Soybean transcription factors GmHSF-01 and GmNF-YC03 regulate the GmSRC7^TN-mediated immune pathway

Abstract

Background

Soybean (Glycine max) is a globally important oilseed and forage crop. Soybean mosaic virus (SMV) infection reduces the yield and quality of soybeans. Previously, our laboratory cloned an atypical TNB (TIR‑NBS‑BSP) type disease‑resistance protein, GmSRC7 (SMV resistance cluster 7) from soybean. Its TIR‑NBS domain (GmSRC7^TN) displays strong antiviral activity, however, the molecular mechanisms underlying this domain‑mediated disease resistance remain to be elucidated.

Results

Using GmSRC7^TN as bait, we screened a soybean cDNA library and identified 42 candidate proteins that potentially interact with GmSRC7^TN. Through yeast two‑hybrid, bimolecular fluorescence complementation, and luciferase complementation assays, the transcription factors GmHSF‑01 and GmNF‑YC03 were confirmed to interact with GmSRC7^TN, with the TIR domain of GmSRC7 playing a critical role in these interactions. Further functional studies showed that GmHSF‑01 possesses intrinsic antiviral activity and enhances the antiviral function of GmSRC7^TN, whereas GmNF‑YC03 interacts with the SMV‑encoded 6K1 protein, thereby interfering with viral replication and movement. Knockdown experiments demonstrated that silencing GmHSF‑01 or GmNF‑YC03 in transgenic soybean plants overexpressing GmSRC7^TN compromises disease resistance, suggesting that both transcription factors cooperate with GmSRC7^TN to regulate host antiviral defense.

Conclusions

Our results demonstrate that upon sensing SMV infection, GmSRC7^TN acts as a central hub, initiating downstream defense programs by specifically recruiting the transcription factors GmHSF-01 and GmNF-YC03. The findings provide a foundation for further dissection of the GmSRC7^TN‑dependent disease resistance signaling pathway and offer potential molecular targets for the genetic improvement of SMV‑resistant soybean varieties.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08298-z.

Introduction

Soybean is a nutrient-rich crop of significant importance as a source of both protein and vegetable oil [1]. Throughout its growth and development, soybean is susceptible to infection by various pathogens, including fungi, bacteria, and viruses. Among these, SMV represents one of the most economically damaging diseases in soybean production. The virus is primarily transmitted through infected seeds and by aphid vectors, leading to severe reductions in both yield and quality of affected varieties. Characteristic symptoms include leaf mottling, leaf curling, and overall plant stunting [2–4]. SMV belongs to the genus Potyvirus within the family Potyviridae. Its genome consists of a single-stranded, positive-sense RNA molecule approximately 9.6 kb in length. The viral polyprotein is processed by three virus-encoded proteases into ten functionally distinct mature proteins, from the N- to C-terminus as follows: potyvirus 1 (P1), helper-component proteinase (HC-Pro), P3, 6 kinase 1 (6K1), cylindrical inclusion (CI), 6K2, viral protein genome-linked (VPg), nuclear inclusion a protease (NIa-Pro), nuclear inclusion b (NIb), and coat protein (CP) [5–7]. Compared to the animal immune system, Jones proposed the zigzag model of the plant immune system in 2006 [8]. The plant immune system is considered to be divided into two layers according to the location of the immune response. The first layer of the immune system is triggered by the recognition of conserved pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to pattern-triggered immunity (PTI) [9]. The second layer of immunity is mediated by nucleotide-binding and leucine-rich repeat receptors (NLRs) in plants, which directly or indirectly recognize specific pathogen effectors and initiate effector-triggered immunity (ETI) [10]. This is usually accompanied by localized cell death in the plant, referred to as the hypersensitive response (HR) [11]. NLR genes represent one of the largest and functionally most significant gene families in plants, with over 140 NLRs reported to counteract host immunity through recognition of known effectors [12]. Studies by Ngou and Yuan have demonstrated that PTI and ETI act synergistically to collectively mount plant defenses against pathogens [13, 14]. Although the two immune tiers differ in their activation mechanisms and early signaling events, they ultimately converge on many similar downstream responses. These findings reveal a cooperative interplay between PTI and ETI in joint pathogen defense, thereby refining and expanding the original “zig-zag” model.

Approximately 80% of identified plant resistance (R) genes belong to the nucleotide‑binding and leucine‑rich repeat (NLR or NB‑LRR) family, which represents one of the most rapidly evolving gene families in plants. Structurally, a typical NLR protein comprises three main domains: an N‑terminal variable domain, a central nucleotide‑binding and oligomerization domain (NOD), and a C‑terminal leucine‑rich repeat (LRR) domain [15]. The NOD itself consists of three sub‑domains—the nucleotide‑binding domain (NBD), helical domain 1 (HD1), and wing‑helix domain (WHD). NBD proteins belong to the branch of the AAA-ATPase superfamily STAND (Signal transduction ATPases with numerous domains) subfamily, STAND proteins regulate molecular switches through nucleotide binding [16], the ADP forms a quiescent off-state and upon recognition by pathogens, conformational changes result in the exchange of ADP for ATP to initiate downstream signaling. Based on the architecture of the N‑terminal domain, NLRs are classified into three major groups: those carrying a coiled‑coil (CC) domain are designated CNLs; those containing a Toll/interleukin‑1 receptor/resistance protein (TIR) domain are termed TNLs [17], and those possessing an RPW8 (powdery mildew resistance 8) domain are referred to as RNLs [18]. The expression of NLR genes varies across plant tissues, species, and environmental conditions [19], reflecting their central role in intracellular signal recognition and the activation of immune responses that restrict pathogen proliferation.

