Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes significant reductions in soybean yield and seed quality. In this study, both tag-free and reporter gene green fluorescent protein (GFP)-containing infectious clones for the SMV N1 strain were constructed by Gibson assembly and with the yeast homologous recombination system, respectively. Both infectious clones are suitable for agroinfiltration on the model host N. benthamiana and show strong infectivity for the natural host soybean and several other legume species. Both infectious clones were seed transmitted and caused typical virus symptoms on seeds and progeny plants. We used the SMV-GFP infectious clone to further investigate the role of key amino acids in the silencing suppressor helper component-proteinase (Hc-Pro). Among twelve amino acid substitution mutants, the co-expression of mutant 2—with an Asparagine→Leucine substitution at position 182 of the FRNK (Phe-Arg-Asn-Lys) motif—attenuated viral symptoms and alleviated the host growth retardation caused by SMV. Moreover, the Hc-Prom2 mutant showed stronger oligomerization than wild-type Hc-Pro. Taken together, the SMV infectious clones will be useful for studies of host–SMV interactions and functional gene characterization in soybeans and related legume species, especially in terms of seed transmission properties. Furthermore, the SMV-GFP infectious clone will also facilitate functional studies of both virus and host genes in an N. benthamiana transient expression system.

GmSRC7 is a broad-spectrum antiviral R gene from soybean, but its downstream and functionally related genes remain unclear. Virus-induced gene silencing (VIGS) assays in Nicotiana benthamiana (Nb) showed that suppression of several gene families—WRKY transcription factors, chaperones, ethylene pathway components, MAPK cascade elements, salicylic acid (SA) signaling genes, calcium-dependent protein kinases, nuclear migration proteins, RNA replication-related genes, and immune regulators—consistently weakened GmSRC7-mediated resistance to Soybean Mosaic Virus (SMV) and Tobacco Mosaic Virus (TMV). Targeted silencing of four regulatory genes—NbEDS1, NbARF1, NbSGT1, and NbCOI1—markedly enhanced GmSRC7-mediated resistance to SMV and TMV in our experiments. Silencing the serine/threonine kinase gene NbPBS1 increased GmSRC7-conferred resistance to SMV but did not significantly alter its resistance to TMV. Transient expression assays showed that NbARF1, NbSGT1, and NbCOI1 antagonize GmSRC7-mediated defense against SMV and TMV, whereas NbPBS1 specifically suppresses anti-SMV activity without affecting TMV resistance. Transient overexpression of SA-degrading enzymes (AtS3H, AtS5H, and NahG) significantly reduced GmSRC7-conferred resistance to SMV, indicating that SA is essential for this R protein-mediated defense. Genes were also grouped by immune pathways and function: co-expression of chaperone family genes inhibited GmSRC7 activity against SMV and TMV, while co-expression of WRKY family genes enhanced anti-SMV activity of GmSRC7. Finally, transient silencing of soybean genes GmEDS1, GmSGT1-1, GmSGT1-2, GmJAR1, and GmSGS3 compromised GmSRC7-mediated resistance to SMV.

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.