Screening and mechanistic analysis of soybean varieties resistant to stay‐green syndrome caused by the soybean stay‐green‐associated virus

Abstract

Soybean, one of the world's most economically significant crops, contributes 25% of global edible oil production and provides two-thirds of the concentrated protein for livestock feed. However, in recent years, the emergence of soybean stay-green syndrome (SGS), or ‘zhengqing’, in the Huang-Huai-Hai region of China, has become a major threat to soybean production. This syndrome has led to severe yield losses, with some areas reporting complete crop failure (Li et al., 2019; Wei et al., 2023; Xu et al., 2019). The issue is expanding and has become a prominent concern across China (Cheng et al., 2023; He et al., 2025; Xu et al., 2019). SGS is characterized by delayed senescence at harvest, with soybean leaves remaining green, pods failing to form, and seed development stalling, leading to few or flat pods (Cheng et al., 2022; Li et al., 2019; Wang et al., 2022; Wei et al., 2023). The profound impact of this syndrome on both yield and quality presents a significant challenge for the soybean industry in China. Recently, our research has identified the aetiology of SGS as a novel geminivirus, named soybean stay-green-associated geminivirus (SoSGV). Through Koch's postulates, we have demonstrated for the first time that SoSGV causes the stay-green symptoms in soybeans (Cheng et al., 2022). SoSGV is a monopartite, single-strand DNA virus, likely resulting from intergeneric recombination of geminiviruses (Cheng et al., 2022). Furthermore, we have identified the common brown leafhopper (Orosius orientalis) as the transmission vector of SoSGV (Cheng et al., 2023). Previous reports have indicated that SGS presents diverse phenotypic expressions in the field, with variations in symptom severity (Cheng et al., 2023; Xu et al., 2019). However, the causes of this variability are unclear, and it is uncertain whether differences in symptom expression are linked to SoSGV infection across various soybean cultivars. Breeding for host-plant resistance is a cost-effective and environmentally friendly strategy to enhance crop yield. However, progress in identifying SoSGV-resistant soybean varieties has been limited. In this study, we utilized a vector-transmitted inoculation system developed in our laboratory to assess SoSGV resistance in 36 elite soybean germplasm from the Huang-Huai-Hai region (Table S1). Our findings revealed that the majority of these varieties were highly susceptible to SoSGV. To determine whether SoSGV induces stay-green symptoms at maturity, sixteen soybean varieties infected with SoSGV and mock controls were transplanted to the field one month after inoculation. These varieties, selected for their relatively weak phenotypes during early-stage SoSGV infection, were monitored from July to October (2022–2024). All mock-treated plants exhibited yellowing and mature seeds, while 15 of the 16 varieties, except Fudou_18, showed delayed leaf senescence and flattened pods (Figure 1a,b). Ten varieties (Jiadou_2, Jidou_12, Xiangfeng_4, Andou_203, Huadou_19, Lindou_11, Xudou_20, Shengdi_1, Lindou_10, and Zhonghuang_13) exhibited dwarfing at maturity. The remaining five varieties (Wandou_38, Tianyikedou_18, Hedou_12, Huaidou_11, and Shidou_1) showed no significant changes in plant height (Figure 1a; Figure S2a). Pod number was reduced in most dwarf varieties, except Xudou_20 (Figure 1b; Figure S2b). In Xudou_20 and Shidou_1, pod number was unaffected, but pod filling was severely reduced, with over 80% flat pods (Figure S2c). These results suggested that phenotypic variation among soybean varieties manifested upon SoSGV infection. To assess the impact of SoSGV infection on yield, we analyzed infected soybean varieties and observed yield losses ranging from 15.8% to 97.05% (Figure 1b,c; Figure S3a). Among these varieties, ten exhibited severe yield reductions exceeding 80%, including Jiadou_2, Wandou_38, Tianyikedou_18, Jidou_12, Xiangfeng_4, Huadou_19, Lindou_11, Xudou_20, Lindou_10, and Zhonghuang_13 (Figure S3a). Interestingly, Huaidou_11 demonstrated the lowest yield loss (36.19%) despite displaying evident stay-green symptoms (Figure 1a). This relatively low yield loss aligns with our observation that SoSGV infection did not significantly affect the total pod number or flat pod rate in Huaidou_11 (Figure S3b). However, infected Huaidou_11 produced more abnormal seeds, marked by smaller size, altered shape and incomplete maturation, severely reducing seed quality (Figure 1b; Figure S3b). Fudou_18 exhibited remarkable tolerance to SoSGV infection, as reflected in its mild symptom severity (Figure 1c; Figure S1). While plants showed symptoms such as leaf curling and dwarfing during the early stages of infection (Figure S1), only mild stay-green symptoms were observed at maturity, with no significant difference in plant height or pod maturation compared to the mock treatment (Figure 1c, Figure S1b). SoSGV infection did not significantly affect the total pod number or flat pod rate in Fudou_18 (Figure 1c). Despite a slight reduction in mean seed width from 7.705 mm to 6.872 mm (Figure 1c), the 100-grain weight decreased by only 