Two Glutathione S‐Transferase Genes Confer Resistance to Gibberella Ear Rot and Stalk Rot in Maize

Maize (Zea mays L.) is a globally important crop, but its yield and quality are significantly reduced by Fusarium graminearum-induced diseases such as Gibberella ear rot (GER) and Gibberella stalk rot (GSR). Beyond yield loss, these diseases cause harmful mycotoxin accumulation, notably deoxynivalenol (DON), threatening food and feed safety (Mesterhazy et al. 2022; Ye et al. 2019). Particularly in the maize-wheat double-cropping area, the F. graminearum fungus overwinters on maize and wheat residues, thereby consecutively causing GER and GSR in maize and Fusarium head blight (FHB) in wheat (Dill-Macky and Jones 2000). Therefore, resistance genes are urgently needed to control these diseases. In wheat, the FHB resistance gene Fhb7 has been identified (Wang et al. 2020; Zhao et al. 2024). It encodes a glutathione S-transferase (GST) that catalyses the conversion of F. graminearum-produced DON to the non-toxic de-epoxidated DON-glutathione adduct and was predicted to be horizontally transferred from fungi to Thinopyrum elongatum, with its close homologues being found in Thinopyrum ponticum and different Epichloë fungi (Wang et al. 2020). To examine whether those GST homologues of Fhb7 could confer resistance to GER and GSR in maize, we expressed and purified 4 representatives, TpGST, EsGST, EuGST, and EgGST2, in E. coli (Table S1), and examined their catalytic activity converting DON to DON-glutathione. All exhibited similar or relatively higher activity compared to that of Fhb7 (Figure 1a,b). We then selected TpGST (from Thinopyrum ponticum) and EuGST (from Epichloë uncinate) to represent plant- and fungal-origin homologues, respectively, and overexpressed their codon-optimised genes (Table S1) in the maize inbred line Zong 31 (Z31). We selected three transgenic lines with high expression levels for each gene (TpGST-10, TpGST-16, and TpGST-17 for TpGST; EuGST-42, EuGST-45, and EuGST-46 for EuGST) to evaluate their disease resistance (Figure S1). The evaluation of GER resistance was performed by artificial inoculation of field-grown transgenic maize ears with F. graminearum in three distinct environments in two consecutive years. Almost all TpGST and EuGST transgenic lines exhibited significantly higher GER resistance compared to the wild-type Z31 and non-transgenic plants across all three environments including 2022E1, 2022E2, and 2023E1, except for EuGST-42 and EuGST-45 in 2022E1 (Figure 1c–f). In summary, our findings indicate that TpGST and EuGST could significantly enhance GER resistance in maize without affecting major agronomic traits (Figure S2), although the resistance may be affected by environments. We also evaluated the GSR resistance of each overexpression line by artificial stem inoculation of the field-grown plants with F. graminearum. All lines except TpGST-17 showed significantly enhanced GSR resistance (Figure S3). Since GSR resistance is highly influenced by environmental conditions, we performed a seedling stage assay under controlled greenhouse conditions. At 5–7 days post inoculation, non-transgenic seedlings displayed severe stem necrosis. In contrast, all transgenic lines showed significantly reduced susceptibility (Figure 1g,h and Figure S4), confirming the enhanced GSR resistance. To determine whether the enhanced resistance was pathogen specific, we challenged plants with three other pathogens: Fusarium verticillioides (causing Fusarium stalk rot, FSR), Curvularia lunata (causing Curvularia leaf spot), and Puccinia polysora Underw (causing southern corn rust). No significant difference in disease severity was observed between transgenic and non-transgenic plants (Figure S5), indicating that TpGST and EuGST confer specific resistance to F. graminearum-caused diseases (GER and GSR). To further evaluate the utility of TpGST and EuGST for maize breeding, we generated hybrid maize carrying these resistance genes by crossing the transgenic lines with the inbred line P138. Control hybrids were similarly produced by crossing wild-type Z31 or non-transgenic (NT) lines with P138. In field trials across three different environments of Henan in 2024, the average GER disease scores were significantly lower in P138 × Z31(TpGST) and P138 × Z31(EuGST) hybrids than in the P138 × Z31 and P138 × Z31(NT) control hybrids (Figure 1i–l). Correspondingly, we observed a significantly lower relative F. graminearum level in kernels of the transgenic hybrids compared to the controls (Figure S6). Consistently, LC–MS analysis showed that DON content was significantly lower in kernels of the transgenic hybrids (Figure S7), indicating enhanced detoxification ability conferred by TpGST and EuGST. To assess DON tolerance, we performed a seedling growth inhibition assay on medium supplemented with 0 (mock) or 25 mg/L DON. The transgenic hybrid seedlings expressing TpGST and EuGST grew significantly taller than the non-transgenic control under DON treatment (Figure 1m–p). Furthermore, in artificial field inoculations, the transgenic hybrids also exhibited significantly greater resistance to GSR compared to non-transgenic hybrids (Figure 1q,r and Figure S8). Collectively, these results demonstrate the considerable potential of utilising the GST homologous genes in maize breeding programmes. In conclusion, our study demonstrates that heterologous expression of TpGST and EuGST in maize confers enhanced resistance to F. graminearum and reduces DON accumulation, offering a direct strategy to control GER and GSR in maize, and presenting a promising approach to mitigate outbreaks of co-occurring Fusarium diseases in wheat-maize double-cropping areas. M.G. and Y.L. conceived the research. Y.W., Y.L., Y.Y., Z.J., Q.W., K.W., and G.L. performed the experiments. M.G. and Y.W. wrote the manuscript. Y.L. revised the manuscript. All authors read and approved the manuscript. This work was funded by National Key Research and Development Program (2022YFD1201801 to M.G.), Rolling Support of Fund for Distinguished Young Scholars in Henan (252300421235 to M.G.), the National Natural Science Foundation of China (U24A20395 and 32372208 to M.G). This work was supported by the National Key Research and Development Program of China (2022YFD1201801), Rolling Support of Fund for Distinguished Young Scholars in Henan (252300421235), National Natural Science Foundation of China (U24A20395, 32372208). The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. Figure S1: Relative transcriptional levels of TpGST and EuGST in transgenic plants. Figure S2: Agronomic traits of TpGST and EuGST transgenic plants. Figure S3: Evaluation of GSR resistance by artificial inoculation of Fusarium graminearum in TpGST and EuGST maize transgenic lines in the field. Figure S4: Evaluation of GSR resistance by artificial inoculation of Fusarium graminearum in TpGST and EuGST maize transgenic lines at the seedling stage. Figure S5: Resistance of TpGST and EuGST transgenic lines against Fusarium verticillioides, Curvularia lunata, and Puccinia polysora Underw. Figure S6: Relative level of Fusarium graminearum in kernels of maize hybrids without or with TpGST and EuGST transgenes. Figure S7: DON content in kernels of maize hybrids without or with TpGST and EuGST transgenes. Figure S8: Evaluation of GSR resistance by artificial inoculation of Fusarium graminearum in stems of maize hybrids without or with TpGST and EuGST transgenes. Table S1: The codon-optimised DNA sequence of GSTs studied in this study. Table S2: Primers used in this study. Material S1. Plant materials and methods. 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.