scholarly journals Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260532
Author(s):  
Jean-Louis Kouadio ◽  
Meiying Zheng ◽  
Michael Aikins ◽  
David Duda ◽  
Stephen Duff ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Protective Antigen ◽  
Domain Architecture ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Insecticidal Proteins ◽  
Amino Terminal ◽  
Carbohydrate Binding Modules

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel β-sheets organized into β-sandwich layers. The amino-terminal domains 1–3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long β-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4–6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.

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

2020 ◽  
Author(s):  
David Bowen ◽  
Yong Yin ◽  
Stanislaw Flasinski ◽  
Catherine Chay ◽  
Gregory Bean ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pathogenic Bacteria ◽  
Insect Pests ◽  
Diabrotica Virgifera Virgifera ◽  
Western Corn Rootworm ◽  
Cross Resistance ◽  
Corn Rootworm ◽  
Diabrotica Virgifera ◽  
Insecticidal Proteins ◽  
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.

scholarly journals Binary Toxins from Bacillus thuringiensis Active against the Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

2004 ◽  
Vol 70 (8) ◽  
pp. 4889-4898 ◽  
Author(s):  
James A. Baum ◽  
Chi-Rei Chu ◽  
Mark Rupar ◽  
Gregory R. Brown ◽  
William P. Donovan ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Insect Pest ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Binary Toxin ◽  
Corn Rootworm ◽  
Diabrotica Virgifera ◽  
Crystal Proteins ◽  
Genes Encoding

ABSTRACT The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.

scholarly journals Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

2002 ◽  
Vol 68 (3) ◽  
pp. 1137-1145 ◽  
Author(s):  
R. Tracy Ellis ◽  
Brian A. Stockhoff ◽  
Lisa Stamp ◽  
H. Ernest Schnepf ◽  
George E. Schwab ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Insecticidal Activity ◽  
Diabrotica Virgifera Virgifera ◽  
Bacillus Sphaericus ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Sequence Comparisons ◽  
Insecticidal Proteins ◽  
Crystal Proteins ◽  
Insecticidal Crystal Proteins

ABSTRACT A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258052
Author(s):  
Jean-Louis Kouadio ◽  
Stephen Duff ◽  
Michael Aikins ◽  
Meiying Zheng ◽  
Timothy Rydel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Receptor Binding ◽  
Functional Characterization ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Insecticidal Proteins

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 (β-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.

scholarly journals Resistance to Bt Maize by Western Corn Rootworm: Effects of Pest Biology, the Pest–Crop Interaction and the Agricultural Landscape on Resistance

Insects ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 136
Author(s):  
Aaron Gassmann
Keyword(s):  
Agricultural Landscape ◽  
Continuous Cultivation ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Agricultural Crop ◽  
Bt Maize ◽  
Bt Toxin ◽  
Resistance Evolution

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is among the most serious pests of maize in the United States. Since 2003, transgenic maize that produces insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) has been used to manage western corn rootworm by killing rootworm larvae, which feed on maize roots. In 2009, the first cases of field-evolved resistance to Bt maize were documented. These cases occurred in Iowa and involved maize that produced Bt toxin Cry3Bb1. Since then, resistance has expanded to include other geographies and additional Bt toxins, with some rootworm populations displaying resistance to all commercially available Bt traits. Factors that contributed to field-evolved resistance likely included non-recessive inheritance of resistance, minimal fitness costs of resistance and limited adult dispersal. Additionally, because maize is the primary agricultural crop on which rootworm larvae can survive, continuous maize cultivation, in particular continuous cultivation of Bt maize, appears to be another key factor facilitating resistance evolution. More diversified management of rootworm larvae, including rotating fields out of maize production and using soil-applied insecticide with non-Bt maize, in addition to planting refuges of non-Bt maize, should help to delay the evolution of resistance to current and future transgenic traits.

