Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed exclusively on artificial diets supplemented with Cry37Aa alone displayed no statistical difference compared to the control. Conversely, when exposed solely to Cry23Aa, larval survival decreased by roughly 69%. However, the combined provision of both Cry23Aa and Cry37Aa in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under the regulation of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. After confirming forty-five transgenic cotton events, we selected the top seven events that exhibited elevated expression levels of Cry23Aa and Cry37Aa toxins in flower buds, 70%, for greenhouse bioassays. The mortality rate of CBW larvae feeding on both T0 and T1 generation transgenic cotton plants ranged from 75 to 100%. Our computational analyses unveiled that Cry23Aa possesses all the hallmark characteristics of a β-pore-forming toxin (β-PFT), specifically binding to sugar components in glycoproteins. Intriguingly, our studies also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Ultimately, our discussion centers on the crucial structural attributes of Cry23Aa that likely play a role in the toxin's mechanism of action. With the observed low LC50 for CBW and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that across successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.