment was conducted in a randomized block design with 1 m row length, 0.65 m row width, 0.06 m plant spacing, and three replications.

2.2. Preparation of Sequencing Materials

Dongnong 50, which exhibits sensitivity to SCN 3, was sown in black pots to facilitate the propagation of SCN 3. Thirty days after sowing, the intact root systems of the plants were carefully extracted and subsequently rinsed with tap water at a slow flow rate. SCN 3 was isolated using 60-mesh sieves, and the SCN 3 retained on the sieves was meticulously and gently ground by hand. The 40-mesh sieve was then thoroughly rinsed with distilled water, and the collected J₄ eggs were transferred to 50 mL centrifuge tubes. After thorough mixing, a fraction of the cyst-containing liquid was retrieved. The J₄ eggs were observed under a microscope to determine their quantity, and the solution was adjusted to a final concentration of 1800 J₄ eggs per milliliter by appropriate dilution.

Dongnong L10 and Heinong 37 were, respectively, inoculated with SCN 3 to form the experimental groups, while those not inoculated with SCN 3 were set as the control groups. The indoor temperature was set at 25 °C, and the humidity was adjusted to 70%. At the V₂ stage of the seedlings, 2 mL of the soybean SCN 3 J₄ egg suspension was inoculated into each plant. The root tissues at 0 d and 14 d were collected and stained with acid fuchsin to ensure successful SCN 3 inoculation at 14 d. These root tissues were then used for preparing RNA-Seq sequencing samples.

2.3. RNA Extraction and cDNA Synthesis from Soybean Roots

The soybean variety Dongnong L10, which is resistant to the SCN, was planted in vermiculite. When the soybeans grew to the stage of the second pair of true leaves (V₂ stage), the vermiculite on the soybean roots was thoroughly rinsed off with clean water, and then 1.5 g of the soybean roots were cut off by a surgical scalpel. RNA from soybean roots was extracted by using the TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA). Use 2% agarose gel electrophoresis to detect the quality of the RNA.

RNA was reverse transcribed using the ReverTra Ace^® qPCR RT Master Mix (TOYOBO, Osaka, Japan). (1) A total of 4 μL of RNA was heat-denatured at 65 °C for 5 min. After denaturation, it was quickly inserted into crushed ice. (2) Add 2 μL of 4 × DN Master Mix, 2 μL of ddH₂O, and 4 μL of the denatured RNA. Remove DNA at 37 °C for 5 min and then take and place it into the denatured solution. (3) Take 2 μL of 5 × RT Master Mix II and add it into the denatured solution in Step 2. Incubate at 37 °C for 15 min, at 50 °C for 5 min, and at 98 °C for 5 min to form cDNA.

2.4. Transcriptome Sequencing and Analysis

In this study, transcriptome sequencing analysis was conducted using the Illumina HiSeq™ 2500 platform to examine the changes in gene expression levels related to synthesis and regulation between soybean varieties exhibiting extreme resistance and susceptibility to SCN. The analysis included both SCN-resistant (Dongnong L10) and SCN-susceptible (Heinong 37) soybean varieties, which were either infected with SCN or not infected.

The same quantity of root tissue from identical parts was collected from both the treatment group and the control group. Subsequently, the samples were rapidly frozen in liquid nitrogen and wrapped in aluminum foil. Total RNA was extracted using the TRNzol Universal kit (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA library was constructed with the assistance of the Illumina TruSeq RNA library preparation kit (Illumina, San Diego, CA, USA), and RNA-Seq sequencing of the cDNA was conducted on the Illumina HiSeq™ 2500. The visualization analysis of transcriptome data can be performed using the DESeq2 R package in Rstudio software V2025.05.1+513.pro3. The up-regulated and down-regulated genes in the transcriptome were utilized to screen for candidate genes through fold change analysis of differential expression and KEGG enrichment analysis. Consequently, the candidate gene GmABI3VP1, which exhibited differential expression under SCN 3 stress and non-stress treatments, was identified.

