Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3’s toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa’s stability and toxicity inside the protease-enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa’s mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development. Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3’s toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa’s stability and toxicity inside the protease-enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa’s mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development. Microbially derived insecticidal proteins are useful substitutes for synthetic pesticides due to their highly targeted insecticidal effects and more sustainable manners (1Tabashnik B.E. Carrière Y. Surge in insect resistance to transgenic crops and prospects for sustainability.Nat. Biotechnol. 2017; 35: 926-935Crossref PubMed Scopus (395) Google Scholar, 2Liu L. Li Z. Luo X. Zhang X. Chou S.-H. Wang J. et al.Which is stronger? A continuing battle between Cry toxins and insects.Front Microbiol. 2021; 12665101Google Scholar, 3Gupta M. Kumar H. Kaur S. Vegetative insecticidal protein (Vip): a potential contender from Bacillus thuringiensis for efficient management of various detrimental agricultural pests.Front Microbiol. 2021; 12: 659736Crossref PubMed Scopus (27) Google Scholar, 4Valtierra-de-Luis D. Villanueva M. Berry C. Caballero P. Potential for Bacillus thuringiensis and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance.Toxins. 2020; 12: 773Crossref PubMed Scopus (35) Google Scholar, 5Silva-Filha M.H.N.L. Romão T.P. Rezende T.M.T. Carvalho K.d.S. Gouveia de Menezes H.S. Alexandre do Nascimento N. et al.Bacterial toxins active against mosquitoes: Mode of action and resistance.Toxins. 2021; 13: 523Crossref PubMed Scopus (36) Google Scholar, 6Chalivendra S. Microbial toxins in insect and nematode pest biocontrol.Int. J. Mol. Sci. 2021; 22: 7657Crossref PubMed Scopus (9) Google Scholar). Bacillus thuringiensis (Bt) is the most broadly used microbial insecticide worldwide due to its production of highly effective Cry insecticidal proteins (7Kumar P. Kamle M. Borah R. Mahato D.K. Sharma B. Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture.Egypt. J. Biol. Pest Control. 2021; 31: 95Crossref Scopus (20) Google Scholar, 8Pinos D. Andrés-Garrido A. Ferré J. Hernández-Martínez P. Response mechanisms of invertebrates to Bacillus thuringiensis and its pesticidal proteins.Microbiol. Mol. Biol. Rev. 2021; 85: e00007-e00020Crossref PubMed Scopus (27) Google Scholar), which account for over 75% of the microbial biopesticide market (9Jurat-Fuentes J.L. Heckel D.G. Ferré J. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis.Annu. Rev. Entomol. 2021; 66: 121-140Crossref PubMed Scopus (120) Google Scholar, 10Olson S. An analysis of the biopesticide market now and where is Pest Scopus Google Scholar). Cry proteins are by and are used for biological control of pest in the of transgenic crops and (7Kumar P. Kamle M. Borah R. Mahato D.K. Sharma B. Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture.Egypt. J. Biol. Pest Control. 2021; 31: 95Crossref Scopus (20) Google Scholar, N. J.L. of Bacillus thuringiensis toxins and mechanism of Scopus Google Scholar, Bacillus mechanism of and a Rev. Biotechnol. PubMed Scopus Google Scholar, L. M. A. Bacillus thuringiensis insecticidal Cry of insect resistance and for Microbiol. Rev. PubMed Scopus Google Scholar, J.L. N. for Cry insecticidal insights from their of PubMed Scopus Google Scholar). However, the application of Cry proteins has its due to their insecticidal and of resistance in pests (1Tabashnik B.E. Carrière Y. Surge in insect resistance to transgenic crops and prospects for sustainability.Nat. Biotechnol. 2017; 35: 926-935Crossref PubMed Scopus (395) Google Scholar, 2Liu L. Li Z. Luo X. Zhang X. Chou S.-H. Wang J. et al.Which is stronger? A continuing battle between Cry toxins and insects.Front Microbiol. 2021; 12665101Google Scholar, 8Pinos D. Andrés-Garrido A. Ferré J. Hernández-Martínez P. Response mechanisms of invertebrates to Bacillus thuringiensis and its pesticidal proteins.Microbiol. Mol. Biol. Rev. 2021; 85: e00007-e00020Crossref PubMed Scopus (27) Google Scholar, J.L. Heckel D.G. Ferré J. