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.

Soybean mosaic virus (SMV) establishes successful infection through the coordinated action of multiple viral proteins, yet how these components are collectively engaged in host processes remains unclear. Using 10 SMV-encoded proteins as baits, we screened a soybean (Glycine max) cDNA library using the yeast two-hybrid (Y2H) method and obtained 200 positive clones corresponding to 147 nonredundant candidate host proteins. Integrative analyses indicated that these interacting proteins were mainly associated with host processes related to photosynthesis and energy metabolism, as well as protein quality control (PQC), and that their promoters were enriched in light- and stress-responsive elements. Photosynthesis-related genes were more strongly perturbed in the susceptible cultivar, whereas PQC-module genes showed overall downregulation in both resistant and susceptible cultivars upon SMV infection. Y2H, Bimolecular Fluorescence Complementation (BiFC), and transient expression assays identified four soybean resistance factors that interact with SMV proteins. These factors delayed systemic spread and continued to restrict viral proliferation after systemic infection was established. Notably, SMV accumulation partially rebounded when viral proteins were co-expressed with GmSTOP1, GmHrBP1, or GmGIP2, while co-expression of GmSiPPase and NIa-Pro further inhibited viral proliferation. In summary, this study maps the host-targeting profile of SMV across multiple viral components and provides clues to the SMV–soybean interaction network and antiviral gene resources in soybean.

Defense responses against pathogens are essential for plant growth, and the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. Besides NBS and LRR domains, NLR proteins also contain TIR, CC, or RPW8 domain at their N-terminus and the functions of these canonical domains are well documented. Recently, some atypical X domains, also named integrated domains (IDs), have been reported to be encoded by NLR genes and to play key roles in effector-triggered immunity. Here, we screened the plant NLR database to identify atypical NLRs in potato. A total of 181 atypical NLR protein entries, identified from 642 candidates as truncated or St-NLR-IDs (potato NLRs encoding ID) and comprising 147 NLR-IDs and 34 truncated NLRs, were classified into four major groups, including T(TIR)-class, C(CC)-class, N(NBS)-class, and X-class, and together with 12 typical CNL/TNL sequences, they were used for phylogenetic analysis. Promoter regions of the majority of these entries contain “stress and defense” elements such as MYB/MYC cis-regulatory element, indicating their involvement in plant immune response. In the Rywal background, the expression of six genes was upregulated and that of nineteen genes was downregulated. In the NahG-Rywal plants, the expression of seventeen genes was upregulated and that of eight genes was downregulated. In the shRBOHD-Rywal background, the expression of eighteen genes was upregulated and that of six genes was downregulated. We also investigated the expression of four C-JID-containing NLR-ID genes, M1BEE2, M1BF53, M1A5Z0, and M1BZB1, upon PVX, PVY infection and SA treatment by RT-qPCR and suggested their potential involvement in plant immunity. Promoter–GUS assays for the above four genes also supported their responses to viral infection and SA treatment. In summary, we identified and classified atypical NLRs in potato, analyzed their expression under PVY^N-Wilga infection using public transcriptome data, and validated the virus- and SA-responsive expression of four C-JID-encoding genes, providing a resource for future functional studies.