2.5. Fluorescence Quantitative PCR

The PrimerQuest™ Tool (https://sg.idtdna.com/PrimerQuest/Home/Index accessed on 1 May 2025) was employed for the design of quantitative primers targeting the GmABI3VP1 candidate gene (see Table S7). The housekeeping gene selected for this study was GmActin4 (GenBank accession number: AF049106). The PCR instrument utilized was the Gene ROCGENE quantitative real-time PCR system (Kunpeng, Beijing, China), and the operating software used was Archimed X6 (ver. 201905v1.08, Microvision Instruments, Evry, Île-de-France, France). For specific procedures, experiments were conducted following the operational guidelines provided with the TaKaRa fluorescent quantitative reagent TB Green kit (TaKaRa, Shiga Prefecture, Japan). Relative expression levels were calculated using the 2^−ΔΔCT method. CT values were determined by averaging three biological replicates.

2.6. Cloning of the GmABI3VP1 Gene

Cloning was performed using the cDNA extracted from the roots of Dongnong L10 as a template. A 20 μL reaction mixture was prepared, consisting of the following: 1 μL of cDNA, 10 μL of KOD One Plus (TOYOBO, Osaka, Japan), 1 μL of GmABI3VP1-F primer, 1 μL of GmABI3VP1-R primer (refer to Table S7), and 6 μL of ddH₂O. The PCR program was configured as follows: initial denaturation at 98 °C for 3 min; followed by denaturation at 98 °C for 10 s; annealing at 58 °C; extension at 68 °C for 5 s; and a further extension step at 68 °C for an additional 10 min. The denaturation–extension cycles were repeated for a total of 38 cycles before storing the samples at 4 °C. The position of the target band was assessed through electrophoresis on a gel containing agarose at a concentration of 2%.

2.7. Bioinformatics Analysis of Tools

The signal peptide of the protein encoded by GmABI3VP1 was predicted using the website (https://novopro.cn/tools/signalp accessed on 1 May 2025). The hydrophilicity and hydrophobicity of the protein were analyzed by Novopro 3.0 (https://www.novopro.cn/tools/protein-hydrophilicity-plot.html accessed on 1 May 2025). The transmembrane domains of the protein were analyzed by TMHMM (https://www.novopro.cn/tools/tmhmm.html accessed on 1 May 2025). The secondary and tertiary structures of the protein were analyzed by SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa%20_sopma.html accessed on 1 May 2025) and SWISS-MODEL (https://swissmodel.expasy.org/ accessed on 1 May 2025), respectively. The promoter elements were analyzed using the online website Plant CARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 1 May 2025). Phosphorylation sites were analyzed using the online website NetPhos (https://services.healthtech.dtu.dk/services/NetPhos-3.1/ accessed on 1 May 2025). Download the genome files as well as the GFF3 annotation files of soybean and A. thaliana via Ensembl Plant (https://plants.ensembl.org/index.html accessed on 1 May 2025). Subsequently, perform a collinearity analysis between these two species using Tbtools II software (ver. 2.322, South China Agricultural University, Guangzhou, China). In regard to the collinearity analysis within the soybean species group, first retrieve the genome file and gene annotation file of soybean from Ensembl Plant, and then utilize Tbtools II to conduct the collinearity analysis within the soybean genome.

2.8. Analysis of the Subcellular Localization of GmABI3VP1

Prepare MS solid medium (Table S2) and pour tobacco seeds into a 1.5 mL sterile EP tube. Add 100 μL of sodium hypochlorite (NaClO) and 900 μL of sterile water. Shake in a vortex oscillator for 8 min, then let it stand still at 25 °C for 4 min. Wash it with 500 μL of sterilized water three times in a sterile operating platform. Use a pipette tip to plant the tobacco seeds in the MS solid medium and cultivate them for 10 d. Transfer the seedlings to black pots filled with a 1:1 mixture of vermiculite and nutrient soil. Conduct the subcellular localization experiment after the tobacco has grown for 28 d. Firstly, pretreat the tobacco. Place the tobacco in the dark for 24 h. Secondly, put the tobacco under normal light conditions 1 h before infection. Then, perform tobacco injection. For the Agrobacterium carrying pCAMBIA1302-GmABI3VP1 (adjust the tobacco resuspension to an OD₆₀₀ value of 0.8), incubate it at a constant temperature for 5 h, then inject it into the abaxial side of tobacco leaves using a sterile syringe. After tobacco injection is completed, place the plants in a dark environment for 12 h, then expose them to light for 24 h. Finally, observe and take pictures under an inverted fluorescence microscope.

