Genes involved in disease resistance are crucial for plant immune systems, yet their transcriptional regulatory mechanisms remain poorly understood. SRC4, a key member of the soybean mosaic virus resistance cluster (SRC), encodes a Ca²⁺-binding EF-hand domain and possesses antiviral activity, but its expression regulation is unclear. Here, we systematically analyzed 4085 soybean (Glycine max) transcriptome datasets and conducted SMV inoculation experiments to characterize SRC4 expression patterns. Cis-acting element analysis identified 12 regulatory elements in the SRC4 promoter, including salicylic acid (SA)-responsive elements. Furthermore, a Pro_SRC4::GUS reporter vector was constructed and functional analysis was performed in tobacco (Nicotiana benthamiana) and transgenic Arabidopsis thaliana. SRC4 exhibited significantly higher basal expression than typical resistance genes (R genes) and was induced by SMV infection, SA treatment, and Ca²⁺ supplementation, with peak expression at 2–5 h post-treatment (hpi). In transgenic tobacco overexpressing NahG, neither SMV nor Ca²⁺ could induce Pro_SRC4::GUS expression, demonstrating that SRC4 transcriptional regulation is mediated through SA signaling pathways. SRC4 showed predominant expression in roots and leaves and responded to temperature stress. Transgenic plants overexpressing SRC4 exhibited enhanced tolerance to both 12 °C and 37 °C temperature stress. This study elucidates the molecular mechanisms underlying SRC4 transcriptional regulation through Ca²⁺ and SA signaling pathways, revealing its dual role in both biotic and abiotic stress responses, especially in temperature stress.

Most of R (resistance) genes encode the protein containing NBS-LRR (nucleotide binding site and leucine-rich repeat) domains. Here, N. benthamiana plants were used for transient expression assays at 3–4 weeks of age. We identified a TNL (TIR-NBS-LRR) encoding gene GmRUN1 that was resistant to both soybean mosaic virus (SMV) and tobacco mosaic virus (TMV). Truncation analysis indicated the importance of all three canonical domains for GmRUN1-mediated antiviral activity. Promoter-GUS analysis showed that GmRUN1 expression is inducible by both salicylic acid (SA) and a transcription factor GmDREB3 via the cis-elements as-1 and ERE (ethylene response element), which are present in its promoter region. Interestingly, GmRUN1 gDNA (genomic DNA) shows higher viral resistance than its cDNA (complementary DNA), indicating the existence of intron-mediated enhancement (IME) for GmRUN1 regulation. We provided evidence that intron2 of GmRUN1 increased the mRNA level of native gene GmRUN1, a soybean antiviral gene SRC7 and also a reporter gene Luciferase, indicating the general transcriptional enhancement of intron2 in different genes. In summary, we identified an antiviral TNL type soybean gene GmRUN1, expression of which was regulated at different layers. The investigation of GmRUN1 gene regulatory network would help to explore the mechanism underlying soybean-SMV interactions.

Plant viral diseases cause severe yield losses in global crop production. SRC7, a novel soybean TIR-NBS-BSP-type resistance (R) protein, confers broad-spectrum antiviral immunity against multiple viruses including soybean mosaic virus (SMV), tobacco mosaic virus (TMV), potato virus Y (PVY), and plum pox virus (PPV). Its TIR-NBS domain (SRC7^TN) mediates full antiviral resistance, making it an ideal bait for screening immune-related proximal candidate proteins. In this study, TurboID-based proximity labeling (PL) was applied to profile the SRC7^TN proximal candidate proteins in N. benthamiana. We identified 262 high-confidence proximal candidate proteins, which were mainly enriched in plant immune signaling, redox regulation, protein modification and nuclear-related pathways. Among them, 21 candidates were functionally characterized, revealing multiple host factors contributing to SRC7-dependent antiviral immunity. Eleven interactions were confirmed via bimolecular fluorescence complementation (BiFC), and two nucleoside/nucleotide hydrolase-family proteins MZN14.5 and RIHA were subjected to further interaction-domain analysis. Collectively, this work constructs a high-confidence SRC7^TN proximal-interaction network and highlights critical host factors in SRC7-mediated broad-spectrum antiviral immunity. It also validates TurboID’s utility for screening for NLR-proximal candidate proteins in heterologous plant systems and provides insights into R-protein-governed plant antiviral defense.