R proteins can co-regulate immune responses through interactions with transcription factors. Major transcription factors involved in plant stress responses include members of the WRKY, MYB, NAC, bZIP, and AP2/ERF families. In rice, the CC-NB-LRR type resistance protein Pb1 interacts with the transcription factor OsWRKY45, leading to activation of the ubiquitin-proteasome system-modulated salicylic acid (SA) signaling pathway and ultimately conferring disease resistance [20]. In Arabidopsis, the TNL mutant snc1 interacts with the transcription factor bHLH84 to enhance immune responses. Simultaneous knockdown of three homologous bHLHs suppresses snc1-mediated autoimmunity, while overexpression of bHLH enhances immunity [21]. The transcription factor PIBP1 and its homologous gene Os06g02240 activate the expression of the downstream immunity genes OsWAK14 and OsPAL1 by directly interacting with the broad-spectrum resistance R gene and promoting its accumulation in the nucleus [22]. The CC domain of the NLR receptor Sw-5b interacts with MED10b, a transcriptional co-activator, and knockdown of MED10b enhances plant responsiveness to Tomato spotted wilt virus (TSWV) [23]. The N. benthamiana RPW8-NLR central immune receptor activated disease resistance 1 (NbADR1), together with the key immune proteins enhanced disease susceptibility 1 (NbEDS1) and phytoalexin deficient 4 (NbPAD4), plays an essential role in bacterial pathogen and flg22-induced stomatal immunity by regulating the expression of SA and abscisic acid (ABA) biosynthesis or response-related genes [24]. Previous studies in our laboratory have shown that GmSRC7, which encodes a nuclear-localized atypical TNB class R protein, and GmSRC7^TN exhibit strong antiviral activity [25]. However, the interaction between GmSRC7^TN and transcription factors in soybean remain to be elucidated.

In this study, we screened a soybean cDNA library using GmSRC7^TN as bait to identify interacting transcription factors and constructed a protein-protein interaction (PPI) network. We then validated the interactions and identified key structural domains involved in these interactions. Furthermore, we investigated two GmSRC7^TN-interacting transcription factors, GmHSF-01 and GmNF-YC03, to analyze their roles in GmSRC7^TN-mediated resistance to SMV. The results will contribute to understanding the disease resistance mechanism of GmSRC7^TN and provide a basis for controlling plant viral diseases and breeding resistant soybean varieties.

Materials and methods

Plant material and soybean cDNA library

Williams 82 was provided by Dr. Bin Zhou from the Crop Research Institute of Anhui Academy of Agricultural Sciences, and N. benthamiana accession 17Wt was maintained in our laboratory. All plants were cultivated under controlled greenhouse conditions (22–25℃, 14 h light/10 h dark photoperiod, 60–70% relative humidity). N. benthamiana 17Wt was employed for transient expression assays. OE-GmSRC7^TN transgenic soybean and OE-GmSRC7 transgenic tobacco were used in virus-induced gene silencing (VIGS) experiments; the OE-GmSRC7^TN soybean line was kindly supplied by Weimi Biotechnology Co., Ltd. Red-nucleus tobacco was provided by Prof. Yingchun Wang (Inner Mongolia University). Total RNA extracted from leaves of Williams 82 was used to construct a soybean cDNA library, which was subsequently prepared by Shanghai OE Biotech Co., Ltd. (Shanghai, China).

Gene cloning, primer design and vector construction

Total RNA was extracted from leaves of Williams 82 soybean and N. benthamiana 17Wt using TRIzol reagent (Vazyme, Nanjing, China). First-strand cDNA was synthesized with the Hifair^® III 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, China). The full-length coding sequence of each target gene was amplified from cDNA using gene-specific primers and subsequently purified by agarose gel extraction. The GmSRC7^TN bait vector was generated by cloning its coding sequence (amplified from soybean cDNA with primers GmSRC7^TN‑F/R) into the appropriate backbone. The linearized vector for pGBKT7 (BD) was prepared by double digestion with SmaⅠ and SalⅠ, and the bait plasmid BD-GmSRC7^TN was assembled using ClonExpress Mix using the “one-step cloning” method. For the yeast two-hybrid (Y2H) assay, target gene fragments were cloned into the linearized pGADT7 (AD) vector, which was prepared by double digestion with EcoRⅠ and BamHⅠ. For LCA plasmids, the following genes were individually cloned into the pCAMBIA1300-cLuc or pCAMBIA1300-nLuc vectors: GmHSF-01, GmNF-YC03, GmSRC7^TIR, GmSRC7^NBS, GmSRC7^BSP, GmSRC7^TN, and GmSRC7. All vectors were linearized by double digestion with SalⅠ and BamHⅠ. To construct the vectors for BiFC experiments, the genes were recombinantly inserted into the Gateway entry vector pQBV3 and then into the destination vector pDEST-vYNE(R)GW (nVenus1-173 aa) or pDEST-vYCE (R)GW (cVenus156-239 aa). For transient expression and VIGS experiments, target genes were cloned into pC-Luc and pTRV2 vectors, respectively. Construction of transgene expression vectors was performed by inserting GmHSF-01 into the overexpression vector pNC-Cam-33HC according to the Nimble Cloning Kit instructions. All recombinant plasmids were verified by DNA sequencing. Primers used in this study are listed in Supplementary Table 2.

Yeast library screening and sequence analysis

A soybean cDNA library was screened with the BD-GmSRC7^TN plasmid as bait following the Nuclear System Yeast Two-Hybrid Mating Screen Library protocol. Bait strains were cultured in tryptophan-dropout liquid medium (-W) of an OD₆₀₀ of 0.8, and cell density was quantified using a hemocytometer. Meanwhile, the AD library aliquot was thawed from − 80℃, diluted appropriately, and plated on leucine-dropout solid medium (-L) to assess library quality. The bait strain culture and the library yeast strain (prey) were mixed for mating in 2 × YPDA medium, followed by incubation at 30℃ with gentle shaking (30–50 rpm) for 20–24 h until mating conjugates (clover shaped structures) were observed microscopically. The mixture was plated on -L, -W, and minimal media lacking leucine and tryptophan (-L-W) to assess mating efficiency. The remaining culture was plated on selective medium lacking leucine, tryptophan, and histidine (-L-W-H) and incubated at 30 °C for 3–5 days. Individual colonies were then transferred to quadruple-dropout medium lacking leucine, tryptophan, histidine, and adenine (-L-W-H-A) supplemented with X-α-Gal for colorimetric screening. Blue monoclonal colonies grown on -L-W-H-A plates containing X-α-Gal were submitted for commercial DNA sequencing. The obtained sequences were analyzed by BLAST comparison via the NCBI website (https://www.ncbi.nlm.nih.gov/).

