Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (ß-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

Author Correction: A transgenic approach for controlling Lygus in cotton.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith).

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.

Development of enzymes for robust aryloxyphenoxypropionate and synthetic auxin herbicide tolerance traits in maize and soybean crops.

BACKGROUND: Effective management of weedy species in agricultural fields is essential for maintaining favorable growing conditions and crop yields. The introduction of genetically modified crops containing herbicide tolerance traits has been a successful additional tool available to farmers to better control weeds. However, weed resistance challenges present a need for additional herbicide tolerance trait options. RESULTS: To help meet this challenge, a new trait that provides tolerance to an aryloxyphenoxypropionate (FOP) herbicide and members of the synthetic auxin herbicide family, such as 2,4-dichlorophenoxyacetic acid (2,4-D), was developed. Development of this herbicide tolerance trait employed an enzyme engineered with robust and specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbial-sourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the higher temperature conditions often found in field environments. CONCLUSION: Optimized herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance for two herbicide families in transgenic maize and soybeans. This herbicide tolerance trait for FOP and synthetic auxin herbicides such as 2,4-D could be useful in weed management systems, providing additional tools for farmers to control weeds. © 2019 Society of Chemical Industry.

A transgenic approach for controlling Lygus in cotton.

Lygus species of plant-feeding insects have emerged as economically important pests of cotton in the United States. These species are not controlled by commercial Bacillus thuringiensis (Bt) cotton varieties resulting in economic losses and increased application of insecticide. Previously, a Bt crystal protein (Cry51Aa2) was reported with insecticidal activity against Lygus spp. However, transgenic cotton plants expressing this protein did not exhibit effective protection from Lygus feeding damage. Here we employ various optimization strategies, informed in part by protein crystallography and modelling, to identify limited amino-acid substitutions in Cry51Aa2 that increase insecticidal activity towards Lygus spp. by >200-fold. Transgenic cotton expressing the variant protein, Cry51Aa2.834_16, reduce populations of Lygus spp. up to 30-fold in whole-plant caged field trials. One transgenic event, designated MON88702, has been selected for further development of cotton varieties that could potentially reduce or eliminate insecticide application for control of Lygus and the associated environmental impacts.