2.9. Over-Expression, Gene Editing and Homologous Recombination

The plasmid was linearized by single enzyme digestion. The pCAMBIA3300 vector was linearized with Hind III enzyme (Biolabs New England Biolabs #R3104, Ipswich, MA, USA), the pCAMBIA1302 vector was linearized with Nco Ι enzyme (Biolabs New England Biolabs #R3193, Ipswich, MA, USA), and the pYLCRISPR/Cas9 vector was linearized with Bas I enzyme (Biolabs New England Biolabs #R3733, Ipswich, MA, USA). The PCR program was set as follows: incubation at 37 °C for 5 h, then at 65 °C for 25 min, and, finally, storage at 12 °C. The enzyme digestion system had a total volume of 20 μL, which consisted of 2 μL of Hind III enzyme, 4 μL of enzyme buffer, 10 μL of plasmid (150 ng/μL), and 4 μL of ddH₂O.

Use the ClonExpress II One Step Cloning Kit (VAZYME, Nanjing, China) to perform homologous recombination ligation between the cloned DNA and the linearized pCAMBIA3300 and pCAMBIA1302, respectively. The homologous recombination system had a total volume of 20 μL, which consisted of 2 μL of homologous recombination enzyme, 4 μL of buffer, 3.5 μL of the linearized pCAMBIA3300 vector, 1.5 μL of the target fragment, and 9 μL of water. Place it in a water bath at 37 °C, 30 min (Tables S4 and S5).

Construction of the gene editing vector; for Reaction 1, specific amplification was carried out (including 1 round-3d, 1 round-3b). Based on Reaction 1, Reaction 2 was performed for the second specific PCR amplification (2 rounds-3d, 2 rounds-3b). After the gel purification of the products from the 2 rounds of amplification, they were recombined with the pYLCRISPR/Cas9 vector.

The pCAMBIA3300 and pCAMBIA1302 plasmids were linearized by the method of single enzyme digestion. The results showed that lanes 1–4 carried the flanking target sequences of the pCAMBIA3300 plasmid, and lanes 5–7 carried the flanking target sequences of the pCAMBIA1302 plasmid. After single enzyme digestion, there was a certain electrophoresis distance between the linearized vectors and the plasmids, indicating that the enzyme digestion was complete (Figure S4C,D). The coding sequence of the target gene was cloned using specific primers. The results demonstrated that an obvious band appeared at 1236 bp (Figure S4E). The linearized gel products and the target gene gel products were ligated using the ClonExpress II One Step Cloning Kit and then transformed into DH5α. PCR specific amplification was carried out (Figure S4I), followed by sequencing analysis. The sequencing results indicated that the target gene sequence was successfully recombined into DH5α.

2.10. Agrobacterium-Mediated Hairy Root Transformation

The recipient used Dongnong 50 for the creation of both gene-edited (KO) hairy roots and overexpressed (OX) hairy roots. Soybean Dongnong 50, which is sensitive to SCN 3, was planted in black pots filled with vermiculite. When the soybeans grew to the bud stage (V_E stage), the main roots of the soybeans were obliquely cut and then were transferred into Agrobacterium K599 containing the plasmid (7.5 μL of 1M As, and 1 mL of 500 mM MgCl₂ with an OD₆₀₀ value ranging from 0.6 to 0.8). The mixture was placed in a shaker at 28 °C with a rotation speed of 50 rpm/min for 30 min. After that, the soybeans were taken out from the shaker and subjected to vacuum treatment using a vacuum pump. The pressure was set at 5–10 kPa, and the vacuum treatment lasted for 1 min. Then, they were left to stand still for 30 s. This process was repeated three times. For the control group, the soybeans were soaked in distilled water, and the operation process was the same as that of the Agrobacterium-mediated hairy root transformation method. The soybean seedlings in the bacterial solution were reinserted into the vermiculite and were cultured under 80% humidity at 25–28 °C until they reached the second trifoliate leaf stage (V₂ stage) for the subsequent identification of SCN.