Bioinformatic analysis

All candidate genes were functionally annotated and pathway-enriched through the DAVID online platform (http://david.ncifcrf.gov). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results were visualized using the bioinformatics online toolkit (https://www.bioinformatics.com.cn). Homologs of the 42 soybean proteins in Arabidopsis thaliana were identified via the TAIR database (https://www.arabidopsis.org). PPI networks for these 42 Arabidopsis proteins were generated using the STRING database (https://cn.string-db.org) and further analyzed with Cytoscape software. Structural domains were predicted using SMART (http://smart.embl-heidelberg.de). Amino acid sequence alignment and phylogenetic analysis were performed in MEGA 7 under default parameters, with evolutionary relationships inferred by the neighbor-joining method (1,000 bootstrap replicates).

Y2H assays

We performed yeast transformation using the PEG/LiAc method. To assess potential bait toxicity, BD‑GmSRC7^TN and the empty BD plasmid were separately co‑transformed into Y2H Gold yeast and plated on -W. To investigate self-activation, BD-GmSRC7^TN was transformed into the Y2H Gold and plated on -W and W + X-α-Gal (BD-53 and AD-T: positive control; BD-lam and AD-T: negative control) for screening. Candidate interacting genes of GmSRC7^TN, along with GmSRC7^TIR, GmSRC7^NBS, GmSRC7^TN, and GmSRC7, were cloned into AD and BD vectors, respectively. Additionally, ORF sequences encoding the 10 SMV component proteins were PCR-amplified from SMV genomic DNA and inserted into the same vectors. Yeast co-transformed with both AD and BD recombinant plasmids were first selected on -L-W medium, followed by stringent selection on -L-W-H + X-α-Gal and -L-W-H-A + X-α-Gal. Plates were incubated at 30 °C for 4–5 days, and well-grown colonies were documented photographically.

LCA, BiFC and subcellular localization

The bimolecular fluorescence complementation (BiFC) and luciferase complementation assay (LCA) related plasmids were separately transformed into Agrobacterium GV3101, resuspended in MES buffer to an OD₆₀₀ of approximately 0.5, and mixed at a 1:1 ratio. The mixed suspensions were infiltrated into red-nucleus tobacco and N. benthamiana leaves, respectively. After 48 h, fluorescence was imaged using either a Nikon AX confocal laser microscope (Tokyo, Japan) or a Zeiss LSM710 confocal laser scanning system (Carl Zeiss AG, Oberkochen, Germany). Red nuclear fluorescence was detected with excitation at 580 nm. Protein interaction was indicated by yellow fluorescence upon excitation at 514 nm. For subcellular localization, GmHSF-01 and GmNF-YC03 were cloned into the pEarleyGate103 vector via Gateway recombination, transformed into Agrobacterium, and similarly infiltrated into N. benthamiana leaves. eGFP signals were captured with 488 nm excitation, and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) were visualized using 405 nm excitation.

Transient expression and DAB staining

Agrobacterium strains carrying the target genes were co‑infiltrated with SMV‑GFP or TMV‑GFP viral inocula into N. benthamiana leaves for transient expression assays [26]. Each experiment included three biological replicates, with 20–30 technical replicates per biological set. At 4–5 days post infiltration (dpi), GFP fluorescence was visualized using a handheld long wavelength (365 nm) UV lamp. For hydrogen peroxide (H₂O₂) detection, leaf tissues were observed after 3 days of transient expression. Infiltrated sites were compared with negative controls (empty vector infiltration) to assess whether hypersensitive response (HR) occurred, and images were recorded. DAB (3,3′-diaminobenzidine) staining was performed using freshly prepared solution protected from light. During vacuum infiltration, care was taken to avoid leaf damage. Staining proceeded for 24–36 h at 37℃ in the dark, followed by decolorization in 70% ethanol to remove excess stain and chlorophyll. Results were observed under white light and photographed.

Transformation, Screening and Functional Validation of OE-GmHSF-01 Transgenic tobacco

Wild‑type tobacco seeds were surface‑sterilized using a chlorine‑based dry sterilization method and subsequently transferred onto ½ MS solid medium. Plates were incubated at 28 °C in a light‑controlled growth chamber. The GmHSF‑01 gene was cloned into the overexpression vector pNC‑Cam‑33HC following the Nimble Cloning Kit protocol [27] and introduced into Agrobacterium for transformation. For tobacco transformation, Agrobacterium carrying the pNC‑Cam‑33HC‑GmHSF‑01 plasmid was cultured to an OD₆₀₀ of 0.5 to prepare the infection solution. Sterilized tobacco leaves were cut into approximately 1 cm² explants, immersed in the infection solution for 10 min, briefly blotted to remove excess liquid, and then cultured on differentiation medium in darkness for 2–4 days. Thereafter, explants were transferred to herbicide‑containing differentiation medium and subcultured every 3–4 weeks until regenerated shoots appeared. When the explants developed clusters of shoots reaching approximately 1–1.5 cm in height, individual shoots were excised and transferred to a rooting medium containing herbicide to promote root formation, yielding T0‑generation OE‑GmHSF‑01 transgenic plants. Positive transgenic lines were confirmed by conventional PCR, RT‑qPCR, and western blot (WB) analyses. T3‑generation OE‑GmHSF‑01 transgenic tobacco plants and wild‑type controls were inoculated with SMV‑GFP or TMV‑GFP viral constructs. Following a 24 h dark incubation post‑inoculation, plants were returned to standard light conditions for an additional 48 h. Viral spread was then visualized under a handheld UV lamp in a darkened environment.

