Soybean, an important economic and oil crop, is frequently threatened by soybean mosaic virus (SMV) disease, which is caused by the soybean mosaic virus (SMV). Previous studies identified a differentially expressed C₂H₂ single zinc finger protein gene, GmSZFP, which positively regulated soybean resistance to SMV infection. Using a yeast two-hybrid library, we screened for proteins that interact with GmSZFP and explored their functions in the soybean-SMV interaction. This research provides a theoretical basis for further elucidating the regulatory network of transcription factors involved in soybean resistance to SMV infection.

In this study, the compatible (Jidou 7 and SMV strain SC-8) and incompatible (Jidou 7 and SMV strain N3) combinations were used to screen the potential interaction proteins of GmSZFP by yeast two-hybrid library. the protein-protein interactions were validated through yeast two-hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC). Real-time quantitative PCR (qPCR) and Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) were employed to analyze the transcriptional expression levels and functions of the identified interaction proteinin the soybean-SMV interaction.

We identified GmERF7 as apotential target protein of GmSZFP through yeast two-hybrid system. The interaction between GmSZFP and GmERF7 was confirmed by Y2H and BiFC. GmERF7 is a 393 -amino acid protein containing an AP2/ERF domain and two nuclear localization signals (NLS). qPCR results showed that the expression levels of GmERF7 were significantly higher in the compatible combination than those in the incompatible combination at 4, 12, and 24 hours after infection, peaking at 24 hours. VIGS-mediated silencing of GmERF7 in Jidou 7 plants infected with SC-8 (compatible combination) results in increased callose deposition at the inoculation site compared to the control. At the 14 days after inoculation, the expression of SMV coat protein CP gene was undetectable in the upper leaves of the GmERF7-silenced plants, and no SMV infection symptoms were observed. In contrast, the CP gene was detected in the control plants, which exhibited typical susceptibility symptoms such as mosaic and green-loss. These results indicate silencing GmERF7 weakened virus transport and spread between cells, thereby enhancing plant resistance to SMV. This suggest that GmERF7 negatively regulates soybean resistance to SMV.

The ERF transcription factor GmERF7 interacts with the zinc finger protein GmSZFP, and negatively regulates soybean resistance to SMV.

The molecular mechanism underlying the resistance to soybean mosaic virus (SMV) infection in soybean is of great importance as soybean mosaic caused by SMV has become one of the major soybean disease worldwide. We have previously performed transcriptome analysis of SMV-inoculated soybean after inhibition of H₂O₂ production and have identified a differentially expressed C₂H₂-type single zinc finger protein gene, Glyma.18G003600.1, named GmSZFP. In this study, we use virus induced gene silencing (VIGS) technique to investigate the function of GmSZFP in soybean-SMV interaction, providing a foundation for further investigation of the molecular mechanism of GmSZFP in soybean-SMV interaction.

Soybean cultivar Jidou 7 and SMV strains SC-8 (susceptive) and N3 (resistance) were used as the materials in this study. Bioinformatic analysis was conducted to predict the protein domains of GmSZFP; its transcription factor activity was measured by transcriptional activation assay in yeast; the expression characteristics of GmSZFP at the transcriptional level in soybean-SMV interaction were verified by real-time quantitative PCR (qPCR); and the function of GmSZFP in soybean-SMV interaction was investigated by VIGS technique.

The CDS region of GmSZFP gene was cloned; amino acid sequence analysis and transcriptional activation assay in yeast revealed that GmSZFP is a C₂H₂-type zinc finger protein transcription factor with transcriptional activation activity; qPCR results showed that GmSZFP was strongly induced by SMV inoculation, and the expression pattern was different between the compatible and the incompatible combinations. The expression of GmSZFP was elevated after SMV inoculation, and then decreased in the incompatible combination, and the expression level of GmSZFP was significantly lower in the compatible combination than that in the former. Moreover, the expression level of GmSZFP was found to be reduced to the level that is similar to the level in the compatible combination that was pre-inoculated with imidazole, indicating that GmSZFP responds to SMV infestation at the transcriptional level and is regulated by H₂O₂; After silencing GmSZFP, we found that callose at the SMV inoculation site was greatly reduced compared to the control, and the expression of callose synthase genes GmGSL7c and GmGSL12b was reduced compared to the control, and the expression of callose hydrolase gene BG was elevated compared to the control; In addition, after GmSZFP was silenced, the virus spread outward to a distance of 2 mm at 72 h and to a distance of 3 mm at 96 h from the central source after SMV was inoculated in a small area, while the expression of SMV capsid protein (CP) gene was not detectable outside the inoculation site in control leaves; 10 d after SMV inoculation, the upper leaves (of the SMV inoculated leaves) in the GmSZFP-silenced plants showed mosaic, greening and curling symptoms, and CP gene was expressed, indicating that silencing of GmSZFP enabled SMV to transport in an unrestricted manner.

GmSZFP is a canonical C₂H₂-type mono-zinc finger protein, and the GmSZFP gene plays a positive regulatory role in soybean resistance to SMV infection.