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis.Annu. Rev. Entomol. 2021; 66: 121-140Crossref PubMed Scopus (120) Google Scholar, in agricultural pest of transgenic Sci. S. A. 2021; Scopus Google Scholar). the of effective insecticidal protein is M. Kumar H. Kaur S. Vegetative insecticidal protein (Vip): a potential contender from Bacillus thuringiensis for efficient management of various detrimental agricultural pests.Front Microbiol. 2021; 12: 659736Crossref PubMed Scopus (27) Google Scholar, 4Valtierra-de-Luis D. Villanueva M. Berry C. Caballero P. Potential for Bacillus thuringiensis and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance.Toxins. 2020; 12: 773Crossref PubMed Scopus (35) Google Scholar, P. Kamle M. Borah R. Mahato D.K. Sharma B. Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture.Egypt. J. Biol. Pest Control. 2021; 31: 95Crossref Scopus (20) Google Scholar, J.L. Heckel D.G. Ferré J. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis.Annu. Rev. Entomol. 2021; 66: 121-140Crossref PubMed Scopus (120) Google Scholar). Vegetative insecticidal protein proteins are secreted by its M. N. Y. B. Ferré J. insecticidal proteins from Mol. Biol. Rev. PubMed Scopus Google Scholar, a Bacillus thuringiensis insecticidal protein a of against Sci. S. A. PubMed Scopus Google Scholar). proteins Cry bind to and efficient insecticidal activity, against as a of insecticidal proteins M. Kumar H. Kaur S. Vegetative insecticidal protein (Vip): a potential contender from Bacillus thuringiensis for efficient management of various detrimental agricultural pests.Front Microbiol. 2021; 12: 659736Crossref PubMed Scopus (27) Google Scholar, M. N. Y. B. Ferré J. insecticidal proteins from Mol. Biol. Rev. PubMed Scopus Google Scholar, M. Z. Li Y. Y. et insights insecticidal proteins as pest 2020; 12: PubMed Scopus Google Scholar, B. et as a for Bacillus thuringiensis insecticidal protein Vip3Aa and the of Vip3Aa via Scopus Google Scholar, X. L. Z. J. a for insecticidal protein PubMed Scopus Google Scholar). proteins been used in transgenic crops in Cry insecticidal and the of resistance in pests has been to M. Kumar H. Kaur S. Vegetative insecticidal protein (Vip): a potential contender from Bacillus thuringiensis for efficient management of various detrimental agricultural pests.Front Microbiol. 2021; 12: 659736Crossref PubMed Scopus (27) Google Scholar, M. Z. Li Y. Y. et insights insecticidal proteins as pest 2020; 12: PubMed Scopus Google Scholar), which their application potential in and insecticide resistance toxins are multidomain proteins of a and a more proteins been N. Berry C. S. R. A for Bacillus thuringiensis and other pesticidal 2021; PubMed Scopus Google Scholar, S. R. Berry C. N. A to and bacterial pesticidal PubMed Scopus Google Scholar). their in a Bacillus thuringiensis insecticidal protein a of against Sci. S. A. PubMed Scopus Google Scholar), the and of action of is that proteins are as by midgut proteases of host into activated toxins H. N. Mode of action of the Bacillus thuringiensis insecticidal protein from that of Microbiol. PubMed Scopus Google Scholar, H. R. Y. B. M. et of a activated Vip3Aa from Bacillus 2017; PubMed Scopus Google Scholar, L. L. B. Z. Li J. et of Bacillus thuringiensis Vip3Aa protein by Spodoptera midgut J. Biol. PubMed Scopus Google Scholar). proteins are into of to the and to the Y. N. M. B. Ferré J. into the of the Vip3Aa insecticidal protein by 2017; PubMed Scopus Google Scholar, L. et insecticidal from Bacillus thuringiensis a that is 2017; PubMed Scopus Google Scholar). the to M. Ferré J. In and in of Vip3Aa to Spodoptera frugiperda midgut and of by Microbiol. PubMed Scopus Google Scholar), which is essential for Vip3’s toxicity Wang X. et of and insecticidal proteins against 2017; PubMed Scopus Google Scholar, Y. Ferré J. domains of the Bacillus thuringiensis protein by of PubMed Scopus Google Scholar). toxicity mechanism of proteins to the midgut of of pest is understood. to host as the mechanism of H. N. Mode of action of the Bacillus thuringiensis insecticidal protein from that of Microbiol. PubMed Scopus Google Scholar, H. R. Y. B. M. et of a activated Vip3Aa from Bacillus 2017; PubMed Scopus Google Scholar), B. et as a for Bacillus thuringiensis insecticidal protein Vip3Aa and the of Vip3Aa via Scopus Google