2.11. Identification of the Disease Phenotype of SCN

The soybeans were extracted from the vermiculite, thoroughly washed with water, and a portion of their root tissues were harvested. Subsequently, both the transgenic plants for validation and the control group plants were transferred into soil inoculated with SCN 3. They were cultured at 25 °C with a soil humidity of 40%. After 14 d, the roots were removed from the infected soil and rinsed extensively with clean water. The identification of SCN 3 in the root tissues of soybeans was conducted using the acid fuchsin staining method. The operational steps are as follows: Firstly, the roots were decolorized by soaking them in a solution of 5% sodium hypochlorite for 80 min. Following this step, they were washed by soaking in double-distilled water (ddH₂O) for an additional 15 min. Next, a working solution of acid fuchsin (1× concentration; Table S1) was prepared and heated in an induction cooker until boiling for approximately 60 s to firm up the roots. Afterward, the roots were carefully removed from the induction cooker and dried gently using clean paper towels. Finally, for microscopic examination, soybean roots were evenly spread on a transparent plastic pressing plate. The number of SCN present was observed and counted under a stereomicroscope at a magnification of ×20.

2.12. Measurement of the Indices Related to the Resistance of Roots to Stress Caused by SCN

In this study, roots of transgenic plants stressed by SCN 3 for 14 d and WT plants were rinsed with water. Then, equal-mass lateral roots were selected to measure Peroxidase (POD) and superoxide dismutase (SOD) activities, malondialdehyde (MDA) content, relative water content (RWC), electrical conductivity (EC), and proline synthetase (Pro) activity. The specific determination method was carried out according to the instructions in the manual [27].

3. Results

3.1. RNA Quality Assessment of Soybean Roots

The results showed that the concentration and purity of RNA were measured by Nano Drop One/One C, and the integrity of RNA was detected by agarose gel electrophoresis. With an OD_260/280 ratio of 2.0, high-quality and intact RNA samples were obtained. The results of 2% agarose gel electrophoresis indicated that the electrophoresis bands were clear without obvious smearing, and the brightness ratio of the 28 S rRNA and 18 S rRNA bands was close to 2:1, suggesting that the RNA had good integrity (Figure S4A) and could be used for subsequent reverse transcription. The concentration of cDNA measured by NanoDrop One/One C was good, and it could be used for the subsequent gene cloning and RNA sequence.

3.2. Transcriptome Sequencing Results and Analysis

Data quality control was carried out on 12 sequenced samples. After filtering, the Q₃₀ ratio ranged from 94.38% to 95.32%, and the GC content after filtering was between 41.75% and 44.15% (Table S3). It was indicated by the results that the base recognition was accurate and could be used for subsequent analysis.

The sequencing data obtained were aligned with the soybean reference genome (Glycine max Wm82.a2.v1) by using the HISAT2 software V_2.2.1. It was found that among the 12 sequenced samples, the mapped rate coverage ranged from 82.48% to 91.82% (Table S6), and a high coverage was shown. It was demonstrated by the results that the sequencing results were reliable and could be further analyzed. The differentially expressed genes were visualized by using volcano plots (Figure 1A). The thresholds of |log₂(Fold change)| > 1 and adjusted p value < 0.05 for screening the fold change in differences; among them, 4185 up-regulated genes and 3195 down-regulated genes were identified.

The screened genes were analyzed by KEGG (Figure 1B). In total, 1127 differentially expressed genes were found to be enriched in KEGG pathways, including metabolic pathways, Phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, and many other pathways. In addition, differentially expressed genes were also significantly enriched in the plant hormone signal transduction pathway, Flavonoid biosynthesis pathway, and MAPK signaling pathway–plant. It appears from the research findings that when soybeans are affected by SCN, the resistance genes within them may exhibit a relatively obvious upward regulation trend and could be involved in various biological metabolic processes. Eventually, candidate genes with large fold changes in differential expression were selected for the research on subsequent genes.

