Soybean mosaic virus (SMV) is a severe soybean (Glycine max) pathogen. Here we characterize a soybean SMV resistance cluster (SRC) that comprises five resistance (R) genes. SRC1 encodes a Toll/interleukin-1 receptor and nucleotide-binding site (TIR-NBS [TN]) protein, SRC4 and SRC6 encode TIR proteins with a short EF-hand domain, while SRC7 and SRC8 encode TNX proteins with a noncanonical basic secretory protein (BSP) domain at their C-termini. We mainly studied SRC7, which contains a noncanonical BSP domain and gave full resistance to SMV. SRC7 possessed broad-spectrum antiviral activity toward several plant viruses including SMV, plum pox virus, potato virus Y, and tobacco mosaic virus. The TIR domain alone was both necessary and sufficient for SRC7 immune signaling, while the NBS domain enhanced its activity. Nuclear oligomerization via the interactions of both TIR and NBS domains was essential for SRC7 function. SRC7 expression was transcriptionally inducible by SMV infection and salicylic acid (SA) treatment, and SA was required for SRC7 triggered virus resistance. SRC7 expression was posttranscriptionally regulated by miR1510a and miR2109, and the SRC7-miR1510a/miR2109 regulatory network appeared to contribute to SMV–soybean interactions in both resistant and susceptible soybean cultivars. In summary, we report a soybean R gene cluster centered by SRC7 that is regulated at both transcriptional and posttranscriptional levels, possesses a yet uncharacterized BSP domain, and has broad-spectrum antiviral activities. The SRC cluster is special as it harbors several functional R genes encoding atypical TIR-NBS-LRR (TNL) type R proteins, highlighting its importance in SMV–soybean interaction and plant immunity.

The recognition of pathogen effectors through the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. In addition to typical domains such as TIR, CC, NBS, and LRR, NLR proteins also contain some atypical integrated domains (IDs), the roles of which are rarely investigated. Here, we carefully screened the soybean (Glycine max) genome and identified the IDs that appeared in the soybean TNL-like proteins. Our results show that multiple IDs (36) are widely present in soybean TNL-like proteins. A total of 27 Gm-TNL-ID genes (soybean TNL-like gene encoding ID) were cloned and their antiviral activity towards the soybean mosaic virus (SMV)/tobacco mosaic virus (TMV) was verified. Two resistance (R) genes, SRA2 (SMV resistance gene contains AAA_22 domain) and SRZ4 (SMV resistance gene contains zf-RVT domain), were identified to possess broad-spectrum resistance characteristics towards six viruses including SMV, TMV, plum pox virus (PPV), cabbage leaf curl virus (CaLCuV), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV). The effects of Gm-TNL-ID^X (the domain of the Gm-TNL-ID gene after the TN domain) on the antiviral activity of a R protein SRC7^TN (we previously reported the TN domain of the soybean broad-spectrum resistance gene SRC7) were validated, and most of Gm-TNL-ID^X inhibits antiviral activity mediated by SRC7^TN, possibly through intramolecular interactions. Yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that seven Gm-TNL-ID^X interacted with SMV-component proteins. Truncation analysis on a broad-spectrum antiviral protein SRZ4 indicated that SRZ4^TIR is sufficient to mediate antiviral activity against SMV. Soybean cDNA library screening on SRZ4 identified 48 interacting proteins. In summary, our results indicate that the integration of IDs in soybean is widespread and frequent. The NLR-ID toolkit we provide is expected to be valuable for elucidating the functions of atypical NLR proteins in the plant immune system and lay the foundation for the development of engineering NLR for plant-disease control in the future.

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.

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.

Aphid infestation constrains goji berry production, yet its defense architecture remains poorly resolved. We integrated comparative transcriptomics, phytohormone profiling, small RNA sequencing, and genome-wide nucleotide-binding leucine-rich repeat (NLR) analysis to dissect aphid defense in Lycium barbarum (cv. Ningqi-10)and susceptible L. chinense. Aphid bioassays confirmed that L. barbarum imposes a significant fitness cost on aphids, reducing survival, longevity, and fecundity. Transcriptomic comparison revealed fewer but more defense-enriched differentially expressed genes in the resistant accession. Weighted gene co-expression network analysis (WGCNA) identified a stress-associated turquoise module (288 genes, r = 0.981) receptor-like kinases and mitogen-activated protein kinase (MAPK) components, bridging extracellular signals sensing and hormone signaling. Phytohormone profiling showed that salicylic acid (SA) accumulated to 72 h, whereas jasmonic acid (JA) peaked transiently at 24 h, producing an increased SA/JA ratio suggesting a shift to SA-dominated signaling. MiR398a-3p and miR319-3p dynamics coincided with this SA/JA shift, suggesting a possible link to hormone-coordinated regulation and candidate targets for validation. NLR analysis identified 151 candidate genes, including 18 L. barbarum-biased NLRs up-regulation under SA-dominated signaling, implying a link to SA signaling. Collectively, these findings support a multi-layered framework integrating SA signaling, transcriptional reprogramming, and coupled miRNA and NLR responses in aphid defense.

SUMMARY: The typical nucleotide‐binding leucine‐rich repeat (NLR) proteins, such as TIR‐NBS‐LRR (TNL) and CC‐NBS‐LRR (CNL), are known to be engaged in effector‐triggered immunity (ETI). However, several atypical resistance (R) proteins with truncated NLR domains or with nonclassical domains are emerging to be key regulators in plant immunity. SMV resistance cluster 4 (SRC4) is an atypical NLR protein in soybean, distinguished by an N‐terminal Domain (NTD)‐TIR‐shortened NBS oligomerization region (SNOR)‐EF‐hand (EFh) domain architecture. SRC4 lacks both NBS and LRR domains but retains basal antiviral activity through its TIR‐SNOR region, with SNOR contributing to TIR‐dependent antiviral activity and SRC4 self‐association. The EFh domain recognizes coat protein (CP) of soybean mosaic virus (SMV) and serves as a Ca ²⁺ ‐responsive module to distinctly regulate TIR‐SNOR‐mediated immune response in soybean. SRC4‐ overexpression (Ox‐ SRC4 ) soybean plants show SMV resistance without obvious growth penalty. Furthermore, several SRC4 high‐expression soybean varieties showed stronger SMV resistance and Ca ²⁺ responsiveness than the SRC4 low‐expression soybean varieties. Our study highlights the essential roles of atypical resistance proteins in plant immunity and growth‐defense balance.