Scholar, Wang J. Vip3Aa in Spodoptera frugiperda PubMed Scopus Google Scholar, P. J. Ferré J. B. in and in Spodoptera to of insecticidal 2017; PubMed Scopus Google Scholar). the and of and toxins been M. C. J. of a insecticidal protein a and a Sci. 2020; PubMed Scopus Google Scholar, R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar, et of insecticidal toxin its mechanism of action the 2021; 12: PubMed Scopus Google Scholar), that proteins of domains and into highly in between and II by proteins are to a from a to a activated toxin R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, et of insecticidal toxin its mechanism of action the 2021; 12: PubMed Scopus Google Scholar). of DI is into that can the which is to be for by proteins et of insecticidal toxin its mechanism of action the 2021; 12: PubMed Scopus Google Scholar). their due to their and biological inside the host insect of the molecular of action of as the between the and functions of and the of toxicity in the insect which significantly their efficient application in biological pest In this we the contributions of the various domains of the Vip3Aa to its toxicity in the midgut of Spodoptera frugiperda larvae. We and III of Vip3Aa can bind to the midgut DI-DIII can be activated by midgut proteases and similar cytotoxicity to activated Vip3Aa DI is essential for the stability and toxicity of Vip3Aa by the of the Vip3Aa to the peritrophic matrix via its glycan-binding which contributes to the insecticidal of Vip3Aa. study the various domains of Vip3Aa and provides multiple insights into its molecular of that the multidomain proteins can bind to and of the insect and this has been to effects B. et as a for Bacillus thuringiensis insecticidal protein Vip3Aa and the of Vip3Aa via Scopus Google Scholar, X. L. Z. J. a for insecticidal protein PubMed Scopus Google Scholar, M. Ferré J. In and in of Vip3Aa to Spodoptera frugiperda midgut and of by Microbiol. PubMed Scopus Google Scholar, Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google Scholar, B. Sharma N. R. of Bacillus thuringiensis insecticidal protein protein in Spodoptera Microbiol. PubMed Scopus Google Scholar). However, is this of proteins to the midgut of insect We and full-length Vip3Aa and its R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google we we their to bind to derived from S. frugiperda that the of and to that of the full-length Vip3Aa However, and bind to that and are for to the for the analysis to S. frugiperda that can bind to the as DI-DIII and while We the of Vip3Aa to the midgut of S. frugiperda larvae. we from S. frugiperda midgut and Vip3Aa and the for and we that and are for Vip3Aa to the midgut the of for of the midgut epithelium, we that the of Vip3Aa in a in the of to the midgut the of the of to the midgut demonstrate that Vip3Aa to the S. frugiperda midgut via that Vip3Aa to the S. frugiperda midgut we by which Vip3Aa are for to the midgut that proteins are as and are by midgut proteases the between DI and R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar). of and the of the which that are to be essential to their and cytotoxicity Y. N. M. B. Ferré J. into the of the Vip3Aa insecticidal protein by 2017; PubMed Scopus Google Scholar, Wang X. et of and insecticidal proteins against 2017; PubMed Scopus Google Scholar, R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar). which Vip3Aa’s effects against the S. frugiperda midgut epithelium, of Vip3Aa of the midgut and midgut from S. analysis that and DI-DIII similar to as to the and the of the protein In and and the stability of and analysis further that the of DI-DIII in and we the toxicity of and DI-DIII of Vip3Aa. Vip3Aa and other control proteins to the the toxicity of activated Vip3Aa to significantly more that of the Vip3Aa and Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google Scholar). cytotoxicity further that activated and Vip3Aa proteins similar toxicity to and and A and cytotoxicity the by et Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google Scholar), that and as to as the activated full-length that the DI-DIII has similar cytotoxicity to the full-length which be to is highly to and is by which that of Vip3Aa has insecticidal Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar). that the and toxin into