VIGS and viral infection

VIGS is a highly effective tool for functional gene analysis [28, 29]. In this study, VIGS was performed on OE-GmSRC7^TN transgenic soybean plants. Fragments targeting GmHSF-01 and GmNF-YC03 were cloned into the pTRV2 vector to generate recombinant plasmids, which were then introduced into Agrobacterium strain GV3101. For infiltration, cultures of pTRV1 and recombinant pTRV2 were mixed and adjusted to a final OD₆₀₀ of 0.6. The mixed suspension was infiltrated into whole tobacco leaves and incubated for 7–10 days to produce viral homogenate. On day 3, corresponding to the peak expression period of GmSRC7^TN, OE-GmSRC7^TN transgenic soybean plants were treated with β-estradiol for induction. The viral homogenate was adjusted to an OD₆₀₀ of 0.8 and delivered via infiltration into the root systems of soybean plants intended for gene silencing. Each plant received approximately 20 mL of the infiltration solution, administered at 6 day intervals throughout a 20 day incubation period. After confirming successful target-gene silencing, SMV was inoculated using the rub-inoculation method, with the SMV-GFP infiltration solution adjusted to a final OD₆₀₀ of 0.5. The plants were incubated for 10 days, and successful infection was confirmed when wild‑type (WT) plants displayed typical SMV susceptibility symptoms. Systemic leaves were collected for viral titer quantification, and the expression level of the SMV CP gene was measured by RT‑qPCR in both OE‑GmSRC7^TN transgenic soybean and WT plants. Cycle threshold (Ct) values obtained from the experiment were compiled in Excel and analyzed using GraphPad software.

Gene silencing assays were conducted in OE‑GmSRC7 transgenic tobacco by first identifying silencing fragments targeting the GmHSF‑01 and GmNF‑YC03 homolog in N. benthamiana using the Sol Genomics Network (https://solgenomics.sgn.cornell.edu). Subsequently, these fragments were ligated into the linearized pTRV2 vector and transformed into GV3101, followed by transient co expression in tobacco leaves via agroinfiltration together with Agrobacterium harboring the pTRV1 plasmid. The three to four lowest leaves of each plant were infiltrated, and plants were subsequently maintained at room temperature. When plants infiltrated with pTRV2‑PDS exhibited bleaching (approximately 2 weeks post‑infiltration), the silencing efficiency was evaluated by RT‑qPCR. Upon successful knockdown of the target gene, systemic upper leaves were agroinfiltrated with TMV‑GFP or SMV‑GFP, with three to four systemic leaves per plant infiltrated again. Systemic leaves were collected 5 days later and observed under a handheld UV lamp for fluorescence imaging. Empty TRV2 and TRV1 vectors were included as negative controls. All silencing sequences used in this study are listed in Supplementary Table 3.

Results

Screening of soybean proteins interacting with GmSRC7^TN

In this study, GmSRC7^TN was used as the bait protein for Y2H mating library screening, which required prior validation of its toxicity and self‑activation potential. The successfully constructed BD‑GmSRC7^TN bait plasmid and the empty plasmid control were separately transformed into Y2H Gold yeast strains and cultured on -W. Comparable growth between the two groups confirmed the absence of bait‑related toxicity (Fig. 1A). To test for self-activation, BD-GmSRC7^TN was introduced into the Y2H Gold strain by transformation. The transformed yeast grew normally on -W but failed to survive on -W+ X‑α‑Gal, demonstrating no self‑activation activity of the bait protein (Fig. 1B). These results indicate that GmSRC7^TN is suitable for cDNA library screening. Using the yeast mating method, we screened the Y2H mating library and obtained 42 candidate interacting proteins, three of which were annotated as transcription factors. Gene identifiers and functional annotations are provided in Supplementary Table S1.

Fig. 1.

Evaluation of the BD-GmSRC7^TN bait vector. A Bait vector toxicity assay. B Bait vector self-activation assay. The dilution gradients of yeast concentration were 10^− 1, 10^− 2, 10^− 3, and 10^− 4. BD-p53/AD-T and BD-Lam/AD-T were the positive and negative controls, respectively

The 42 candidate genes were functionally annotated and pathway-enriched using the DAVID online platform. GO and KEGG enrichment results were visualized via the Bioinformatics online toolkit. KEGG enrichment analysis revealed 29 enriched interactions, which were primarily associated with photosynthesis, metabolic pathways, and amino acid biosynthesis. GO enrichment analysis indicated that the interacting proteins were predominantly localized in the cytoplasm, chloroplasts, and mitochondria. Their major molecular functions included fructose-bisphosphate aldolase activity, inositol-3-phosphate synthase activity, methyltransferase activity, and proton-transporting ATP synthase activity (Fig. S1). To examine the interaction relationships among GmSRC7^TN-interacting proteins, homologs of the 42 soybean proteins were identified in A. thaliana (Table S1). PPI networks were constructed using the STRING database (42 A. thaliana entries). Seventeen proteins displayed connections with other proteins in the network and could be broadly categorized into groups involved in energy synthesis, metabolism, photosynthesis, and transmembrane transport (Fig. S1). The remaining 25 proteins, which showed no detectable interactions, were not represented in the network diagrams.

Validation of GmSRC7^TN interactions with candidate interacting proteins

Based on bioinformatics analysis, we selected ten candidate proteins, including three transcription factors, for validation using Y2H assays. The results demonstrated that two transcription factors Glyma.01G185800 (GmHSF-01) and Glyma.04G196200 (GmNF-YC03) as well as two non-transcription factors, Glyma.17G101100 and Glyma.20G206400, interacted with GmSRC7^TN (Fig. 2A). The interactions of GmHSF-01 and GmNF-YC03 with GmSRC7^TN were further confirmed by BiFC and LCA (Fig. 2B, C and D). GmSRC7 comprises three structural domains: TIR, NBS, and BSP. To identify the major interacting regions of GmSRC7, we performed Y2H and LCA analyses. The results revealed that GmHSF-01 interacted with GmSRC7^TIR, GmSRC7^NBS, and full length GmSRC7, whereas GmNF-YC03 interacted only with GmSRC7^TIR. Neither transcription factor showed interaction with GmSRC7^BSP, and GmNF-YC03 did not interact with full length GmSRC7 (Fig. S2). These results indicate that the BSP domain is not involved in the interaction between transcription factors and GmSRC7. Further analysis of the LCA results showed that the fluorescence intensity of GmHSF-01 was greater when co-expressed with GmSRC7^TIR than co-expressed with other structural domains, suggesting that this structural domain may play a key role in the interactions of GmSRC7^TN with GmHSF-01 and GmNF-YC03.

