Studies on the insecticidal mechanism of bacillus thuringiensis vip3a and cry proteins
- Autores: Yudongquan Quan
- Directores de la Tesis: Juan Ferré (dir. tes.), Patrica Hernández Martínez (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2022
- Idioma: español
- Tribunal Calificador de la Tesis: Primitivo Caballero (presid.), Baltasar Escriche (secret.), Souad Rouis (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
The improvement in the performances of global agricultural or food systems significantly affects by the control of pests and pathogens. The more healthy, environmental and sustainable integrated pest management (IPM) strategies are demanded in the modern agriculture. One of the most important strategy or biotechnology rely on Bacillus thuringiensis (Bt) and its secreted insecticidal proteins has been the most economically successful use to control pests to date. Recently, a new subfamily of vegetative insecticidal proteins (Vip3) produced during the vegetative growth phase of Bt was considered as the combined use with the other Bt proteins, especially that have been reported evolving distinct resistant (Cry toxins) with long-term commercial use. Despite Vip3 proteins have been revealed that no sequence or homology similarity with Cry proteins and are toxic to a wide variety of Lepidoptera, its mode of action is yet not completely elucidated. To better apply and understand the Vip3 proteins, in this doctoral we investigate the potential interaction of combination use (insecticidal spectrum, cross resistance, interaction), then we analyze the critical residues on the structure and toxicity through mutagenesis, and use these critical mutations to shed the light on the mode of action of Vip3 proteins.
Firstly we investigate ten Bt toxins (Cry1Ab, Cry1Ac, Cry1Ah, Cry1Fa, Cry2Aa, Cry2Ab, Cry1Ie, Vip3Aa19, Vip3Aa16, and Vip3Ca) toxicities and their combination of use at lab against Mythimna separata, which is a destructive pest of agricultural crops in East Asia. The bioassays results revealed that LC50 (lethal concentration for 50% mortality) values (Cry1Ac/Vip3Aa19/Vip3Ca < Cry1Ab/Cry2Aa/Vip3Aa16 < Cry2Ab/Cry1Fa/Cry1Ah < Cry1Ie) ranged from 1.6 to 78.6 µg/g (toxin/diet) for those toxins. In addition, the interactions between Vip3 and Cry proteins in this study indicated the synergism was tested only in the combination group Vip3Aa16 and Cry1 toxins, that the significant groups (Vip3Aa16 and Cry1Fa, Vip3Aa16 and Cry1Ie) showed around 6.3 to 9.2 fold (or synergetic factors) than expected. We also tested the toxicities of Vip3 (Vip3Aa and Vip3Ca) proteins against different Cry1A-, Cry2A-, Dipel- and Vip3-resistant insect species. Comparing the toxicities of Vip3 proteins between resistant Cry1A proteins, Dipel (Helicoverpa armigera, Trichoplusia ni, Ostrinia furnacalis and Plodia interpunctella) or Cry2Ab (H. armigera and T. ni) and susceptible strains, there were not cross-resistant detected in these insects. In contrast, the strong cross-resistance to the Vip3Ca protein was observed in Vip3Aa (or Vip3Aa/Cry2Ab) resistant H. armigera colonies. Moreover, Vip3Aa protein was tested almost lost the toxicity in O. furnacalis (only showed moderate growth inhibition at the highest concentration tested (100 µg/g)), but Vip3Ca protein was highly toxic. Besides, a colony of Vip3Aa resistant M. separata was obtained and also tested the susceptibility to Cry1Ab and Cry1F, it also suggested no cross resistance between Cry1 and Vip3Aa proteins.
To shed light on the structure of Vip3 proteins, we analyzed the trypsin fragmentation of several critical mutants (or patterns) of the Vip3Af protein obtained through alanine scanning mutagenesis. Based on protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains: domain I ranges amino acids (aa) 12–198, domain II aa199–313, domain III aa314–526, domain IV aa527–668, and domain V aa669–788. The effect of some mutations on the ability to form a tetrameric molecule revealed that domains I–III are required for tetramerization, while domain V is not, domain IV is not clear.
In addition, 12 mutants of the Vip3Af protein were generated by site-directed mutagenesis, which are from critical residues to constitute the integrity of oligomer and unique mutation (at N-terminal) only observed in some Vip3 proteins. Finally ten of these mutants were successfully expressed and tested for stability and toxicity against three insect pests (Spodoptera frugiperda, Spodoptera littoralis and Grapholita molesta). Regarding toxicity, only the mutant M34L (change of Met34 to Lys34) significantly increased the toxicity in S. littoralis, whereas the other mutants (or substitutions) did not improve, or even decreased. The profiles of these site- directed mutagenesis upon trypsin treatmen in the SDS-PAGE (stability) and toxicity showed that, residue 483 required an acidic residue, and residue 552 an aromatic residue, the others are still not clear.
The mode of action of Vip3 proteins is still debatable currently. To clear the details, we firstly analyzed the stability of proteolytic fragments (activated Vip3Af protein) digested by commercial trypsin and S. frugiperda midgut juice in vitro. The results revealed a misleading degradation patterns of Vip3Af was observed with trypsin or midgut juice in the SDS-PAGE. However, the gel filtration chromatography indicated that, under native conditions (incubated with inhibitor before heat for SDS-PAGE), Vip3Af is stable and as a tetramer for protoxin or activation. The identification of the proteolytic fragments suggested a cleavage site (aa 198/199) renders two fragments of approximately 20 kDa and 65 kDa (activation) which still strongly remain together (as tetramer) and that are no further processed even at high protease concentrations.
The understanding of the role of specific binding to membrane receptors is so critical to determine their specificity. In this part, we have set up a new binding condition of 125I-Vip3Af to S. frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV), and the specific binding was observed to BBMVs. Heterologous competitions revealed that, Cry1Ac and Cry1F did not compete for Vip3Af binding sites; Vip3Aa shares the same binding sites with Vip3Af, but that Vip3Ca merely shares partially. Using the truncated Vip3Af molecules (DI-III, DI-IV, DIV-V, and DIV) by trypsin treatment of selected critical alanine-mutants as competitors (compete with 125I-Vip3Af), the results showed 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 experiments using 125I-DI-III. In addition, cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af (or DI-V), DIV-V is totally lost the toxic (at the concentration C=100 ug/ml) suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells. But the domains IV and V is necessary to the toxicity in vivo, and the function need further research.
Finally, a field population of M. separata was collected and subjected to laboratory selection with either Vip3Aa, Cry1Ab or Cry1F proteins. And only the Vip3Aa resistant (>3061-fold) M. separata strain was rapidly obtained after 8 or 9 generations, no remarkable Cry1Ab or Cry1F resistance was observed at the same selected time. Analysis of the difference of labeled 125I-Vip3Aa binding to BBMVs from larvae from the susceptible and resistance insects revealed no any qualitative or quantitative binding difference. It suggests that altered binding to midgut membrane receptors is not the main mechanism of resistance to Vip3Aa protein.
The results we obtained are helpful to the applied strategy of Bt toxins and give to a better understanding of the protein structure and function of Vip3A proteins, which will be guided for the strategies in pest management or resistance management. In addition, the identification of the binding domains of Vip3A is so critical to shed light on the mode of action of Vip3 proteins.