a and this is for Vip3’s toxicity Wang X. et of and insecticidal proteins against 2017; PubMed Scopus Google Scholar, Y. Ferré J. domains of the Bacillus thuringiensis protein by of PubMed Scopus Google Scholar, R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar). DI has been to be essential for the of the Vip3Aa R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar). this and the full-length Vip3Aa a and we that DI of Vip3Aa functions in the of the Vip3Aa which is for its stability and we that the of DI is the protein the of proteins and which are to be a potential Wang X. et of and insecticidal proteins against 2017; PubMed Scopus Google Scholar, J. R. Wang Y. Caballero J. Caballero P. et between Vip3Aa and stability and insecticidal against and 2020; 12: PubMed Scopus Google Scholar). analysis of the Vip3Aa that to from the DI are into the by DI and and We and and their toxicity against S. frugiperda larvae. the Vip3Aa and of which toxicity against S. frugiperda the toxicity of the protein significantly and toxicity for and and analysis of Spodoptera frugiperda as by Vip3Aa and the insecticidal in a insecticidal determine the of the to to Vip3Aa’s insecticidal activity, we a Vip3Aa from S. analysis that to and the and which the insecticidal of Vip3Aa In addition, that the of and significantly from that of Vip3Aa which that their distinct from that of Vip3Aa. further their in we protein Vip3Aa in the which are to the to and DI-DIII and A and that is by the of DI-DIII and from of the to in and and that which further that significantly the stability of in the of midgut proteases from insect further the of of the Vip3Aa to its stability and we a and from DI to be in between Vip3Aa and facilitate the of the Vip3Aa and in which that has is to the of and the other is to the of that the of in and significantly that of Vip3Aa which that their distinct from that of Vip3Aa. further that a and due to of the of In addition, is by S. frugiperda and its insecticidal and which further that this of DI is for the of Vip3Aa and is for Vip3Aa’s stability and that DI is essential for the stability and toxicity of Vip3Aa in the protease-enriched host insect midgut by the of the Vip3Aa the of et Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google that the DI-DIII similar cytotoxicity to full-length and However, that DI-DIII toxicity against S. frugiperda that DV is for the insecticidal of Vip3Aa inside the insect a more that DV a glycan-binding which facilitate of by proteins in the midgut of M. C. J. of a insecticidal protein a and a Sci. 2020; PubMed Scopus Google Scholar, R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar). However, that DV is in of the host midgut has been that the highly the which is by the midgut be by Cry toxins Y. H. of Bacillus thuringiensis the peritrophic of PubMed Scopus Google Scholar, A. de C. J. et of detrimental effects of Bacillus thuringiensis Cry toxins the insect a and Microbiol. PubMed Scopus Google Scholar). We that DV of Vip3Aa bind to the we the from S. frugiperda used to the potential of Vip3Aa protein and to the and DV bind the for DI-DIII that Vip3Aa to the via We glycan-binding the the of the is of of and its by which for proteins and in the of M. matrix PubMed Scopus Google Scholar, X. Zhang J. and of in the and of peritrophic matrix in PubMed Scopus (35) Google Scholar). We the of Vip3Aa to and that Vip3Aa bind to and and that to Vip3Aa A and the glycan-binding of DV bind to and and used in to the of DV in we a molecular to the A DV the that are in the and the potential of the we into DV and and their to bind that the of of the to that of and and In the glycan-binding that of and to the to DV the of to the significantly the of DV to the that is by glycan-binding the of DV to the the toxicity of we in which S. frugiperda Vip3Aa and A and the toxicity of and significantly Vip3Aa by and and that DV to the of S. frugiperda its glycan-binding and demonstrate that this contributes to the insecticidal of Vip3Aa. to various detrimental insect pests is sustainable biological control alternative to synthetic pesticides M.H.N.L. Romão T.P. Rezende T.M.T. Carvalho K.d.S. Gouveia de Menezes H.S. Alexandre do Nascimento N. et al.Bacterial toxins active against mosquitoes: Mode of action and resistance.Toxins. 