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.
Subject(s)
Amino Acid Substitution , Bacillus thuringiensis/metabolism , Bacterial Proteins/pharmacology , Spodoptera/drug effects , Alanine/genetics , Amino Acid Motifs , Animals , Bacillus thuringiensis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Models, Molecular , Molecular Docking Simulation , Protein Stability , Protein Structure, Tertiary , Spodoptera/growth & developmentABSTRACT
AIMS: To identify known vip genes and to detect potentially novel vip genes in a collection of 507 strains of Bacillus thuringiensis. METHODS AND RESULTS: Following a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy, four restriction patterns were found within the vip1 family: vip1Aa1, vip1Ba1/vip1Ba2 and vip1Ca. In the screening of vip2 genes, patterns similar to those of vip2Aa1, vip2Ba1/vip2Ba2 and vip2Ac1 genes were observed. Patterns for vip3Aa1, vip3Ae2 and vip3Af1 were found among vip3 genes. Two new patterns revealed novel vip1 and vip3A genes. The observed frequency of genes belonging to vip1 and vip2 families was around 10%, whereas 48.9% of the strains showed amplification of vip3 genes. A tendency of vip and cry genes to occur together has been observed in this collection of B. thuringiensis strains. CONCLUSIONS: Ten different patterns of vip genes belonging to the three vip families and two novel vip genes have been identified in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that vip1 and vip2 genes have been identified by PCR-RFLP. Furthermore, the results show that the strategy used in this study can lead to the classification of known vip genes as well as the identification of novel vip genes.
Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , DNA Primers/genetics , Gene Frequency , Polymerase Chain Reaction/methods , Sequence Analysis, DNAABSTRACT
The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5'-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions and junction sequences, suggesting a common origin for these genes. Recombinant baculoviruses carrying the firefly luciferase gene under the control of different 5'-upstream regions of the repat1 gene were constructed to elucidate the influence of these regions in gene expression. Infection of Helicoverpa zea gut-derived cells with the recombinant baculoviruses revealed the upstream regions of the repat1 gene which are involved in gene transcription and demonstrated the role of an intron located in the 5'-untranslated region in the enhancement of gene expression.