3.3. qRT-PCR Analysis

It was shown by the results that a relative expression level of the GmABI3VP1 gene existed in the soybean roots. When compared with the non-stressed Dongnong 50 soybeans, the GmABI3VP1 gene in the Dongnong 50 soybeans under SCN 3 stress treatment was found to be up-regulated in expression. The average relative expression level in the root tissues of Dongnong 50 under SCN 3 stress treatment was 2.064, while that in the non-stressed ones was 0.509. For SN 14, the average relative expression level in the root tissues under SCN 3 stress treatment was 3.495, while that in the non-stressed ones was 0.481. As for Dongnong L10, the average relative expression level in the root tissues under SCN 3 stress treatment was 8.520, while that in the non-stressed ones was 1.927. Regarding Y16, the average relative expression level in the root tissues under SCN 3 stress treatment was 7.491, while that in the non-stressed ones was 2.243. Under SCN 3 stress, when compared with the non-stressed soybean roots, the GmABI3VP1 gene in the root parts under SCN 3 stress treatment was observed to be significantly up-regulated. Moreover, the up-regulation of the relative expression level in the disease-resistant germplasms was noticed to be more significant than that in the disease-susceptible germplasms under SCN 3 stress (Figure 2A). The qRT-PCR results for SCN stress in soybean roots, stems, and leaves showed that the relative expression level was highest in the disease-resistant Dongnong L10 variety, with an average of 8.33, while the average relative expression level in the disease-resistant Y16 variety was 7.3 (Figure 2B). In the gene expression analysis, the relative expression level in the root system was 8.336, consistent with the experimental results (Figure 2C). It was proved that the GmABI3VP1 gene could respond to the stress response under SCN 3 stress, and it was considered that this gene might be involved in the response after SCN stress. In summary, through qRT-PCR validation of two disease-resistant materials (Dongnong L10, Y16) and two susceptible materials (Suinong 14, Dongnong 50) subjected to SCN 3 stress, it was observed that during the peak stage of SCN 3 infestation in root systems (J₂ stage), the relative expression level of the GmABI3VP1 gene in disease-resistant varieties was extremely significantly higher than that in susceptible ones. Thus, this gene is regarded as a potential candidate gene to be prioritized for further validation.

The gene expression levels of OX-GmABI3VP1 and KO-GmABI3VP1 hairy roots of soybean under SCN 3 stress were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results indicated that the relative expression level of the GmABI3VP1 gene in wild-type (WT) was approximately 1.0, while it was about 3.7 in overexpression (OX) hairy roots and around 0.4 in gene-edited (KO) hairy roots. This gene showed a significant upregulation trend after overexpression. The overexpression mutant exhibited upregulation due to the inserted overexpression cassette and was involved in regulating the response of soybean-to-soybean cyst nematode (SCN) disease.

3.4. Vector Construction for Over-Expression and Gene Editing

The results of the construction of the gene editing vector showed that in the first round of specific PCR amplification, obvious electrophoresis bands of 3d DNA were observed to appear near 250–500 bp and those of 3b DNA were observed to appear near 140 bp in gel electrophoresis (Figure S4F). Based on the first round of PCR, the second round of specific PCR amplification was conducted. It was indicated by the electrophoresis results that an obvious electrophoresis band was observed to appear near 500 bp (Figure S4G). The purified products of the second round of specific PCR and the Bas I linearized pYLCRISPR/Cas9 (Figure S4B) were connected by using the ClonExpress II One Step Cloning Kit and then were transformed into DH5α. Sequencing analysis was carried out, and it was shown by the sequencing results that the target gene sequence was successfully recombined into DH5α (Figure S4H). The correct recombinant plasmids, pCAMBIA3300-GmABI3VP1 and pYLCRISPR/Cas9-GmABI3VP1, were transformed into Agrobacterium tumefaciens K599.

