Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions.

Danqi Chen,Xiaoran Fu,Graham P Head,Timothy J Rydel,William J Moar,Victor Guzov,Jason S Milligan,Karen Gabbert,Eric C Bretsynder,James A Baum,Agoston Jerga,Jeffrey A Haas,Anilkumar Gowda,Altair Semeao,Allah Bakhsh

doi:10.1371/journal.pone.0249150

Abstract

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP’s) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

Highlights

Soybean, Glycine max (L.) is one of the most important oilseed crops globally and was cultivated on 121 million hectares (MHa) producing 334 million metric tons in 2019 [1]
Using the recently developed disabled insecticidal protein (DIP) assay [42], we demonstrate that Cry1A.2 and Cry1B.2 differ in receptor utilization from each other, commercially available and soon to be commercialized Bacillus thuringiensis (Bt) proteins in soy including Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A, demonstrating their utility in future insect protected soybean products
Luria Broth (LB), agar, chloramphenicol, Q sepharose1, HIS-Select1, sodium carbonate, sodium chloride (NaCl), tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), hydrochloric acid (HCl), phenylmethylsulfonyl fluoride (PMSF), benzamidine, tris(2-carboxyethyl) phosphine (TCEP), Triton X-100, polyethylene glycol 8000 (PEG8000), magnesium chloride (MgCl2), trypsin from bovine pancreas, and chloramphenicol were from Millipore Sigma

Summary

Introduction

Glycine max (L.) is one of the most important oilseed crops globally and was cultivated on 121 million hectares (MHa) producing 334 million metric tons in 2019 [1]. Receptor utilization assessment of new Bacillus thuringiensis proteins active against soybean insect pests which amounts to almost a third of the global market. Important lepidopteran insect pests of soybean in Brazil include soybean looper (SBL, Chrysodeixis includens Walker), velvetbean caterpillar (VBC, Anticarsia gemmatalis Hubner), southern armyworm (SAW, Spodoptera eridania), black armyworm (BLAW, Spodoptera cosmioides) and old-world bollworm (OWB, Helicoverpa armigera) [5,6,7,8,9]. Since 2013, farmers have adopted the cultivation of genetically modified soybeans expressing proteins from the common soil bacterium Bacillus thuringiensis (Bt, Bt soybean) to control lepidopteran pests [14, 15]

Methods

Results

Conclusion