Fig. 2.

The transcription factors GmHSF-01 and GmNF-YC03 interact with GmSRC7^TN. (A) Y2H analysis of candidate proteins interacting with GmSRC7^TN. (B) BiFC assay. Agrobacteria carrying recombinant constructs (OD₆₀₀ = 0.5) were mixed 1:1 and co-infiltrated into tobacco leaves. After 48 h, samples were examined by confocal laser scanning microscopy. RFP fluorescence (red-nucleated tobacco) marked the nucleus; yellow fluorescence (YFP) indicated protein-protein interaction. Scale bars are shown in the figure. (C) LCA assay. Agrobacterial suspensions (OD₆₀₀ = 0.5) harboring recombinant genes or empty vectors (negative control) were co-infiltrated into tobacco leaves. After 48 h, luminescence was detected using a plant live-imaging system. Each experiment included three biological replicates with ≥30 technical repeats. For RT-qPCR analysis, leaf tissues from the same infiltration zones used in LCA were collected. Samples coinfiltrated with the target gene served as the experimental group, while those infiltrated with empty vector were the control. Data are presented as mean ± SD of three independent biological experiments (three technical replicates each). Asterisks denote statistically significant differences between groups (t-test, ***p < 0.001)

Bioinformatics analysis of GmHSF-01 and NF-YC03

GmHSF-01 encodes a 283 amino-acid protein (849 bp full-length) containing a conserved HSF domain. GmNF-YC03 encodes a 223 amino-acid protein (669 bp full-length) with a conserved HFD-NFYC-like domain (Fig. 3A). Self-activation assays showed that GmHSF-01 exhibited no autonomous transcriptional activity, consistent with prior reports [30], whereas GmNF-YC03 displayed self-activation (Fig. 3B), indicating its potential role as a transcription regulator. Phylogenetic analysis classified GmHSF-01 into the HSFB1 family (Fig. 3C). Sequence alignment using DNAMAN predicted that the N-terminus of GmHSF-01 contains a typical DNA-binding domain (DBD) and oligomerization domain (OD), along with a -LFGV- tetrapeptide motif (Fig. 3D). The -LFGV motif represents a core element of the repression domain [31] and may mediate interactions with repressor proteins. Notably, GmHSF-01 lacks the C-terminal AHA trans-activation motif characteristic of class A heat-stress factors, further supporting its classification as an HSFB1 family member. Subcellular localization analysis revealed that GmHSF-01 localizes exclusively to the nucleus, while GmNF-YC03 is present in both the nucleus and the endoplasmic reticulum (Fig. 3E). GmSRC7^TN also localizes to the nucleus, suggesting that it may cooperate with GmHSF-01 and GmNF-YC03 in transcriptional regulation.

Fig. 3.

Bioinformatic analysis of GmHSF-01 and GmNF-YC03. (A) Conserved domain analysis of GmHSF-01 and GmNF-YC03 proteins. (B) Self activation assay for GmHSF-01 and GmNF-YC03. (C) Phylogenetic analysis of GmHSF-01. The tree includes HSFB1 homologs from diverse plant species: DoHSFB1 (OEL26872.1); SiHSFB1 (XP_004957012.1); PhHSFB1 (XP_025802216.1); PmHSFB1 (RLM85842.1); SbHSFB1 (XP_002460330.1); ZmHSFB1 (PWZ12676.1); OsHSFB1 (XP_015611859.1); TaHSFB1 (AHZ44770.1); BdHsSFB1 (XP_003578229.1); PeHSFB1 (XP_020596949.1); DcHSFB1 (XP_020671903.2); AcHSFB1 (XP_020089914.1); EgHSFB1 (XP_010943066.1); PdHSFB1 (XP_008808108.1); AoHSFB1 (XP_020244153.1); AtHSFB1 (AT4G36990); SlHSFB1 (NP_001308379.1). (D) Amino acid sequence alignment of GmHSF-01 with HSFB1 proteins from representative species. Annotated regions include the DBD and OD. (E) Subcellular localization of GmHSF-01 and GmNF-YC03. Proteins were fused to eGFP, transiently expressed in tobacco leaves via Agrobacterium infiltration (final OD₆₀₀= 0.6), and visualized by confocal microscopy at 36-48 h post-infiltration. Nuclei were counterstained with DAPI

Antiviral activity studies of GmHSF-01 and GmNF-YC03

To investigate the roles of GmHSF-01 and GmNF-YC03 in GmSRC7^TN-mediated immunity, we first employed a transient expression system. The results demonstrated that full-length GmHSF-01 conferred resistance to both SMV and TMV, whereas its N-terminal HSF domain (1-110 aa) and C-terminal fragment lacking defined domains (111–180 aa) did not exhibit antiviral activity. In contrast, neither full-length GmNF-YC03 nor its truncated fragments (1-65aa and 66-165aa) showed detectable resistance (Fig. 4A). Using the Nicotiana tabacum genetic transformation system, we generated OE-GmHSF-01 transgenic tobacco lines. Successful integration and stable expression of GmHSF-01 were confirmed by PCR, RT-qPCR, and WB (Fig. S3). We then assessed viral replication using a transient expression-based antiviral activity assay by monitoring the spread of fluorescence in leaves. For this assay, we used a laboratory-developed SMV infectious clone expressing green fluorescent protein (eGFP) [32]. Virus-resistance assays indicating that GmHSF-01 confers antiviral activity (Fig. 4B), consistent with the transient expression results (Fig. 4A). Together, these data suggest that GmHSF-01 positively regulates SMV resistance in tobacco. Y2H results showed that none of the ten component proteins of SMV interacted with GmHSF-01, indicating that GmHSF-01 was unable to directly recognize SMV (Fig. 4C). GmNF-YC03 specifically interacted with SMV 6K1, but not with other viral components (Fig. 4D). Subcellular localization analysis revealed that 6K1 localizes to both the nucleus and the endoplasmic reticulum (Fig. 4E). BiFC further confirmed the interaction between GmNF-YC03 and 6K1 at the endoplasmic reticulum (Fig. 4F). Given the established role of 6K1 in viral replication [33], we propose that GmNF-YC03 may competitively bind or sequester 6K1, thereby interfering with viral replication and movement, and ultimately restricting viral spread.