3.5. Bioinformatics Analysis of GmABI3VP1

The bioinformatics results indicated that, in terms of the physicochemical properties of the protein encoded by GmABI3VP1, 421 amino acids were encoded. Its molecular weight was calculated to be 103.45 Ku, the isoelectric point (PI) of the amino acids was determined as 5.02, and its chemical formula was identified as C₃₇₅₇H₆₂₅₃N₁₂₆₃O₁₅₆₁S₂₉₂, with the aliphatic index being 27.32. It was found that this protein had no signal peptide and was classified as a non-secretory protein (Figure S5A). Through the hydrophilicity analysis, it was observed that the protein was generally a hydrophilic one (Figure S5B). In the analysis of whether the protein was involved in transmembrane transport, it was noticed that the encoded protein at 0–200 bp might be involved, as was shown (Figure S5C). Regarding the secondary conformation of the protein, it was demonstrated that there were three spatial configurations (Figure S5D), namely α-helix (9.29%), random coil (77.52%), and extended strand (11.19%) and (3.14%). Among them, the random coil accounted for the largest proportion. There were 36 potential phosphorylation sites in the protein (Figure S5E), including 7 threonine (Thr) sites, 27 serine (Ser) sites, and 2 tyrosine (Tyr) sites. It was shown that the tertiary conformation of the protein was highly consistent with the secondary conformation, and when compared with the homologous gene in A. thaliana, a similarity as high as 92.54% was obtained (Figure S5F). Collinearity analysis was conducted on the GmABI3VP1 gene within and between groups. The results indicated that there was collinearity for the GmABI3VP1 gene between soybean and A. thaliana. For the between-group analysis, Chromosome 3 of soybean exhibited a collinear relationship with Chromosomes 1, 2, and 3 of A. thaliana (Figure S2). For the within-group analysis, Chromosome 3 of soybean exhibited a collinear relationship with Chromosomes 7, 16, and 19 (Figure S3). The results of the promoter element analysis were presented as follows: multiple elements responsive to hormonal substances were found (Figure S1), such as abscisic acid-responsive elements like ABRE/ABRE3a/ABRE4a, anaerobic induction elements like ARE, and elements related to environmental stress response such as Myb, MYC, and MYc, as well as cis-acting elements for salicylic acid like as-1. Phylogenetic tree analysis showed that the gene was divided into three branches, I–III (Figure 3). The gene had the highest homology with soybean Glyma.19G261300 (Supplementary S1). It was inferred that the GmABI3VP1 gene was closely related to biotic and abiotic stresses, and it was speculated that this gene might be involved in the SCN stress response.

3.6. Subcellular Localization Analysis of GmABI3VP1

The distribution of the GFP (Green fluorescent tag) protein in plants was observed under a confocal laser scanning microscope (Figure 4). The results showed that in the tobacco-containing pCAMBIA1302-GFP in the control group, green fluorescence was present in both the nuclear envelope and the cytoplasm, demonstrating that there was certain expression in the cell membrane, cytoplasm, and nucleus. In the case of pCAMBIA1302-GmABI3VP1, the brightest fluorescence was observed in the nucleus of tobacco, indicating that the protein encoded by GmABI3VP1 was mainly expressed in the nucleus and belonged to a nuclear-localized protein. The experimental results were highly consistent with those predicted by bioinformatics.

3.7. Agrobacterium Transformation and SCN Disease Identification and Analysis

The DNA of soybean roots was detected for transgenic hairy roots by using specific PCR amplification and PAT/bar EPSPS LFD Strips (Figure 5A).The results showed that for the roots of OX₁–OX₃, the electrophoresis bands of the BAR gene amplified specifically from the root DNA appeared near 552 bp, and the BAR test strips for the root tissue samples showed positive results, indicating that pCAMBIA3300-GmABI3VP1 had been successfully transformed into the roots of soybeans. For the roots of KO₁-KO₃, the electrophoresis bands of the BAR gene amplified specifically from the root DNA also appeared near 552 bp, and the PAT/bar EPSPS LFD Strips (LS) for the root tissue samples showed positive results, meaning that pYLCRISPR/Cas9-GmABI3VP1 had been successfully transformed into the roots of soybeans (Figure 5B). The PAT/bar EPSPS LFD Strips (LS) for the root tissue samples of WT₁-WT₃ showed negative results.

The roots of OX₁-OX₃, KO₁-KO₃, and WT₁-WT₃ were planted in the soil infected with SCN 3. Meanwhile, in order to increase the number of SCN 3 per unit area (per cm of lateral root length), SCN 3 J₄ eggs (100) were sown around the roots of each plant. After 14 d later, the results of acid fuchsin staining identification showed that the average number of nematodes per unit area in the OX lines was 2.86, the average number of SCN per unit area in the KO lines was 8.22, and the number of SCN 3 per unit area in the WT was 5.26. The SPSS V25.0 software was used to conduct statistical analysis on the data. It was found that there was an extremely significant difference between the OX lines and the WT lines, and also between the KO lines and the WT lines. The results proved that the OX-GmABI3VP1 gene had the function of inhibiting SCN 3. Graphd Prism 9 was used to conduct statistical analysis on the data regarding the average number distribution of SCN in the roots of OX, WT, and KO in the Dongnong 50 genetic background. The gene-edited hairy roots exhibited an AT base deletion and an A base insertion, which resulted in subsequent amino acid disorder, and successfully obtained gene-edited hairy roots (Figure 5H). The results indicated that the number of SCN per unit area presented a normal distribution, and the differences in the number of SCN 3 between OX and WT as well as between KO and WT were extremely significant (Figure 5C–F).

