| Bacillus thuringiensis(Bt)plays a crucial role in pest control in crops and has become the most rapidly developing,extensively researched,and widely applied microbial insecticide.Previous studies have identified Cry proteins produced by Bt during the dormant period,which,upon entering the insect gut with food,perforate midgut cells’ surface,causing insect mortality.Recent research has revealed that Bt also secretes two toxic insecticidal proteins,Vegetative Insecticidal Proteins(Vip3)and Secreted Insecticidal Proteins(Sip),both harmful to pests.Nutritional phase insecticidal proteins,produced and secreted by Bt during its nutritional growth phase,exhibit efficient insecticidal activity against lepidopteran pests such as Spodoptera Frugiperda.Vip3 is classified into three major classes:Vip3A,Vip3B,and Vip3C.Among them,Vip3A is the most extensively studied and the only Vip3 family protein applied in genetically modified insect-resistant crops,demonstrating significant research and application value.Secreted Insecticidal Proteins represent another class of insecticidal proteins secreted by Bt.Current research indicates that Sip primarily affects Coleoptera,including the Colaphellus bowringi Baly.Due to the lack of structural information,further investigation is required to understand the insecticidal mechanism of Sip.In this study,we conducted a series of investigations into the structure-function relationship and insecticidal mechanism of these two types of secreted insecticidal proteins from Bt.The main research contents and results are outlined as follows:Firstly,employing structural biology,we deeply explored the structure and function of Vip3Aa.We elucidated the high-resolution crystal structure of the Cterminal domain of Vip3Aa(Vip3Aa200-end),revealing the spatial arrangement of its four domains,named Domain Ⅱ,Domain Ⅲ,Domain Ⅳ,and Domain Ⅴ.Through structural analysis,we predicted the potential functions of each domain.Subsequently,utilizing a combination of biophysical,biochemical,and cell biology approaches,we investigated the main functions of Vip3Aa’s major domains during its toxicity against Spodoptera Frugiperda larvae at the protein,cellular,and organism levels.We demonstrated that(1)Domains Ⅰ-Ⅲ are the core domains responsible for Vip3Aa cytotoxicity;(2)Domains Ⅱ-Ⅲ are primarily involved in the binding of Vip3Aa to insect midgut epithelial tissues;(3)Domain Ⅰ is indispensable for maintaining the correct tetramerization of Vip3Aa,and stable tetramerization is essential for Vip3Aa stability in the protease-rich insect midgut environment;(4)Domain Ⅴ,through its sugar-binding ability,binds to the peritrophic membrane enveloping food in the insect midgut,a crucial process for Vip3Aa’s insecticidal activity;(5)Upon entering the insect midgut,multiple cleavage sites between Domains Ⅰ and Ⅱ allow efficient conversion of Vip3Aa from the pro-toxin state to the activated toxin state.These results provide a preliminary model outlining the mechanism of action of Vip3Aa,from entering the insect midgut to exerting insecticidal toxicity.To further study the insecticidal proteins produced by Bt and promote their better application,we also elucidated the three-dimensional structure of Sip1Ab.Through evolutionary analysis,we found that Sip1Ab shares high similarity with Bt ε-toxin family proteins,belonging to the β-pore-forming toxin.By comparing and analyzing the structure with ETX_MTX2 family proteins,we proposed the potential mechanism of action of Sip1Ab when exerting insecticidal toxicity.Additionally,we compared the resolved protein structures with those predicted by four artificial intelligence models and briefly discussed the advantages and disadvantages of structural predictions.In conclusion,this paper comprehensively used structural biology,biochemistry,molecular biology,and cell biology research methods to deeply explore the structure and function of Vip3Aa,a nutritional phase insecticidal protein from Bt.We deciphered the high-resolution crystal structure of Vip3Aa200-end,further investigating the main functions of Vip3 Aa’s major domains during its toxicity against Spodoptera Frugiperda larvae.We proposed a model for Vip3Aa’s insecticidal mechanism and analyzed its cleavage activation mechanism,providing a theoretical basis for the subsequent rational redesign of Vip3 for enhanced insecticidal activity.Additionally,we resolved the monomeric structure of SiplAb and suggested its potential insecticidal mechanism.These research results significantly contribute to elucidating the molecular insecticidal mechanisms of two secretion-type insecticidal proteins from Bacillus thuringiensis,providing an important theoretical foundation for the scientific and rational application of these two types of insecticidal proteins.Simultaneously,this study holds great significance for the development of biopesticides based on microbial insecticidal proteins. |
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