2021; 13: 523Crossref PubMed Scopus (36) Google Scholar, 6Chalivendra S. Microbial toxins in insect and nematode pest biocontrol.Int. J. Mol. Sci. 2021; 22: 7657Crossref PubMed Scopus (9) Google Scholar). of bacterial pesticidal proteins been N. Berry C. S. R. A for Bacillus thuringiensis and other pesticidal 2021; PubMed Scopus Google Scholar, S. R. Berry C. N. A to and bacterial pesticidal PubMed Scopus Google Scholar). However, Cry proteins been and used worldwide (7Kumar P. Kamle M. Borah R. Mahato D.K. Sharma B. Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture.Egypt. J. Biol. Pest Control. 2021; 31: 95Crossref Scopus (20) Google Scholar, 8Pinos D. Andrés-Garrido A. Ferré J. Hernández-Martínez P. Response mechanisms of invertebrates to Bacillus thuringiensis and its pesticidal proteins.Microbiol. Mol. Biol. Rev. 2021; 85: e00007-e00020Crossref PubMed Scopus (27) Google Scholar, N. J.L. of Bacillus thuringiensis toxins and mechanism of Scopus Google Scholar, Bacillus mechanism of and a Rev. Biotechnol. PubMed Scopus Google Scholar, L. M. A. Bacillus thuringiensis insecticidal Cry of insect resistance and for Microbiol. Rev. PubMed Scopus Google Scholar). proteins and mode-of-action mechanisms distinct from Cry proteins and insecticidal against pests and are as a of insecticidal proteins that can be broadly Cry proteins in the M. Kumar H. Kaur S. Vegetative insecticidal protein (Vip): a potential contender from Bacillus thuringiensis for efficient management of various detrimental agricultural pests.Front Microbiol. 2021; 12: 659736Crossref PubMed Scopus (27) Google Scholar, M. N. Y. B. Ferré J. insecticidal proteins from Mol. Biol. Rev. PubMed Scopus Google Scholar, M. Z. Li Y. Y. et insights insecticidal proteins as pest 2020; 12: PubMed Scopus Google Scholar, B. et as a for Bacillus thuringiensis insecticidal protein Vip3Aa and the of Vip3Aa via Scopus Google Scholar, X. L. An B. J. by Vip3Aa has a in Spodoptera frugiperda 2021; 12: PubMed Scopus Google Scholar). However, the of of the molecular mechanisms underlying their insecticidal their application and rational development. Here, we the functions of the multiple domains of the Vip3Aa of the are highly and insights can be to the other of this protein DI of to in host by which is to their toxicity H. N. Mode of action of the Bacillus thuringiensis insecticidal protein from that of Microbiol. PubMed Scopus Google Scholar, H. R. Y. B. M. et of a activated Vip3Aa from Bacillus 2017; PubMed Scopus Google Scholar, R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar). in to the DI a in the stability of the Vip3Aa in the of midgut proteases and from by the of the Vip3Aa analysis of the that in to and are in the of R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, et of insecticidal toxin its mechanism of action the 2021; 12: PubMed Scopus Google Scholar). et N. J. Ferré J. for the insecticidal of from Bacillus PubMed Scopus Google a of the of significantly the insecticidal of in as and been to the of N. J. Ferré J. for the insecticidal of from Bacillus PubMed Scopus Google and the stability of to Y. Ferré J. domains of the Bacillus thuringiensis protein by of PubMed Scopus Google Scholar), further that is for Vip3Aa’s stability in the protease-enriched host insect midgut. In to a in the between Vip3Aa and Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar), and DI-DIII of to bind to Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google Scholar). that the of to bind and the midgut of S. frugiperda similar to that of the full-length Vip3Aa of Vip3Aa and of bind to that and are the domains of In addition, as in proteins are into is that Vip3Aa and bind to the of However, for the we that in to to the Vip3Aa proteins are in midgut midgut are do In to the the of we that the for are the effects and the of Vip3Aa’s in the that Cry toxins can be into midgut S. do Nascimento M. A. J. S. et analysis of the of Bacillus thuringiensis and toxins in 2021; PubMed Scopus Google Scholar, J. C. et and are for by bacterial PubMed Scopus Google Scholar). Vip3Aa can into midgut and functions further We that full-length be activated by and the activated DI-DIII toxicity against similar to activated full-length Vip3Aa. et Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google the