The SPSS V25.0 software was used to conduct a T-test difference analysis on the number of SCN 3 per unit area in the GmABI3VP1 overexpressed roots treated with SCN 3, the number of SCN 3 per unit area in the roots edited by trans-pYLCRISPR/Cas9-GmABI3VP1, and the number of SCN 3 per unit area in the WT roots. The results showed that among the three groups of data, the averages of WT, KO, and OX were 5.26, 8.22, and 2.86, respectively; the standard deviations of OX-WT and OX-KO were 0.32 and 0.75, respectively; the p-values (two-tailed) of the means of OX-WT and OX-KO were 6.1998 × 10⁻⁹ and 2.2641 × 10⁻⁷, respectively. The results indicated that there was an extremely significant difference in the average number of SCN 3 per unit area between the OX group and the WT group, and there was also an extremely significant difference in the average number of SCN 3 per unit area between the KO group and the WT group. It suggested that GmABI3VP1 might play a positive regulatory function and participate in regulating the resistance of soybeans to SCN (

Table 1. Paired samples T-test results.

Treatments	Average	Variance	Observed Value	Standard Deviation	Standard ERROR of the Mean	p (T ≤ t) Double Tail	p (T ≤ t) Double Tail Criticality
GmABI3VP1-KO	8.22	0.13	15	0.32	0.31	6.1998 × 10−9	9.30
GmABI3VP1-OX	2.86	0.13	15	0.75	0.19	2.2641 × 10−7	12.39
WT	5.26	0.12	15	-	-	-	-

).

3.8. Root Physiological and Biochemical Indicator Identification

After 14 d of being stressed by SCN 3, in the roots of both the over-expression (OX) group and the wild-type (WT) group, the plants in the OX group were found to have a more vigorous growth trend compared to those in the WT group. The plants in the WT group were shown to have a more vigorous growth trend than those in the knockout (KO) group (Figure 6A–F). In terms of relative water content, the over-expression group was observed to have a higher water content than the WT and KO groups. After being stressed by SCN, regarding relative electrical conductivity, POD activity, and SOD activity, a trend was presented where the values were in the order of the OX group > the WT group > the KO group. After being stressed by SCN, a trend was shown in the content of MDA, with the order being the OX group < the WT group < the KO group. When not stressed by SCN, no obvious changes were detected in the physiological and biochemical indexes. The data suggest that the GmABI3VP1 gene is considered to be involved in the stress response to SCN, and a positive regulatory function is played by it, which can significantly improve the stress resistance of plants.

4. Discussion

Currently, most of the research mainly focuses on genes near the major resistance loci, Rgh1 and Rgh4. However, Rgh1 and Rgh4 can only account for 60% of the genetic variation, and a large portion remains to be further explored and developed. Thus, how to identify and discover new genes resistant to SCN disease has become of paramount importance.

ABI3/VP1 was initially detected as being part of the seed-specific transcription factors and was utilized as an intermediary in the regulation of ABA-responsive genes throughout the seed development stage. Nowadays, it is acknowledged to play a role beyond the scope of seed physiology, particularly when non-biological stress occurs. Currently, in the numerous literature, it has been frequently documented that under adverse situations like drought, dehydration, temperature variations and salt-alkali stress, the functions of ABI3/VP1 proteins are activated and engaged. The drought resistance of plants and the photosynthetic efficiency can be enhanced and improved simultaneously when the AtABI3 gene in A. thaliana is overexpressed in cotton (Gossypium spp.) [28]. The growth and differentiation of poplar (Populus) plumule leaves can be affected by the overexpression of the PtABI3 gene [29]. Through next-generation sequencing of the ABI3 deletion mutants and wild type, it was indicated in the degradome analysis that the ABI3 gene might be regulated by the plant-specific miR536, with desiccation tolerance being potentially affected [30]. In Brassica napus L., the seeds can be made to have higher desiccation tolerance under dehydration stress by BnABI3 through the regulation of seed-coat development so as to adapt to the stressful environment [31]. In Brassica napus, it was found that the plant’s cold tolerance system could be triggered by the excessive accumulation of ABI3 in seeds and pods, and the decomposition of chlorophyll could be effectively promoted by plant embryos and pods even when they were exposed to frost, and the economic value of oil crops could be ensured to a certain extent [32]. The salt tolerance of the loss-of-function mutants of the A. thaliana transcription factor DIV2-encoding gene AtDIV2 was improved, the sensitivity to exogenous ABA was enhanced, and the ABA content was significantly increased. In the DIV2 mutants, the transcriptional levels of ABA-related genes and stress-related genes such as ABA1, ABI3, and P5CS2 were all up-regulated. It was indicated that the activity of ABI3 might be appropriately regulated by AtDIV2 in wild-type plants to participate in the salt stress response [33,34].

