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