Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects

N Banyuls,J Van Rie,C S Hernández-Rodríguez,J Ferré

doi:10.1038/s41598-018-25346-3

Abstract

Vip3 vegetative insecticidal proteins from Bacillus thuringiensis are an important tool for crop protection against caterpillar pests in IPM strategies. While there is wide consensus on their general mode of action, the details of their mode of action are not completely elucidated and their structure remains unknown. In this work the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. From the 558 residue substitutions, 19 impaired protein expression and other 19 substitutions severely compromised the insecticidal activity against Spodoptera frugiperda. The latter 19 substitutions mainly clustered in two regions of the protein sequence (amino acids 167–272 and amino acids 689–741). Most of these substitutions also decreased the activity to Agrotis segetum. The characterisation of the sensitivity to proteases of the mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of most selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.

Highlights

Vegetative insecticidal proteins (Vip) are entomopathogenic proteins of increasing importance in the framework of sustainable pest management and crop protection strategies
The sequence positions that were not changed to Ala were (Table 1): (i) the Met[1] corresponding to the start codon; (ii) the N-terminus region because, at the start of this project, this region was thought not to be involved in the insecticidal activity of the toxin; (iii) the 30 Ala residue positions in the Vip3Af1(WT), and (iv) 48 positions for which mutagenesis failed and no mutant protein could be obtained
As a first step to determine the positions critical for the insecticidal activity of the Vip3Af1(WT), a quick screening was carried out on S. frugiperda neonates with E. coli cells expressing each of the 588 clones (Table 1)

Summary

Introduction

Vegetative insecticidal proteins (Vip) are entomopathogenic proteins of increasing importance in the framework of sustainable pest management and crop protection strategies. Of particular interest are Vip3A proteins, which were first described in the late 90’s and were found to be active against lepidopteran species with potencies different to those of the widely used Cry proteins, such as the Cry-tolerant Agrotis ipsilon (Lepidoptera: Noctuidae) or of many other species from the Spodoptera genus[1,2,3]. Alanine scanning is a successful technique for mapping crucial positions or epitopes in a protein and allows a greater insight into protein structure-function relationships It consists of a systematic change, one by one, of the native amino acid residues to alanine residues. A total of 558 out of the 788 residues of the Vip3Af1 sequence were changed to alanine and studied in detail This is a first step in a better understanding of the Vip3A protein structure and the relationship to its biochemical hallmarks insecticidal activity

Methods

Results

Conclusion