DI-DIII of from to be as to as the full-length study further that DI-DIII is the and of proteins that is active against insect that DV a glycan-binding M. C. J. of a insecticidal protein a and a Sci. 2020; PubMed Scopus Google Scholar, R. J. Y. Ferré J. P. and of the insecticidal protein Vip3Aa from Bacillus 2020; PubMed Scopus Google Scholar, Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar). However, the functions of DV We that DV is in of midgut and the cytotoxicity of the insecticidal toxicity of DV that in DV the toxicity of N. J. Ferré J. for the insecticidal of from Bacillus PubMed Scopus Google Scholar). that DV is essential to the insecticidal of Vip3Aa in analysis of DV that to the of multidomain Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar), which is a to B. S. S. et for the and of by the from Biol. PubMed Scopus Google Scholar). that DV be a glycan-binding However, which of the insect midgut and of DV to In this we that Vip3Aa bind to the via the glycan-binding of in the glycan-binding of DV significantly the and insecticidal of Vip3Aa. that DV contributes to the Vip3Aa’s inside the host insect midgut and functions by to the of the we that DV of Vip3Aa is a that DV other potential functions in the toxicity of as in the for Vip3Aa’s toxicity M. Y. R. P. et and of for the insecticidal of the Vip3Aa protein from Bacillus Biotechnol. PubMed Scopus Google and the stability of in the N. J. Ferré J. for the insecticidal of from Bacillus PubMed Scopus Google Scholar). that DV multiple in the insecticidal of which further the of the are and its which is a of its M. matrix PubMed Scopus Google Scholar, X. Zhang J. and of in the and of peritrophic matrix in PubMed Scopus (35) Google Scholar). that Vip3Aa bind to to which is of most J. X. and of the PubMed Scopus Google Scholar, for toxins and Rev. PubMed Scopus Google Scholar, X. Z. X. X. J. et insights into the and of Google Scholar). However, multiple of the are the the the between toxin and can be significantly T.P. D. et the of a potential against the Sci. S. A. PubMed Scopus Google Scholar, D. of and PubMed Scopus Google Scholar, toxins are by PubMed Scopus Google Scholar). for this is that the of the the we used the for Vip3Aa. the be to the of Vip3Aa to from the and further the of midgut has been to bind to the this is to be a resistance mechanism by to Cry protein from further to midgut Y. H. of Bacillus thuringiensis the peritrophic of PubMed Scopus Google Scholar, A. de C. J. et of detrimental effects of Bacillus thuringiensis Cry toxins the insect a and Microbiol. PubMed Scopus Google Scholar, M. C. S. Y. of peritrophic matrix provides into its in resistance of PubMed Scopus Google Scholar). In we that the between Vip3Aa and the facilitate Vip3Aa toxicity to S. are to for insecticidal toxicity of Vip3Aa to the by its to is for the insecticidal of Vip3Aa in Y. M. Hernández-Martínez P. Ferré J. domains in the of insecticidal protein to from Spodoptera and insect Microbiol. 2021; Scopus Google Scholar), its is to a glycan-binding the and of the glycan-binding between and DV are Zhang Y. Z. D. J. X. and insights into the of insecticidal toxin of Bacillus 2020; 12: PubMed Scopus Google Scholar). In this we that Vip3Aa’s to the midgut is of and which further that functions from that is to the by the biological of in the of further that the of the midgut is in for J. Google Scholar), and further that is a from midgut to and the midgut in Biol. PubMed Google Scholar). In of proteins in and similar Y. N. M. B. Ferré J. into the of the Vip3Aa insecticidal protein by 2017; PubMed Scopus Google Scholar, M. Y. R. P. et and of for the insecticidal of the Vip3Aa protein from Bacillus Biotechnol. PubMed Scopus Google Scholar, of a is by protein stability in the midgut of Spodoptera PubMed Scopus Google Scholar). the we that Vip3Aa similar the In addition, the in can to the that the can the of Vip3Aa in the midgut. of the action mechanism of has been of Vip3Aa to the S. frugiperda to be the as the in the from to is to the and of the and that a in is to and cytotoxicity in a However, that in can the of proteins in the midgut of and to the in the midgut more In the study the functions of and DV of the Vip3Aa protein and their contributions to its toxicity against S. frugiperda larvae.