Abstract
Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.
Highlights
Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda
Cry1B proteins exhibiting significantly higher insecticidal activities than the buffer negative control in bioassays at concentrations between 50 and 3,500 ng/cm2 were further characterized by bioassays following sucrose gradient purification of the crystalline inclusions, and the highest specific insecticidal activity was observed for Cry1B.868, which comprises domain 1 (D1) and domain 2 from Cry1Be2 (M1 to I503), domain 3 from Cry1Ca1 (N468 to N633), and the C-terminal protoxin moiety from Cry1Ab3 (E626 to E1155)
We identified Cry1Da_7[V108C,E128C] (Fig. 1B), Cry1B.868[A160N,N167D] (Fig. 1C), and Cry1F.842[I108C,D128C] as disabled insecticidal protein (DIP) variants based on the selection criteria that these variants (i) had no significant insecticidal activity toward FAW, (ii) displayed similar processing with trypsin in vitro, (iii) exhibited similar insecticidal activities in bioassays mixed with their native counterparts at a 1:1 molar concentration, and (iv) competed against their native counterparts in feeding assays with multiple lepidopteran species, including FAW, when presented in a molar excess of Ͼ10
Summary
Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A These two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cellbased assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A Pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture. We report the development of two new modified insecticidal proteins for use against FAW, Cry1B.868 and Cry1Da_7, with enhanced specific activity against FAW and corn earworms (CEW), Helicoverpa zea (Boddie), respectively, and our comprehensive assessment of their FAW receptor preferences based on available resistant colonies, DIP assays, and cell-based receptor screens
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