Soybean mosaic virus (SMV), an RNA virus, is the most common and destructive pathogenic virus in soybean fields. The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to viruses; hence, this study aimed to engineer SMV resistance in soybean using this system. Specifically, multiple sgRNAs were designed to target positive- and/or negative-sense strands of the SMV HC-Pro gene. Subsequently, the corresponding CRISPR/CasRx vectors were constructed and transformed into soybeans. After inoculation with SMV, 39.02%, 35.77%, and 18.70% of T₁ plants were confirmed to be highly resistant (HR), resistant (R), and mildly resistant (MR) to SMV, respectively, whereas only 6.50% were identified as susceptible (S). Additionally, qRT-PCR and DAS-ELISA showed that, both at 15 and 30 d post-inoculation (dpi), SMV accumulation significantly decreased or was even undetectable in HR and R plants, followed by MR and S plants. Additionally, the expression level of the CasRx gene varied in almost all T₁ plants with different resistance level, both at 15 and 30 dpi. Furthermore, when SMV resistance was evaluated in the T₂ generation, the results were similar to those recorded for the T₁ generation. These findings provide new insights into the application of the CRISPR/CasRx system for soybean improvement and offer a promising alternative strategy for breeding for resistance to biotic stress that will contribute to the development of SMV-immune soybean germplasm to accelerate progress towards greater soybean crop productivity.

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.

Soybean mosaic virus (SMV) is a member of the genus Potyvirus that extensively impairs global soybean production. The full-length coding sequence of the MADS-box transcription factor GmCAL was cloned from the SMV-resistant soybean cultivar Kefeng 1. SMV-induced expression analysis indicated that GmCAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2. GmCAL was expressed at high levels in flowers and pods but at lower levels in leaves. The gene was localized to the nucleus by subcellular localization assay. Virus-induced gene silencing did not increase the accumulation of SMV in GmCAL-silenced Kefeng 1 plants (with silencing efficiency ∼ 80%) after SC8 inoculation. GmCAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression. SMV content decreased significantly in GmCAL-overexpressing NN1138-2 plants after SMV-SC3, SMV-SC7, and SMV-SC8 inoculation in comparison with a vector control, showing that overexpression of GmCAL conferred broad-spectrum resistance to multiple SMV strains. These results confirm that GmCAL, a key regulator but not a specific SC8 resistance gene (Rsc8), is a positive regulatory transcription factor involved in soybean resistance to SMV.