Cry75Aa (Mpp75Aa) Insecticidal Proteins for Controlling the Western Corn Rootworm, Diabrotica virgifera virgifera, (Coleoptera: Chrysomelidae), Isolated from the Insect Pathogenic Bacteria Brevibacillus laterosporus.

This study describes three closely related proteins, cloned from Brevibacillus laterosporus strains, that are lethal upon feeding to Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR). Mpp75Aa1, Mpp75Aa2 and Mpp75Aa3 were toxic to WCR larvae when fed purified protein. Transgenic plants expressing each mMpp75Aa protein were protected from feeding damage and showed significant reduction in adult emergence from infested plants by both susceptible and Cry3Bb1 and Cry34Ab1/Cry35Ab1-resistant WCR. These results demonstrate that proteins from B. laterosporus are as efficacious as the well-known Bacillus thuringiensis (Bt) insecticidal proteins in controlling major insect pests such as WCR. The deployment of transgenic maize expressing mMpp75Aa along with other active molecules lacking cross-resistance have the potential to be a useful tool for control of WCR populations resistant to current Bt traits.IMPORTANCE Insects feeding on roots of crops can damage the plant roots resulting in yield loss due to poor water and nutrient uptake and plant lodging. In maize the western corn rootworm (WCR) can cause severe damage to the roots resulting in significant economic loss for farmers. Genetically modified (GM) expressing Bacillus thuringiensis (Bt) insect control proteins, has provided a solution for control of these pests. In recent years populations of WCR resistant to the Bt proteins in commercial GM maize have emerged. There is a need to develop new insecticidal traits for the control of WCR populations resistant to current commercial traits. New proteins with commercial level efficacy on WCR from sources other than Bt are becoming more critical. The Mpp75Aa proteins, from B. laterosporus, when expressed in maize, are efficacious against the resistant populations of WCR and have the potential to provide solutions for control of resistant WCR.

A new Bacillus thuringiensis protein for Western corn rootworm control.

The Western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte is one of the most economically important insect pests in North America. Since 2003, transgenic maize expressing WCR-active proteins from Bacillus thuringiensis (Bt) have been widely adopted as the main approach to controlling WCR in the U.S. However, the emergence of field resistance to the Bt proteins in current commercial products has been documented in recent years, highlighting the need to develop additional tools for controlling this devasting pest. Here we report the discovery of Vpb4Da2 (initially assigned as Vip4Da2), a new insecticidal protein highly selective against WCR, through high-throughput genome sequencing of a Bt strain sourced from grain dust samples collected in the eastern and central regions of the US. Vpb4Da2 contains a sequence and domain signature distinct from families of other WCR-active proteins. Under field conditions, transgenic maize expressing Vpb4Da2 demonstrates commercial-level (at or below NIS 0.25) root protection against WCR, and reduces WCR beetle emergence by ≥ 97%. Our studies also conclude that Vpb4Da2 controls WCR populations that are resistant to WCR-active transgenic maize expressing Cry3Bb1, Cry34Ab1/Cry35Ab1 (reassigned as Gpp34Ab1/Tpp35Ab1), or DvSnf7 RNA. Based on these findings, Vpb4Da2 represents a valuable new tool for protecting maize against WCR.

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.

Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS.

BACKGROUND: Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T102 I, P106 S or TIPS). These same mutations combined with EPSPS overexpression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS Ki ∼ 0.5 µM) was engineered to express a variant AtEPSPS-T102 I, P106 A (TIPA Ki = 150 µM) to determine the resistance magnitude for a more potent variant EPSPS that might evolve in weeds. RESULTS: Transgenic A. thaliana plants, homozygous for one, two or four copies of AtEPSPS-TIPA, had resistance (IC50 values, R/S) as measured by seed production ranging from 4.3- to 16-fold. Plants treated in reproductive stage were male sterile with a range of R/S from 10.1- to 40.6-fold. A significant hormesis (∼ 63% gain in fresh weight) was observed for all genotypes when treated at the initiation of reproductive stage with 0.013 kg ha-1 . AtEPSPS-TIPA enzyme activity was proportional to copy number and correlated with resistance magnitude. CONCLUSIONS: A. thaliana, as a model weed expressing one copy of AtEPSPS-TIPA (300-fold more resistant), had only 4.3-fold resistance to glyphosate for seed production. Resistance behaved as a single dominant allele. Vegetative tissue resistance was 4.7-fold greater than reproductive tissue resistance and was linear with gene copy number. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of 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.

Cotton plants expressing a hemipteran-active Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs.

The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterization of a 35 kDa crystal protein from Bacillus thuringiensis, designated TIC807, which causes reduced mass gain and mortality of L. hesperus and L. lineolaris nymphs when presented in an artificial diet feeding assay. Cotton plants expressing the TIC807 protein were observed to impact the survival and development of L. hesperus nymphs in a concentration-dependent manner. These results, demonstrating in planta activity of a Lygus insecticidal protein, represent an important milestone in the development of cotton varieties protected from Lygus feeding damage.

Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation.

Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O(2) into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (alpha(3)) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co(2+), which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 A, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.