18%. Total yield per plant was reduced by approximately 15.8% a relatively minor impact compared to other varieties that experienced greater yield losses (Figure 1c). Furthermore, the seeds of SoSGV-infected Fudou_18 maintained a uniform shape, consistent with those from the control (Figure 1c). To explore whether the mild symptoms observed in Fudou_18 were associated with lower virus titre, we performed quantitative polymerase chain reaction using infected leaves to quantify SoSGV accumulation at the R5 stage in Zhonghuang_13, Huaidou_11, Wandou_38, and Fudou_18. The results indicated that SoSGV accumulation was lowest in Fudou_18 compared to the other three cultivars (Figure 1d), suggesting that Fudou_18 exhibits resistance to SoSGV infection, potentially through mechanisms that restrict virus accumulation. Next, to elucidate the molecular mechanisms underlying the resistance of Fudou_18 to SoSGV, we performed transcriptomic and metabolomic analyses on Fudou_18 and two susceptible cultivars (Williams 82 and Zhonghuang 13) following SoSGV infection. Transcriptome analysis first revealed genes that were differentially expressed (DEGs) between infected versus mock-treated plants (Figure S4a; Table S2) with KEGG analysis revealing that metabolic genes (particularly for specialized metabolism) are significantly enriched (Figure S4b). We further conducted non-targeted metabolomics analysis on the same samples Fudou_18, Williams 82, and Zhonghuang 13 used for transcriptome analysis. Principal component analysis (PCA) demonstrated a clear separation in the global metabolic profiles among cultivars (Figure S4c). While SoSGV-infected Fudou_18 and the susceptible cultivars exhibited only a minor metabolome change compared to controls (Figure S4c). Differentially accumulated metabolites (DAMs) analysis revealed that there is a clear differentiation of metabolic profiles among the three cultivars and a large number of DAMs were significantly upregulated in Fudou_18 compared with the susceptible cultivars (Figure S4d; Table S3). Enrichment pathway analysis of DAGs identified that those significantly enriched KEGG pathways were involved in phenylpropanoid and starch and sucrose metabolism in Fudou_18 post-infection, indicating that phenolics and sugars might be subjected to metabolic reprogramming after SoSGV infection (Figure S4e,f). We further conducted a comparative analysis between Fudou_18 and the two susceptible cultivars by plotting significant differences versus fold changes of each mass feature (Figure 1e). Notably, kaempferol glucoside (m/z 595.16 @6.4 and 6.7 min, respectively) and apigenin glucoside (m/z 741.22 @5.4 min) were highly accumulated in Fudou_18, less accumulated in Williams 82 and merely accumulated in Zhonghuang 13 (Figure 1e,f), a pattern highly consistent with SoSGV resistance of the three cultivars. To validate the role of flavonoid/isoflavonoid pathways in Fudou_18 resistance to SoSGV, chalcone synthase 1 (CHS1, Glyma.19G105100 and Glyma.06G118550), a key enzyme in this pathway, was silenced using apple latent spherical virus-induced gene silencing (VIGS). Silencing CHS1 expression in Fudou_18 significantly compromised plant resistance to SoSGV, as evidenced by increased SoSGV DNA and coat protein (CP) accumulations, along with severe plant dwarfing at both early and late stages of infection (Figure S5). Moreover, silencing CHS1 expression also increased the flat pod rate following SoSGV infection compared to the control (Figure S5f). We also used a soybean line that stably overexpressing soybean isoflavone synthase 2 (IFS2), a key enzyme involved in isoflavone accumulation (Jung et al., 2000). After SoSGV inoculation, overexpression of IFS2 in soybean cultivar ‘Jack’ significantly enhanced plant resistance to SoSGV compared to ‘Jack’ control plants, as evidenced by decreased SoSGV DNA and CP accumulations (Figure S6). These findings highlight the pivotal role of the flavonoid/isoflavonoid pathway in soybean resistance to SoSGV. Collectively, through large-scale screening, Fudou_18 as a SoSGV-tolerant variety. Elucidating its resistance mechanisms lays a promising foundation for developing soybean cultivars with enhanced resistance to this critical viral disease. This work was supported by the National Key Research and Development Program of China (2022YFC2602000), Biological Breeding-National Science and Technology Major Project 2030 (2023ZD04037), and the Outstanding Youth Fund of Jiangsu Province (BK20231534). The author(s) declare that they have no conflict of interest. H.A., R.C., Y.R., Y.W., K.X., W.Y., W.Y., H.C., and D.Y. performed experiments and analysed the data. Y.X., D.L., Z.L., and X.T. designed the project. Y.X., Z.L., and D.L. supervised the study and wrote the manuscript. All authors revised and approved the manuscript. The data that supports the findings of this study are available in the supplementary material of this article. The raw transcriptome sequencing data have been deposited in the China National GeneBank (CNGB) under accession number PRJCA039705. Figure S1-S6 Supplementary Figures. Table S1-S4 Supplementary Tables. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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