Although the responsive reactions of the ABI3 gene can be observed under various adverse stress conditions, there have been no relevant reports regarding its role in SCN disease to date. The potential function of this target gene in SCN disease requires further exploration, and ongoing efforts should be made to identify and investigate new genes that confer resistance to SCN disease. Dongnong 50 does not contain the rhg1 and Rhg4 loci, while Dongnong L10 contains both the rhg1 and rhg4 loci. Further research and exploration are still needed on the GmABI3VP1 gene. Currently, whether allelic genotypes exist in the GmABI3VP1 promoter remains to be further investigated, and it is also necessary to explore whether upstream effector factors regulate the GmABI3VP1 protein.

Current management of soybean cyst nematode (SCN) resistance predominantly relies on genes such as Rhg1 and Rhg4, which account for only 60% of the observed genetic variation [35]. Our study identifies GmABI3VP1 as a novel contributor to SCN resistance, thereby expanding the genetic toolkit available for breeding efforts. The association of the gene with various stress-responsive pathways, including plant hormone signaling and MAPK pathways, indicates that it may interact with existing resistance loci to synergistically enhance defense mechanisms. Future research should explore its interplay with Rhg1 and Rhg4, as well as its role in resisting other SCN physiological races, to validate its broad applicability. While our results highlight GmABI3VP1′s role in SCN 3 resistance, several questions remain. The downstream target genes of GmABI3VP1 in the SCN response pathway require identification, as do the specific molecular mechanisms by which it regulates antioxidant enzyme activity and stress signaling. Additionally, validating its function in stable transgenic soybean lines and under field conditions will be critical for translational applications. Finally, investigating its role in cross-resistance to other pathogens or abiotic stresses could further broaden its utility. In conclusion, GmABI3VP1 emerges as a key regulator of soybean resistance to SCN 3, acting through transcriptional modulation and physiological adaptation. Its characterization provides a foundation for developing SCN-resistant soybean varieties and deepens our understanding of plant-nematode interactions.

In this study, the Dongnong L10, which is resistant to SCN, and the Heinong 37, which is susceptible to SCN, were adopted. After being subjected to SCN 3 stress for 14 d, the root tissues were sent for RNA-Seq sequencing. The differentially expressed gene GmABI3VP1 was identified through transcriptome data (RNA-Seq) screening. Then, the overexpression recombinant plasmid pCAMBIA3300-GmABI3VP1, the subcellular localization recombinant plasmid pCAMBIA1302-GmABI3VP1, and the gene editing recombinant plasmid pYLCRISPR/Cas9-GmABI3VP1 were constructed. The successfully constructed recombinant plasmids were, respectively, transferred into the competent cells of Escherichia coli DH5α and Agrobacterium tumefaciens K599. Bioinformatics analysis was carried out on the GmABI3VP1 protein, including phosphorylation, secondary and tertiary structures of the protein, and promoter elements. It was shown by the results that multiple gene elements related to responding to adverse stress were contained in this gene, and the plant adverse stress was involved with them. It was also confirmed by the SCN phenotype identification that this gene was involved in resistance to SCN 3, and this gene was classified as a positive regulatory factor. It was preliminarily proved that obvious resistance to SCN 3 disease was possessed by the GmABI3VP1 gene (Figure 7).

5. Conclusions

This study shows that the differentially expressed GmABI3VP1 gene was mined through the transcriptomes of SCN-resistant and SCN-susceptible soybean plants. RT-qPCR was used to verify that the gene exhibits a response SCN disease. The GmABI3VP1 gene was cloned, and the overexpression and gene editing vectors were introduced into soybean roots for the verification of its disease resistance function. The physiological indicators Pro, MDA, and SOD exhibited significant increases, whereas EC demonstrated a notable decrease. The results indicate that the GmABI3VP1 gene is a gene that positively regulates SCN resistance.