Soybean mosaic virus (SMV) poses a substantial threat to the yield and quality of soybean (Glycine max (L.) Merr.), leading to significant economic losses in soybean production. However, the mining of SMV-resistance loci and the exploration of the underlying disease resistance mechanisms remain relatively limited. MicroRNAs (miRNAs) are a class of post-transcriptional regulators that play a pivotal role in modulating plant growth, development and responding to various stresses. In this study, we demonstrated the function of the “miR398c/d-GmCSDs” module between soybean resistant and susceptible varieties, focusing on its differential regulatory roles in SMV infection. Specifically, SMV infection downregulated gma-miR398c/d expression in the resistant variety (Qihuang 1, QH), while upregulated them in the susceptible variety (Nannong 1138-2, NN). Transient expression assay in N. benthamiana confirmed that gma-miR398c/d can target six superoxide dismutase (SOD) family genes, which responded to SMV infection in both varieties. Stable overexpression of Gma-MIR398c/d in soybean or inhibition of the corresponding target genes’ expression via Bean pod mottle virus (BPMV)-induced gene silencing (VIGS) led to reduced H₂O₂ content and thereby promoted SMV infection. Conversely, plants overexpressing the target genes exhibited the opposite phenotypes. The functions of gma-miR398c/d and their target genes were further validated in N. benthamiana through transient co-expression with SMV infectious clone (pSC7-GFP), indicating that gma-miR398c/d negatively regulated soybean resistance to SMV, while the target genes positively contributed to disease resistance. Collectively, our findings provide novel insights into the regulatory mechanisms underlying soybean resistance to SMV.

Drought-induced protein 19 (Di19) is a Cys2/His2 zinc-finger protein that functions in plant growth and development and in tolerance to abiotic stresses. GmPUB21, an E3 ubiquitin ligase, negatively regulates drought and salinity response in soybean. We identified potential interaction target proteins of GmPUB21 by yeast two-hybrid cDNA library screening, GmDi19-5 as a candidate. Bimolecular fluorescence complementation and glutathionine-S-transferase pull-down assays confirmed the interaction between GmDi19-5 and GmPUB21. GmDi19-5 was induced by NaCl, drought, and abscisic acid (ABA) treatments. GmDi19-5 was expressed in the cytoplasm and nucleus. GmDi19-5 overexpression conferred hypersensitivity to drought and high salinity, whereas GmDi19-5 silencing increased drought and salinity tolerance. Transcripts of ABA- and stress response-associated genes including GmRAB18 and GmDREB2A were down-regulated in GmDi19-5-overexpressing plants under drought and salinity stresses. ABA decreased the protein level of GmDi19-5 in vivo, whereas GmPUB21 increased the decrease of GmDi19-5 after exogenous ABA application. The accumulation of GmPUB21 was also inhibited by GmDi19-5. We conclude that GmPUB21 and GmDi19-5 collaborate to regulate drought and salinity tolerance via an ABA-dependent pathway.