Proteolysis activation of Cry1Ac and Cry2Ab protoxins by larval midgut juice proteases from Helicoverpa armigera.

Shaoyan Liu,Yidong Wu,Shuo Wang,Shuwen Wu,Yihua Yang,Youjun Zhang

doi:10.1371/journal.pone.0228159

Abstract

Proteolytic processing of Bacillus thuringiensis (Bt) Cry protoxins by insect midgut proteases is critical to their insecticidal activities against target insects. Although transgenic Bt cotton expressing Cry1Ac and Cry2Ab proteins have been widely used for control of the cotton bollworm (Helicoverpa armigera) in the field, the proteolytic cleavage sites in the two protoxins targeted by H. armigera midgut proteases are still not clear. In this study, the proteolysis of Cry1Ac and Cry2Ab protoxins by midgut juice prepared from midgut tissue of H. armigera larvae was investigated. Cleavage of Cry1Ac protoxin by midgut proteases formed a major protein fragment of ~65 kDa, and N-terminal sequencing revealed that cleavage occurred at Arg28 in the fore-end of helix α-1 in domain I of Cry1Ac. Cleavage of Cry2Ab protoxin by midgut juice proteases produced a major protein fragment of ~50 kDa, and the cleavage occurred at Arg139 between helices α-3 and α-4 in domain I of Cry2Ab. The amino acids Arg28 of Cry1Ac and Arg139 of Cry2Ab were predicted as putative trypsin cleavage sites. Bioassay data showed that the toxicities (LC50s) of Cry1Ac and Cry2Ab protoxins were equivalent to those of their respective midgut juice-activated toxins in the susceptible SCD strain of H. armigera. Identification of the exact sites of N-terminal activation of Cry1Ac and Cry2Ab protoxins will provide a basis for a better understanding of the mode of action and resistance mechanisms based on aberrant activation of these protoxins in H. armigera.

Highlights

Bacillus thuringiensis (Bt) is a ubiquitous gram-positive bacterium, and during sporulation, Bt strains produce crystal proteins (Cry toxins) that are toxic to a variety of insects, such as lepidopterans, coleopterans, dipterans and hemipterans [1]
We investigated the proteolysis of Cry1Ac and Cry2Ab protoxins by H. armigera midgut juice in vitro and detected the proteolysis by SDS-PAGE
Protoxins were digested with H. armigera midgut juice at 37 ̊C using different midgut juice/ protoxin ratios

Summary

Introduction

Bacillus thuringiensis (Bt) is a ubiquitous gram-positive bacterium, and during sporulation, Bt strains produce crystal proteins (Cry toxins) that are toxic to a variety of insects, such as lepidopterans, coleopterans, dipterans and hemipterans [1]. Bt Cry toxins have been extensively used in sprays and transgenic plants, which has contributed to the efficient control of different agricultural pests. They have reduced the use of chemical insecticides and increased farmer profits [2,3,4,5]. The majority of Cry toxins are produced in insoluble and inactive forms as crystal inclusions composed of protoxins. After ingestion by target insect larvae, the crystals are solubilized in the alkaline environment of the larval midgut and are activated by midgut proteases [6].

Methods

Results

Conclusion