Fig. 4.

Antiviral activity analysis of GmHSF-01 and GmNF-YC03. (A) Analysis of antiviral activity of GmHSF-01 and GmNF-YC03 and their truncated fragments. EV: empty vector is the negative control, the final OD₆₀₀ of gene and truncated fragment infestation solution is 1, and the final OD₆₀₀ of infestation clone solution SMV-GFP and TMV-GFP is 0.5. (B) OE-GmHSF-01 transgenic tobacco and WT whole leaves were infiltrated with TMV-GFP and SMV-GFP viruses. The final OD₆₀₀ of SMV-GFP and TMV-GFP bacterial fluids was 0.5. Observations were made and photographed three days later in a dark room using hand-held UV light irradiation. (C) Interaction validation of GmHSF-01 with ten components of SMV. (D) Interaction validation of GmNF-YC03 with ten components of SMV. (E) Subcellular localization of 6K1 in N. benthamiana. (F) BiFC assay for self-interaction of GmNF-YC03 and SMV 6K1 in N. benthamiana

Effect of GmHSF-01 and GmNF-YC03 on disease resistance of GmSRC7^TN and recognition of SMV components

GmSRC7^TN exhibits antiviral activity, and the expression efficiency of the target gene is directly influenced by its concentration in the infiltration solution. A concentration gradient assay revealed that OD₆₀₀ = 0.001 represents the critical concentration at which GmSRC7^TN confers disease resistance. When co-expressed with GmHSF-01, the green fluorescence associated with viral diffusion disappeared, indicating that GmHSF-01 enhances the resistance of GmSRC7^TN to both SMV and TMV (Fig. 5A). In contrast, co-infection of GmSRC7^TN with GmNF-YC03 showed that GmNF-YC03 does not affect the antiviral activity of GmSRC7^TN (Fig. 5B). DAB staining demonstrated that GmHSF-01 promotes the accumulation of H₂O₂ and O²⁻, and co-expression of GmSRC7^TN with GmHSF-01 resulted in more pronounced yellow precipitate formation compared to GmSRC7^TN alone (Fig. 5C), consistent with the findings presented in Fig. 5A. RT-qPCR analysis further revealed that GmHSF-01 expression was significantly upregulated, while GmNF-YC03 expression decreased following SMV infection in soybean (Fig. 5D), suggesting that SMV induces GmHSF-01 expression but suppresses GmNF-YC03 expression. Collectively, these results indicate that GmHSF-01 enhances the antiviral function of GmSRC7^TN.

Fig. 5.

Role of GmHSF-01 and GmNF-YC03 in GmSRC7^TN-mediated antiviral mechanisms. A-B Effect of GmHSF-01 and GmNF-YC03 on GmSRC7^TN antiviral activity. Left panels: GmSRC7^TN was co‑expressed with SMV‑GFP or TMV‑GFP at OD₆₀₀ values of 0.5, 0.05, 0.005, and 0.001 (top to bottom) while viral OD₆₀₀remained constant at 0.5. Right panels: GmHSF‑01 or GmNF‑YC03 (OD₆₀₀ = 1) was added to the corresponding left‑side mixtures. EV, empty plasmid control. CGmHSF‑01 enhances reactive oxygen species (ROS) accumulation. Treatments included EV (pC‑Luc empty plasmid), GmSRC7^TN overexpression, GmHSF‑01 overexpression, and co‑expression of GmSRC7^TN + GmHSF‑01. Single infiltrations used OD₆₀₀ = 0.5; co‑infiltrations used OD ₆₀₀ = 0.25 for each construct. After 3 days of transient expression, leaves were photographed under white light, subjected to DAB staining (24 h), destained in 70% ethanol (72 h), and re‑photographed. D RT‑qPCR analysis of GmHSF‑01 and GmNF‑YC03expression following SMV infection. Data are presented as mean ± SD; statistical significance was assessed by t‑test

GmHSF-01 and GmNF-YC03 genes have important roles in the GmSRC7 antiviral pathway

In this study, we constructed an inducible expression vector pER8::GmSRC7^TN and generated transgenic positive seedlings from the soybean Williams cultivar. GmSRC7^TN expression was induced by spraying β-estradiol, reaching peak levels at 72 h post-induction (Fig. 6A), indicating that estrogen effectively activates the XVE system in soybean and drives downstream gene expression. Three days after β-estradiol treatment, plants were inoculated with SMV. WT plants developed typical susceptibility symptoms including yellowing and mosaic patterns, whereas OE-GmSRC7^TN transgenic plants showed no disease symptoms (Fig. 6B). RT-qPCR analysis revealed that GmSRC7^TN expression in OE-GmSRC7^TN plants was approximately 17-fold higher than in WT, while the viral CP gene expression was about 20-fold lower (Fig. 6C), indicating that GmSRC7^TN overexpression enhances plant resistance to SMV. To further examine the roles of GmHSF‑01 and GmNF‑YC03 in GmSRC7^TN‑mediated antiviral activity, we performed gene silencing in OE‑GmSRC7^TN transgenic soybean. Silencing of GmHSF‑01 or GmNF‑YC03 did not cause obvious growth defects (Fig. 6D), but reduced their transcript levels by 44.7% and 54.2%, respectively (Fig. 6E). Following SMV inoculation, WT plants displayed clear disease symptoms (Fig. 6F). RT‑qPCR showed that CP gene expression in OE‑GmSRC7^TN plants was significantly lower than in WT, confirming that estradiol‑induced GmSRC7^TN expression enhances viral resistance. However, when GmHSF‑01 or GmNF‑YC03 was silenced in OE‑GmSRC7^TN plants, CP gene levels were higher than in unsilenced OE‑GmSRC7^TN plants, though still lower than in WT (Fig. 6G), indicating that down‑regulation of GmHSF‑01 or GmNF‑YC03 partially compromises the estradiol‑induced antiviral resistance conferred by GmSRC7^TN. These results are consistent with our observations in OE‑GmSRC7 transgenic tobacco (Fig. S4).

