Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin‐activated form involves a major conformational change of the N‐terminal Domain I, which is remodelled into a tetrameric coiled‐coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.

https://digital.csic.es/bitstream/10261/276532/1/Microbial%20Biotechnology%20_L%c3%a1zaro%e2%80%90Berenguer%202022_%20Structural%20and%20functional%20role%20of%20Domain%20I%20for%20the%20insecticidal.pdf

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

http://www.jbc.org/article/S0021925823001321/pdf

Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity

Due to their different specificity, the use of Vip3 proteins from Bacillus thuringiensis in combination with the conventionally used Cry proteins in crop protection is being essential to counteract the appearance of insect resistance. Therefore, understanding the mode of action of Vip3 proteins is crucial for their better application, with special interest on the binding to membrane receptors as the main step for specificity. Derived from in vitro heterologous competition binding assays using 125I-Vip3A and other Vip3 proteins as competitors, it has been shown that Vip3 proteins share receptors in Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). In this study, using 125I-Vip3Aa, we have first extended the in vitro competition binding site model of Vip3 proteins to Spodoptera littoralis. With the aim to understand the relevance (in terms of toxicity) of the binding to the midgut sites observed in vitro on the insecticidal activity of these proteins, we have performed in vivo competition assays with S. littoralis larvae, using disabled mutant (non-toxic) Vip3 proteins as competitors for blocking the toxicity of Vip3Aa and Vip3Af. The results of the in vivo competition assays confirm the occurrence of shared binding sites among Vip3 proteins and help understand the functional role of the shared binding sites as revealed in vitro. 

/pdf/in-vivo-competition-assays-between-vip3-proteins-confirm-the-1xzbxkd2.pdf

In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis

Different Bacillus thuringiensis (Bt) strains produce a broad variety of pore-forming toxins (PFTs) that show toxicity against insects and other invertebrates. Some of these insecticidal PFT proteins have been used successfully worldwide to control diverse insect crop pests. There are several studies focused on describing the mechanism of action of these toxins that have helped to improve their performance and to cope with the resistance evolved by different insects against some of these proteins. However, crucial information that is still missing is the structure of pores formed by some of these PFTs, such as the three-domain crystal (Cry) proteins, which are the most commercially used Bt toxins in the biological control of insect pests. In recent years, progress has been made on the identification of the structural changes that certain Bt insecticidal PFT proteins undergo upon membrane insertion. In this review, we describe the models that have been proposed for the membrane insertion of Cry toxins. We also review the recently published structures of the vegetative insecticidal proteins (Vips; e.g. Vip3) and the insecticidal toxin complex (Tc) in the membrane-inserted state. Although different Bt PFTs show different primary sequences, there are some similarities in the three-dimensional structures of Vips and Cry proteins. In addition, all PFTs described here must undergo major structural rearrangements to pass from a soluble form to a membrane-inserted state. It is proposed that, despite their structural differences, all PFTs undergo major structural rearrangements producing an extended α-helix, which plays a fundamental role in perforating their target membrane, resulting in the formation of the membrane pore required for their insecticidal activity.

/pdf/structural-changes-upon-membrane-insertion-of-the-1dr5m8jg.pdf

Structural changes upon membrane insertion of the insecticidal pore-forming toxins produced by Bacillus thuringiensis

Transgenic corn and cotton expressing Cry and Vip insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt), have been a valuable tool for the management of lepidopteran pests. In 2019, a Vip3Aa-resistant strain of Helicoverpa zea (CEW-Vip-RR) was isolated from F2 screens of field populations in Texas. Characterizing the resistance mechanism in this strain is important for predicting the sustained efficacy of current commercial Bt traits and guiding the development of future transgenic traits. Resistance to insecticidal proteins in Bt traits is commonly associated with reduced toxin binding, with the exception of Vip3Aa resistance being associated to altered proteolytic processing in the insect host gut. Therefore, Vip3Aa protoxin processing was tested by incubation with midgut fluids from CEW-Vip-RR relative to a susceptible strain (CEW-SS). Finding no significant processing differences, alterations in Vip3Aa binding were tested by comparing binding of radiolabeled and biotinylated Vip3Aa toxin to midgut brush border membrane vesicles (BBMV) from CEW-Vip-RR and CEW-SS larvae. Specific Vip3Aa binding to CEW-Vip-RR BBMV in these experiments was consistently reduced when compared with CEW-SS BBMV. These results support that an altered Vip3Aa- receptor is associated with resistance in CEW-Vip-RR. Understanding this resistance mechanism could have important implications for resistance management decisions considering widespread Cry1 and Cry2 resistance in H. zea populations. IMPORTANCE Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton producing insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant incorporated protectant in Bt crops consistently providing excellent control of H. zea. The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.

/pdf/reduced-binding-associated-with-resistance-to-vip3aa-in-the-1ij24amw.pdf

Reduced binding associated with resistance to Vip3Aa in the corn earworm (Helicoverpa zea)

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125I-DI-III. In addition, the truncated protein 125I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo.IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for ex vivo toxicity to Sf21 cells.

Critical Domains in the Specific Binding of Radiolabeled Vip3Af Insecticidal Protein to Brush Border Membrane Vesicles from Spodoptera spp. and Cultured Insect Cells.

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect’s resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore–crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of “functional mortality” was used to discriminate between “nontoxic” and “toxic” isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore–crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130 kDa whereas the Bt isolate Bl5NA expresses a protein of 65–70 kDa. The LC–MS/MS results indicate that the major peptides in the 130 kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70 kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce β-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

Maria Lázaro-Berenguer

Papers

Critical Domains in the Specific Binding of Radiolabeled Vip3Af Insecticidal Protein to Brush Border Membrane Vesicles from Spodoptera spp. and Cultured Insect Cells.

Structural and functional role of Domain I for the insecticidal activity of the Vip3Aa protein from Bacillus thuringiensis

In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis

Evaluation of the Toxicity of Supernatant Cultures and Spore–Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria