Overexpression of modified Bacillus thuringiensis toxin Cyt2Aa in wheat strongly enhances aphid resistance

Overexpression of modified Bacillus thuringiensis(Bt)-derived toxin fused with gut-binding peptides strongly enhanced aphid resistance in wheat. Wheat (Triticum aestivum L.) is a major staple crop globally, contributing approximately 20% of the total food calories worldwide (Zörb et al., 2018). However, stable wheat production faces significant challenges due to the prevalence of various diseases and pests. Among these, aphids are the major insect pest of wheat worldwide. Within wheat-producing regions of China, Sitobion miscanthi is the predominant aphid species, leading to significant annual yield reductions (Li et al., 2023). Insect-resistant transgenic plants expressing insect-specific toxins derived from the bacterium Bacillus thuringiensis (Bt) have provided effective suppression of lepidopteran and coleopteran pests, leading to widespread adoption worldwide (Sanahuja et al., 2011; Jouzani et al., 2017). Currently, crystal (Cry) toxins have been widely used in commercial insect-resistant crops. Although cytolytic (Cyt) toxins are less frequently applied in transgenic insect-resistant crops, the mode of action of Cyt toxins is less complex than that of Bt Cry toxins (Butko, 2003; Chougule et al., 2013). Notably, Cyt toxins have been successfully used worldwide in the control of mosquitoes, demonstrating their recognized safety profile (Cantón et al., 2011). However, the sap-sucking aphids exhibit limited susceptibility to Bt-derived toxins (Porcar et al., 2009), rendering the application of Bt toxins ineffective in wheat cultivation. Previous research has demonstrated that the incorporation of gut-binding peptide GBP3.1 with Bt toxin Cyt2Aa fusion can enhance its toxicity against aphids (Chougule et al., 2013). However, the practical viability of modified Cyt2Aa toxins for aphid resistance in transgenic plants remains uncertain. In this study, we substituted the aphid GBP3.1 at a loop (159th to 164th site, SLSAHN) of Cyt2Aa (also named CGSL4; Chougule et al., 2013), as depicted in Figure 1A, resulting in a mCyt2Aa (modified Cyt2Aa) structure akin to that of Cyt2Aa. The mCyt2Aa was shown to be highly toxic to both the pea aphid and green peach aphid, while its toxicity to Sitobion miscanthi remains undetermined. The median lethal dose (LC50) value of mCyt2Aa protein against Sitobion miscanthi is approximately 15.39 μg/mL (Figure S1), indicating the strong toxicity of mCyt2Aa toward Sitobion miscanthi. Subsequently, we developed transgenic wheat lines (Fielder cultivar) overexpressing mCyt2Aa with BASTA resistance. Compared with wild-type (WT), overexpression of mCyt2Aa (mCyt2AaOE) had no obvious effects on wheat growth and development (Figure 1B) but resulted in enhanced BASTA resistance (Figure 1C). Transgenic lines were selected based on BASTA resistance and confirmed using genomic PCR (Figure S2). In total, ten independent lines overexpressing mCyt2Aa were identified. Among these lines, mCyt2AaOE 1#, 7#, and 8#, with the highest transcript level measured by qRT-PCR, were selected as representatives for further detailed analysis (Figure 1D). To determine the content of mCyt2Aa in transgenic wheat, we generated polyclonal antibodies against mCyt2Aa. Three independent wheat transgenic lines (1#, 7#, and 8#) were confirmed to be overexpressed using immunoblotting with this anti-mCyt2Aa antibody (Figure 1E). Enzyme-linked immunosorbent assay (ELISA) assays showed high levels of mCyt2Aa protein in the three transgenic lines (1#, 7#, and 8#), and relatively lower expression of mCyt2Aa in transgenic lines 3#, 4#, and 9#, but no protein expression was detected in other transgenic lines and WT (Figure 1F). Notably, these transgenic lines displayed growth and developmental traits similar to WT plants, including growth phenotype, seed size and 1,000-grain weight in the glasshouse and the yield in the field (Figures 1G–I, S3, S4). These findings suggested that overexpression of mCyt2Aa had no effect on wheat growth. Engineering transgenic wheat with enhanced aphid Sitobion miscanthi resistance (A) Ribbon structures of Cyt2Aa and mCyt2Aa. Aphid gut-binding peptide GBP3.1 replaces the loop of Cyt2Aa. (B, C) Phenotypes of 30-d-old mCyt2AaOE transgenic plants (B) and after BASTA treatment (C). Bars = 10 cm. (D) Relative expression levels of Cyt2Aa in mCyt2AaOE transgenic plants. (E) Western blot detection of mCyt2Aa protein expression in mCyt2AaOE transgenic wheat (1#, 7#, and 8#). (F) mCyt2Aa protein assay from young leaves of mCyt2AaOE transgenic lines. Data represent means ± SD from at least three plants from each line. (G, H) Growth phenotype and 1,000-grain weight of mCyt2AaOE plants in the greenhouse. Bar = 10 cm. (I) The yield of wild-type (WT) and mCyt2AaOE plants in the field. (J) A comparison of leaves from WT and mCyt2AaOE inoculated Sitobion miscanthi after 7 d. Bar = 1 cm. (K) Mortality data obtained for third-instar Sitobion avenae feeding on WT, and mCyt2AaOE leaves. Data represent means ± SD from three biological replicates of each line. (L) Statistics of the population of Sitobion avenae in WT and mCyt2AaOE plant spikes grown in the field 45 d after inoculation. Statistical differences were determined using Duncan's test, different letters indicate significant differences at P < 0.05. Next, we aimed to determine the efficacy of the mCyt2Aa toxin in suppressing aphids in wheat. To achieve this, we conducted leaf inoculation experiments on 40-d-old wheat seedlings with third-instar aphids Sitobion miscanthi larvae. As illustrated in Figure 1J, WT leaves were covered in aphids after 7 d, whereas few aphids were observed in the leaves of mCyt2AaOE plants. Subsequently, third-instar Sitobion miscanthi larvae were fed with the leaves derived from WT and mCyt2AaOE plants. Following 7 d of larval feeding, mCyt2AaOE lines demonstrated much stronger resistance than WT. The mortality rate of the larvae feeding on mCyt2AaOE leaves was substantially higher than that of those feeding on WT leaves, with a clear positive correlation observed between larval resistance and mCyt2Aa protein content (Figure 1K). We then assessed the larvae population on leaves of the field-grown transgenic wheats when inoculated with Sitobion miscanthi for 45 d. As shown in Figures 1L and S5, Sitobion miscanthi predominantly congregated on the spike of WT, while being almost invisible on the mCyt2AaOE spike. These results indicated that mCyt2Aa in wheat is sufficiently toxic to Sitobion miscanthi. To further investigate the toxicology mechanism of mCyt2Aa on Sitobion miscanthi, we conducted RNA-seq to detect the changes in the digestive and immune systems of Sitobion miscanthi fed with wheat leaves of WT and mCyt2AaOE transgenic lines. As anticipated, the expression of a series of digestion-related, absorption-related, and immunity-related genes significantly changed in Sitobion miscanthi after feeding with leaves of mCyt2AaOE wheat compared with WT, indicating the high toxicity of mCyt2Aa toward Sitobion miscanthi (Figures S6–S8; Table S2). We further detected the accumulation of mCyt2Aa in aphids fed on mCyt2AaOE transgenic wheat. The results demonstrated a gradual increase in mCyt2Aa protein accumulation compared with the aphids fed on WT wheat, reaching peak levels after 3 d of feeding (Figure S9). Previous research has highlighted the potential of enhancing toxin affinity to aphid gut by incorporating a gut-binding peptide as a promising strategy for agricultural pest management (Chougule et al., 2013). Our results demonstrated that this strategy can effectively be utilized for the development of aphid-resistant transgenic plants. In summary, we demonstrated that overexpression of the Bt toxin Cyt2Aa fused with gut-binding peptides in wheat strongly enhanced aphid resistance. This research underscores the critical role of modified Bt toxins in the effective suppression of various types of plant pests. This work was supported by grants from Taishan Scholars Program (tsqn202306306), the Key Research and Development Program of Shandong Province (2022LZGC001 and 2022LZGC005), the National Natural Science Foundation of China (U24A20389), the Key R&D Program of Shandong Province (2025CXPT149), the Shandong Youth Innovation Team Plan (2022KJ168), and Yellow River Delta Scholars (DYRC20220109). The authors declare that they have no conflicts of interest. D.W., G.Q., Yi.L., and G.L. designed the research, analyzed data and wrote the manuscript. Z.S., Yu.L. and G.L. performed wheat transformation. D.W., Z.C., W.C., and H.Y. performed the experiment. All authors read and approved the final manuscripts. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70038/suppinfo Figure S1. The LC50 of mCyt2Aa against Sitobion miscanthi Figure S2. PCR amplification of Cry3A in wild-type (WT) and mCyt2AaOE transgenic lines Figure S3. Seed size of wild-type (WT) and mCyt2AaOE transgenic lines Figure S4. The phenotype of wild-type (WT) and mCyt2AaOE transgenic lines grown in the field Figure S5. Aphid population statistics in field-grown wild-type (WT) and mCyt2AaOE transgenic lines Figure S6. RNA-seq analysis of absorptive system alterations in Sitobion miscanthi fed on wild-type (WT) versus mCyt2AaOE transgenic wheat Figure S7. RNA-seq analysis of digestive system alterations in Sitobion miscanthi fed on wild-type (WT) versus mCyt2AaOE transgenic wheat Figure S8. RNA-seq analysis of immune system alterations in Sitobion miscanthi fed on wild-type (WT) versus mCyt2AaOE transgenic wheat Figure S9. Detection of mCyt2Aa accumulation in Sitobion miscanthi fed on mCyt2AaOE transgenic wheat Table S1. Primers used in this study 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.