Fig. 6.

Use of VIGS to study the function of GmHSF-01 and GmNF-YC03 in the GmSRC7^TN immune pathway. A β-estradiol induces the expression of GmSRC7^TN gene in transgenic plants. The vertical axis represents the expression amount of GmSRC7^TN, and the abscissa represents the induction time of β-estradiol, the asterisk indicates that there is a significant difference in the expression level of the gene after estradiol treatment (t-test, *** p <0.001). B Phenotype of leaves of WT and OE-GmSRC7^TN transgenic soybeans after SMV virus infection. C RT-qPCR was used to detect GmSRC7^TN and CP genes expression levels in OE-GmSRC7^TN transgene and WT plants after SMV infection. The asterisk indicates a significant difference in the expression levels of genes in WT and OE-GmSRC7^TN transgenic soybeans. D Phenotypes of plants after interference with GmHSF-01 and GmNF-YC03 in OE-GmSRC7^TN transgenic soybeans. E Expression of the gene of interest after GmHSF-01 and GmNF-YC03 interference. F Phenotypes of SMV-infected plants after successful interference with GmHSF-01 and GmNF-YC03 in OE-GmSRC7^TN transgenic soybean. G RT-qPCR detection of CP gene expression changes in SMV virus-infested WT, OE-GmSRC7^TN transgenic soybean, and OE-GmSRC7^TN transgenic soybean with target gene silencing (t-test, * p <0.05，** p < 0.01，*** p < 0.001). TUA of soybean was used as a control gene and the results were analyzed using TBtools software. Three biological replicates and three technical replicates were set up for each reaction sample. H A working model of GmSRC7^TN-GmHSF-01 and GmNF-YC03 module in antiviral regulation. GmSRC7^TN recruits GmHSF-01 and GmNF-YC03 for interaction, initiating the plant's systemic disease resistance response. Meanwhile, GmNF-YC03 also directly interacts with the SMV encoded 6K1 protein, potentially interfering with viral replication and movement to inhibit virus spread by competing with or sequestering 6K1. GmHSF-01 possesses antiviral activity, and both it and GmSRC7^TN can produce reactive ROS to enhance the plant's antiviral activity. Solid lines indicate established interactions, while dashed lines indicate unknown connections

Discussion

GmSRC7 exhibits broad-spectrum antiviral activity against SMV, tobacco mosaic virus (TMV), potato virus Y (PVY), and plum pox virus (PPV). Domain truncation experiments indicate that both GmSRC7^TN and GmSRC7 can confer disease resistance in plants [25]; however, the underlying molecular mechanisms remain unclear. Since GmSRC7^TN lacks distinct transcription factor domains and is unable to directly regulate downstream gene expression, it is hypothesized that it may modulate downstream genes through interactions with other transcription factors. High-quality cDNA libraries enable the analysis of protein functions and interactions, facilitating the screening of interacting partners for target proteins and the construction of protein-protein interaction networks [34]. In this study, a cDNA library was constructed from RNA extracted from Williams 82 seedling leaves to screen for candidate proteins interacting with GmSRC7^TN. The interactions between GmSRC7^TN and GmHSF-01 as well as GmNF-YC03 were further confirmed. Domains are the core functional units of proteins. For instance, the wheat zinc finger protein TaZF interacts with AvrPm2 through its zinc finger domain, thereby promoting pathogen recognition mediated by Pm2a [35]; similarly, PM3b interacts with AVRPM3b2/c2 within plants via its LRR domain [36]. GmSRC7 comprises three domains: TIR, NBS, and BSP. In this study, Y2H and LCA experiments revealed that its TIR domain plays a critical role in protein interactions. The research team led by Professor Chai Jijie systematically elucidated the molecular mechanism by TIR domain to activates the downstream EDS1-PAD4/EDS1-SAG101 complexes through allosteric effects, revealing that the catalytic products of the TIR domain can activate plant immune responses [37, 38]. Yu reported that the TIR protein of the Arabidopsis TNL protein RPS4 can activate the EPA signaling pathway, thereby enhancing plant disease resistance [39]. In summary, we hypothesize that the TIR domain plays an essential role in the pathway through which GmSRC7 interacts with GmHSF-01 and NF-YC03 to regulate antiviral activity in plants.

Heat shock transcription factors (HSFs) play pivotal roles in plant responses to both biotic and abiotic stresses [40]. They participate in the regulation of plant disease resistance through signaling pathways involving ROS, SA, abscisic acid (ABA), ethylene (ET), and jasmonic acid (JA) [41–44]. HSFs specifically bind to heat shock elements (HSEs) in the promoters of target genes, thereby regulating the transcription and expression of heat shock-responsive genes [45]. GmHSF-01 belongs to the transcription repressor HsfB1 gene family [46], is localized to the nucleus, and functions within the nuclear compartment. Previous studies have shown that LlHSFA4 enhances resistance to Botrytis cinerea by forming a transcriptional cascade with LlWRKY33 and LlCAT2, which reduces cell death and ROS accumulation [47]. Overexpression of the nucleus-localized GhHSFB2a in Arabidopsis has been shown to enhance resistance to Verticillium dahliae [48]. The HR leads to the accumulation of ROS at infection sites [49], and excessive ROS levels can trigger apoptosis or even necrosis [50]. Research indicates that HSFs can influence the expression of ROS-related genes by activating heat shock protein (HSP) chaperones [51]. The accumulation of H₂O₂ and O²⁻ in the leaves of this tobacco was investigated using DAB. In this study, we found that the resistance gene GmHSF-01 induces ROS accumulation and enhances the HR response mediated by GmSRC7^TN. Furthermore, SMV failed to spread in transgenic tobacco overexpressing GmHSF-01, suggesting that GmHSF-01 positively regulates antiviral activity in plants.

