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Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis
- Source :
- Toxins; Volume 12; Issue 9; Pages: 543, Academica-e. Repositorio Institucional de la Universidad Pública de Navarra, instname, Academica-e: Repositorio Institucional de la Universidad Pública de Navarra, Universidad Pública de Navarra, Toxins, Vol 12, Iss 543, p 543 (2020)
- Publication Year :
- 2020
- Publisher :
- Multidisciplinary Digital Publishing Institute, 2020.
-
Abstract
- The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee. This research was funded by the Spanish Ministry project AGL2017-83498-C2-1-R.
- Subjects :
- Biopesticides
lepidopteran cells
Health, Toxicology and Mutagenesis
Vegetative pesticidal proteins
lcsh:Medicine
Sf9
Entomopathogen
Toxicology
law.invention
03 medical and health sciences
chemistry.chemical_compound
law
Recombinant baculovirus
Bacillus thuringiensis
Consensus sequence
Actin
030304 developmental biology
Cytochalasin D
recombinant baculovirus
0303 health sciences
biology
030306 microbiology
lcsh:R
Transfection
biology.organism_classification
vegetative insecticidal proteins
Vpb/Vpa
entomopathogen
ADP-ribosyltransferase
biopesticides
broad spectrum
Vip1/Vip2
Molecular biology
Lepidopteran cells
Nocodazole
Broad spectrum
chemistry
Recombinant DNA
Vpa
vegetative pesticidal proteins
Subjects
Details
- Language :
- English
- ISSN :
- 20726651
- Database :
- OpenAIRE
- Journal :
- Toxins; Volume 12; Issue 9; Pages: 543
- Accession number :
- edsair.doi.dedup.....5b58dc585c255ee2084c6d0bcb3fd9ab
- Full Text :
- https://doi.org/10.3390/toxins12090543