Domain Shuffling between Vip3Aa and Vip3Ca: Chimera Stability and Insecticidal Activity against European, American, African, and Asian Pests

Abstract

The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Helicoverpa armigera, Mamestra brassicae, Anticarsia gemmatalis, and Ostrinia furnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S. frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189–509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostrinia furnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.

Insecticidal Activity of 11 Bt toxins and 3 Transgenic Maize Events Expressing Vip3Aa19 to Black Cutworm, Agrotis ipsilon (Hufnagel)

“…Vip3_ch4 is a chimeric protein with a central domain; i.e., domains II and III, as proposed for the Vip3Af domains by Quan and Ferré (2019) [41] from Vip3Aa45, and domain I as well as domains IV and V from Vip3Ca2. It demonstrates insecticidal activity against O. furnacalis, but does not show toxicity to S. frugiperda, H. armigera, Anticarsia gemmatalis, and Mamestra brassicae [19]. In this study, Vip3_ch4 was nontoxic to BCW, confirming a loss of toxicity to Noctuidae pests.…”

“…The lethal dose (LC 50 ) ratio of Vip3_ch1 was 15-fold higher than Cry1F, but it was 12-fold lower than Vip3Aa19. Similar to Vip3Aa16 [24], Vip3_ch1 was nontoxic to O. furnacalis [19].…”

“…Vip3Aa19 was obtained by IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction and affinity column chromatography (GE Healthcare). Vip3_ch1 and Vip3_ch4, chimeric proteins from Vip3Aa45 and Vip3Ca2, were generated by overlap PCR (Polymerase Chain Reaction) [19]. Vip3Ca2, Vip3_ch1 and Vip3_ch4 were provided by J. Gomis-Cebolla and J. Ferré from the Institute of Biotechnology and Biomedicine, Universitat de València, Spain.…”

Structural and Functional Insights into the C-terminal Fragment of Insecticidal Vip3A Toxin of Bacillus thuringiensis

“…Vip3A and Vip3B share about 65% sequence similarity and have different insecticidal specificity [42], and recently the C-terminal fragment was found to be related to insecticidal specificity of Vip3 [42,43]. Our structure provides a good opportunity to further study the mechanism of insecticidal specificity between Vip3A and Vip3B.…”

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

“…In the Special Issue, five papers analyze different aspects of its biology. They cover aspects ranging from its crystal structure [ 14 ] and structural–functional domain analyses [ 15 ] to different aspects in the mode of action, such as a study of a possible receptor (the alkaline phosphatase) in a resistant strain [ 16 ], the role of oligomerization in toxicity [ 17 ], and the study of intracellular events promoted by Vip3A intoxication in Spodoptera frugiperda Sf9 cells [ 18 ].…”