scholarly journals RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions

Insects ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 57
Author(s):  
Molly Darlington ◽  
Jordan D. Reinders ◽  
Amit Sethi ◽  
Albert L. Lu ◽  
Partha Ramaseshadri ◽  
...  
Keyword(s):  
Environmental Protection Agency ◽  
Management Strategies ◽  
Housekeeping Genes ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Lessons Learned ◽  
Corn Rootworm ◽  
In Planta ◽  
Commercial Use

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1–2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

scholarly journals Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm

Toxins ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 162 ◽  
Author(s):  
Jingtong Hou ◽  
Ruth Cong ◽  
Michi Izumi-Willcoxon ◽  
Hana Ali ◽  
Yi Zheng ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
3D Structure ◽  
Diabrotica Virgifera Virgifera ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Diabrotica Virgifera ◽  
Cry Protein ◽  
Neutral Ph ◽  
Highly Active ◽  
Cry Toxin

A novel Bacillus thuringiensis Cry protein, Cry8Hb, active against Diabrotica virgifera virgifera (Western corn rootworm, WCRW) was discovered. Unexpectedly, the anti-rootworm activity of the Cry8Hb toxin was enhanced significantly by fusing Escherichia coli maltose binding protein (MBP) to this Cry toxin. While the exact mechanism of the activity enhancement remains indefinite, it is probable that the enhancement is a result of increased solubility of the MBP-Cry8Hb fusion in the rootworm midgut. This hypothesis was examined using a synthetic Cry3 protein called IP3-1, which was not soluble at a neutral pH like Cry8Hb and marginally active to WCRW. When IP3-1 was fused to MBP, its anti-WCRW activity was enhanced 13-fold. To further test the hypothesis, DNA shuffling was performed on IP3-1 to increase the solubility without MBP. Screening of shuffled libraries found six new IP3 variants showing very high anti-WCRW activity without MBP. Sequence and 3D structure analysis of those highly active, shuffled IP3 variants revealed several charge-altering mutations such as Lys to Glu on the putative MBP-attaching side of the IP3 molecule. It is likely that those mutations make the protein acidic to substitute the functions of MBP including enhancing the solubility of IP3 at a neutral pH.

scholarly journals A new Bacillus thuringiensis protein for Western corn rootworm control

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242791
Author(s):  
Yong Yin ◽  
Stanislaw Flasinski ◽  
William Moar ◽  
David Bowen ◽  
Cathy Chay ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Insect Pests ◽  
Diabrotica Virgifera Virgifera ◽  
Field Resistance ◽  
Western Corn Rootworm ◽  
Transgenic Maize ◽  
Corn Rootworm ◽  
The Us ◽  
Central Regions ◽  
Root Protection

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.

EFFECTS OF AZADIRACHTIN ON THE WESTERN CORN ROOTWORM, DIABROTICA VIRGIFERA VIRGIFERA (LECONTE) (COLEOPTERA: CHRYSOMELIDAE)

1991 ◽  
Vol 123 (3) ◽  
pp. 707-710 ◽  
Author(s):  
Y.S. Xie ◽  
D. Gagnon ◽  
J.T. Arnason ◽  
B.J.R. Philogène ◽  
J.D.H. Lambert ◽  
...  
Keyword(s):  
United States ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Crop Damage ◽  
Corn Rootworm ◽  
Diabrotica Virgifera ◽  
Corn Production ◽  
Main Method ◽  
Diabrotica Virgifera Virgifera Leconte

Corn rootworm (Diabrotica spp., Coleoptera: Chrysomelidae) is a serious pest insect of corn production. It is estimated that farmers in the United States have losses of over $1 billion each year as a result of crop damage and treatment costs for this pest (Metcalf 1986). Chemical control is the main method of suppressing corn rootworm populations and the amount of insecticide used against Diabrotica spp. is greater than for any other pests of corn in the United States (Suguiyama and Carlson 1985). The development of nontoxic and biodegradable alternatives to chemical insecticides is highly desirable.

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