GmNF-YC03 belongs to the NF-YC subunit gene family. Its HFD dimer can bind to the DNA phosphate backbone, suggesting that NF-Y can recognize and bind to the 25-bp CCAAT nucleotide sequence [52]. NF-Y is composed of three subunits: NF-YA, NF-YB, and NF-YC, and is involved in responses to abiotic stresses such as drought, salinity, nutrient deficiency, and temperature fluctuations [53]. Studies have found that overexpression of OsHAP2E enhances photosynthetic rate, improves drought and salt tolerance, and increases resistance to rice blast [54]. GmNF-YC4-2 confers broad-spectrum resistance and promotes early maturation in soybean by elevating protein levels [55]. Members of the heterotrimeric NF-Y transcription factor complex, such as MeNF-YA1/3, MeNF-YB11/16, and MeNF-YC11/12, are localized to the nucleus, possess CCAAT motifs and transcriptional activation activity, and their overexpression or silencing enhances or reduces plant resistance to Xam, respectively [56]. YB2 and YB3 are functionally redundant and jointly positively regulate plant resistance to the necrotrophic pathogen Botrytis cinerea [57]. NLR proteins initiate antiviral defense mechanisms in plants upon recognition of pathogen-secreted effectors [58]. Our previous research showed that only GmSRC7^BSP strongly interacts with 6K2, whereas GmSRC7^TN does not directly interact with any of the ten components of SMV. This study reveals that GmNF-YC03 localizes to the nucleus and endoplasmic reticulum, while SMV 6K1 localizes to the nucleus and endoplasmic reticulum (Fig. 4E), consistent with previous findings [59–62]. GmNF-YC03 interacts with 6K1 in both the nucleus and endoplasmic reticulum (Fig. 4F). Studies have shown that the 6K1 protein is associated with viral replication [32]. We speculate that GmNF-YC03 may interfere with viral replication and movement by competitively binding to or sequestering 6K1, providing a novel perspective for plant antiviral defense.

VIGS is a widely used method for studying the functions of plant disease resistance-related genes. For instance, silencing the NDR1, RAR1, and HSP90 genes in Arabidopsis leads to the loss of RPS2-mediated resistance [63]. Similarly, Steven employed BSMV carrying fragments of RAR1, SGT1, and HSP90 to silence these genes, revealing their essential roles in Lr21-mediated resistance to wheat leaf rust [64]. Using TurboID-based proximity labeling, the Alfin-like7 (NbAL7) protein was identified as an interactor of the N protein [65]. Subsequent investigations revealed that NbAL7 is a nuclear-localized transcription factor, and knockdown of NbAL7 significantly attenuates N gene-mediated resistance to TMV [66]. In this study, utilizing a TRV-based VIGS system, we found that silencing GmHSF-01 and GmNF-YC03 in transgenic tobacco overexpressing GmSRC7 and in transgenic soybean overexpressing GmSRC7^TN, respectively, after viral infection, led to a significant reduction in virus resistance in these transgenic plants. This indicates that the GmHSF-01 and GmNF-YC03 genes play crucial roles in the antiviral pathway mediated by GmSRC7. Therefore, we propose that GmSRC7^TN functions upstream of the transcription factors GmHSF-01 and GmNF-YC03, and that recruiting these two factors is essential for fully activating downstream defense responses, ultimately resulting in the inhibition of SMV replication.

Conclusions

In this study, using GmSRC7^TN as a bait protein, 42 candidate interacting proteins were screened. These genes were found to be associated with photosynthesis, plasma membrane transport, and mRNA binding functions. Further validation confirmed the interaction between GmSRC7^TN and GmHSF-01/GmNF-YC03, and revealed that GmSRC7^TIR plays a critical role in this interaction process. Experiments involving transient overexpression in tobacco and stable overexpression in OE-GmHSF-01 transgenic tobacco demonstrated the antiviral activity of GmHSF-01. Notably, GmNF-YC03 not only interacts with GmSRC7^TN but also with the SMV-encoded 6K1 protein, suggesting that GmNF-YC03 may inhibit viral replication and movement by competitively binding to or sequestering 6K1. Subsequent VIGS assays further confirmed that GmHSF-01 and GmNF-YC03 negatively regulate SMV infection in plants. In summary, GmSRC7^TN interacts with the transcription factors GmHSF-01 and GmNF-YC03 to jointly regulate the plant’s disease resistance mechanisms (Fig. 6H). These findings provide important insights for further exploration of the GmSRC7^TN-mediated disease resistance signaling pathway and offer potential molecular targets for the genetic improvement of resistant soybean varieties.

Authors’ contributions

Investigation, Q.C.G, Z.B and Z.K.Z; formal analysis, Q.C.G, Z.B. and N.N. ; writing—original draft preparation, Q.C.G,; writing—review and editing, Hada.; funding acquisition, Hada. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Jilin Province (YDZJ202601ZYTS272), Science and Technology Program of Inner Mongolia (2020GG0045), Innovative Research Team in Universities of Inner Mongolia Autonomous Region (NMGIRT2329), Natural Science Foundation of Inner Mongolia Autonomous Region (2020MS03023), National Natural Science Foundation of China (31570142) and National Natural Science Foundation of China (32260079).

Data availability

All datasets supporting the conclusions of this study are included in the article and its supplementary information files. Soybean gene sequences were retrieved from the National Center for Biotechnology Information (NCBI), and the corresponding accession numbers are provided in Supplemental Table S1. Specifically, the soybean TUA gene (Accession No.: XM_006584532.1) and the Nicotiana benthamiana Actin (Tac9) gene (Accession No.: X69885.1) were obtained from the NCBI database.

Declarations

Ethics approval and consent to participate

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Competing interests

The authors declare no competing interests.