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

Abstract

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.

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

“…Mpp75Aa1.1 (formerly known as Cry75Aa1.1), discovered by mining the genome of Brevibacillus laterosporus , provides effective control of WCR larvae when expressed in transgenic maize [ 7 ]. Furthermore, Mpp75Aa1.1 exhibits significant activity toward WCR that are resistant to Cry3Bb1 and Gpp34Ab1/Tpp35Ab1 (formerly known as Cry34Ab1/Cry35Ab1) [ 7 ], suggesting a distinct receptor specificity in WCR compared to Cry3Bb1 and Gpp34Ab1/Tpp35Ab1. Bioinformatic analysis identified that Mpp75Aa1.1 belongs to the aerolysin-like superfamily of bacterial beta-pore forming proteins (β-PFPs) found across all kingdoms of life [ 8 , 9 ].…”

Safety assessment of insecticidal proteins: A case study utilizing His-tagged protein to determine the safety of Mpp75Aa1.1, a new ETX_MTX2 protein that controls western corn rootworm

“…Two strains of B. laterosporus were isolated following methods previously described (11). The EG4227 strain was isolated from grain dust while the BOD strain was isolated from the powder of a commercially available probiotic capsule.…”

“…The proteins were electro-transferred from the gel to 0.2 M nitrocellulose membrane using Bio-Rad Turbo Blot TM device for 7 minutes. The membrane was probed with a primary anti-mMpp75Aa2 (mature form of Mpp75Aa2, (11)) polyclonal antibody diluted at 1 g/ml in the 1X phosphate buffered saline with 0.1% Tween ® 20 with PBST + 2% non-fat milk (NFMK) overnight (~12 hours at 4 o C). The membrane was washed 3 times with 1X PBST to remove primary antibody.…”

“…Notably, the top alignment, which represents the self-identification of Mpp75Aa1.1, is labeled as "insecticidal protein" from B. laterosporus (NCBI Accession ASY04853.1). This annotation is unsurprising as Mpp75Aa1.1 is a known insecticidal member of the ETX_MTX2 family of proteins (11).…”

Western Corn Rootworm, Plant and Microbe Interactions: A Review and Prospects for New Management Tools

“…These germplasm will be widely planted for the first time in 2022. Bayer also recently discovered an additional Bt protein [194] and a protein from Brevibacillus laterosporus [195], each with strong activity against western corn rootworm larvae and no cross resistance to current rootworm toxins. Corteva has a new transgenic maize line producing a toxin derived from Pseudomonas chlororaphis pyramided with dsRNA available [151], but the feasibility for large scale application has yet to be evaluated.…”