Vip3Aa domain IV and V mutants confer higher insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera

Abstract

BACKGROUND The fall armyworm Spodoptera frugiperda and cotton bollworm Helicoverpa armigera are major insect pests of corn and cotton worldwide. Genetically engineered crops producing Vip3Aa, a potent endotoxin, from the bacterium Bacillus thuringiensis (Bt) are effective in controlling these two harmful pests. However, Vip3Aa efficacy is relatively weak compared to that of other Bt proteins such as Cry1A and Cry1F. This study sought to modify Vip3Aa for increased insecticidal activity and determine the cause of elevated activity. RESULTS The two triple Vip3Aa mutants in domains IV and V (Vip3Aa‐S543N/I544L/E627A and Vip3Aa‐S543N/I544L/S686R) exhibited 7.3‐fold and 2.8‐fold increased toxicity against S. frugiperda, respectively, compared with the wild type while the toxicity of Vip3Aa‐S543N/I544L/S686R was 3.2 times that of wild‐type protein in H. armigera. The mutants had enhanced stability in midgut juice and 2.6–5.1 times higher binding affinity against S. frugiperda and H. armigera compared with wild type protein. CONCLUSIONS The enhanced toxicity of Vip3Aa mutants was due to increased stability and binding affinity during infection. The amino acids S543 and I544 combined with E627 or S686 in domains IV and V of Vip3Aa are important for maintaining structural stability and receptor binding. The results match insecticidal activity (LC50) with binding activity (Kd), which provides novel clues for the rational design of Bt insecticidal proteins. © 2022 Society of Chemical Industry.

N-Terminal α-Helices in Domain I of Bacillus thuringiensis Vip3Aa Play Crucial Roles in Disruption of Liposomal Membrane

“…Similar to Vip3A domain I, Cry1Ac domain I is responsible for pore formation [ 27 , 28 ]. Additionally, domains II and III of Cry1Ac are involved in receptor binding, target selection, and structural maintenance, analogous to the proposed roles of domains III–V in Vip3A [ 7 , 29 , 30 , 31 ]. According to the individual insecticidal activity of Vip3Aa and Cry1Ac against S. litura and P. xylostella larvae [ 12 , 32 , 33 , 34 ], we fused domain I and additional α-helices in domain II of Vip3Aa with domains II and III of Cry1Ac to generate multiple chimeric proteins ( Figure 3 a).…”

Bacillus thuringiensis Cry9Aa Insecticidal Protein Domain I Helices α3 and α4 Are Two Core Regions Involved in Oligomerization and Toxicity

“…The interaction of Cry insecticidal proteins with BBMV proteins is considered a fundamental step in the mode of action of Cry toxins . Defects of Cry proteins on binding to BBMVs correlate with the resistance of some insect pests to Cry proteins .…”

“…The interaction of Cry insecticidal proteins with BBMV proteins is considered a fundamental step in the mode of action of Cry toxins. 40 Defects of Cry proteins on binding to BBMVs correlate with the resistance of some insect pests to Cry proteins. 41 To determine if the binding of Cry9Aa mutants was affected, binding analyses were performed by ELISA binding assays, as described in Materials and Methods.…”

Domain III β4–β5 Loop and β14–β15 Loop of Bacillus thuringiensis Vip3Aa Are Involved in Receptor Binding and Toxicity

“…Replacement of M34 in domain I with leucine increased the insecticidal activity against S. exigua and S. littoralis [ 12 , 26 ]. Mutants in domains IV and V showed improved structural stability and affinity for BBMVs and the insecticidal activity of these mutants against S. frugiperda and Helicoverpa armigera was also significantly improved [ 27 ]. In this study, we obtained single mutants that exhibited higher affinity for S. frugiperda BBMVs and showed increased insecticidal activity against S. frugiperda ( Figure 2 ).…”

“…Finally, the results of at least three independent trials were used to evaluate the mortality rate at each toxin dose. GraphPad Prism v.8.0 (GraphPad, San Diego, USA) was used to calculate the lethal concentration (LC 50 ) values [ 27 ]. The toxicity of the proteins was quantified by probit analysis using SPSS Statistics (IBM, Chicago, IL, USA).…”