Cry1Ac Toxin Binding in the Velvetbean Caterpillar : Study of Midgut Aminopeptidases N

Overview

Journal Front Physiol

Date 2024 Nov 13

PMID 39534858

Authors

M D Lanzaro

I Padilha

L F C Ramos

A P G Mendez

A Menezes

Y M Silva

M R Martins

M Junqueira

F C S Nogueira

C D AnoBom

G M Dias

F M Gomes

D M P Oliveira

Affiliations

Soon will be listed here.

Abstract

The velvetbean caterpillar is one of the main soybean defoliators in Brazil. Currently, the main biopesticide used to control insect pests worldwide is the bacteria (Bt), which produces entomopathogenic Crystal toxins (Cry) that act in the midgut of susceptible insects, leading them to death. The mode of action of Cry toxins in the midgut involves binding to specific receptors present on the brush border of epithelial cells such as aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, and others. Mutations in these receptors, among other factors, may be involved in the development of resistance; identification of functional Cry receptors in the midgut of is crucial to develop effective strategies to overcome this possible scenario. This study's goal is to characterize APNs of and identify a receptor for Cry1Ac in the midgut. The interaction of Bt spores with the midgut epithelium was observed by immunohistochemistry and total aminopeptidase activity was estimated in brush border membrane vesicle (BBMV) samples, presenting higher activity in challenged individuals than in control ones. Ten APN sequences were found in a ' transcriptome and subjected to different analysis, such as phylogenetic tree, multiple sequence alignment and identification of signal peptide, activity domains and GPI-anchor signal. BBMV proteins from 5th instar larvae were submitted to a ligand blotting using activated Cry1Ac toxin and a commercial anti-Cry polyclonal antibody; corresponding bands of proteins that showed binding to Cry toxin were excised from the SDS-PAGE gel and subjected to mass spectrometry analysis, which resulted in the identification of seven of those APNs. Quantitative PCR was realized to compare expression levels between individuals subjected to sublethal infection with Bt spores and control ones, presenting up- and downregulations upon Bt infection. From these results, we can infer that aminopeptidases N in could be involved in the mode of action of Cry toxins in its larval stage.

References

Gomez I, Arenas I, Benitez I, Miranda-Rios J, Becerril B, Grande R . Specific epitopes of domains II and III of Bacillus thuringiensis Cry1Ab toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta. J Biol Chem. 2006; 281(45):34032-9. DOI: 10.1074/jbc.M604721200. View

Palma L, Munoz D, Berry C, Murillo J, Caballero P . Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins (Basel). 2014; 6(12):3296-325. PMC: 4280536. DOI: 10.3390/toxins6123296. View

Erlanger B, KOKOWSKY N, Cohen W . The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys. 1961; 95:271-8. DOI: 10.1016/0003-9861(61)90145-x. View

Rajagopal R, Agrawal N, Selvapandiyan A, Sivakumar S, Ahmad S, Bhatnagar R . Recombinantly expressed isoenzymic aminopeptidases from Helicoverpa armigera (American cotton bollworm) midgut display differential interaction with closely related Bacillus thuringiensis insecticidal proteins. Biochem J. 2002; 370(Pt 3):971-8. PMC: 1223210. DOI: 10.1042/BJ20021741. View

Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R . Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim Biophys Acta. 2004; 1667(1):38-46. DOI: 10.1016/j.bbamem.2004.08.013. View

Chen J, Brown M, Hua G, Adang M . Comparison of the localization of Bacillus thuringiensis Cry1A delta-endotoxins and their binding proteins in larval midgut of tobacco hornworm, Manduca sexta. Cell Tissue Res. 2005; 321(1):123-9. DOI: 10.1007/s00441-005-1124-6. View

Gomes F, Carvalho D, Machado E, Miranda K . Ultrastructural and functional analysis of secretory goblet cells in the midgut of the lepidopteran Anticarsia gemmatalis. Cell Tissue Res. 2013; 352(2):313-26. DOI: 10.1007/s00441-013-1563-4. View

Xie Y, Li H, Luo X, Li H, Gao Q, Zhang L . IBS 2.0: an upgraded illustrator for the visualization of biological sequences. Nucleic Acids Res. 2022; 50(W1):W420-W426. PMC: 9252815. DOI: 10.1093/nar/gkac373. View

Bel Y, Zack M, Narva K, Escriche B . Specific binding of Bacillus thuringiensis Cry1Ea toxin, and Cry1Ac and Cry1Fa competition analyses in Anticarsia gemmatalis and Chrysodeixis includens. Sci Rep. 2019; 9(1):18201. PMC: 6890801. DOI: 10.1038/s41598-019-54850-3. View

10.

Chauhan V, Dhania N, Lokya V, Bhuvanachandra B, Padmasree K, Dutta-Gupta A . Midgut aminopeptidase N expression profile in castor semilooper () during sublethal Cry toxin exposure. J Biosci. 2021; 46. View

11.

Bravo A, Gill S, Soberon M . Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon. 2007; 49(4):423-35. PMC: 1857359. DOI: 10.1016/j.toxicon.2006.11.022. View

12.

Pigott C, Ellar D . Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol Mol Biol Rev. 2007; 71(2):255-81. PMC: 1899880. DOI: 10.1128/MMBR.00034-06. View

13.

Lin P, Cheng T, Jin S, Jiang L, Wang C, Xia Q . Structural, evolutionary and functional analysis of APN genes in the Lepidoptera Bombyx mori. Gene. 2013; 535(2):303-11. DOI: 10.1016/j.gene.2013.11.002. View

14.

Chattopadhyay P, Banerjee G . Recent advancement on chemical arsenal of toxin and its application in pest management system in agricultural field. 3 Biotech. 2018; 8(4):201. PMC: 5874219. DOI: 10.1007/s13205-018-1223-1. View

15.

Hughes A . Evolutionary diversification of aminopeptidase N in Lepidoptera by conserved clade-specific amino acid residues. Mol Phylogenet Evol. 2014; 76:127-33. PMC: 4372138. DOI: 10.1016/j.ympev.2014.03.014. View

16.

Qiu L, Cui S, Liu L, Zhang B, Ma W, Wang X . Aminopeptidase N1 is involved in Bacillus thuringiensis Cry1Ac toxicity in the beet armyworm, Spodoptera exigua. Sci Rep. 2017; 7:45007. PMC: 5361178. DOI: 10.1038/srep45007. View

17.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

18.

Pardo-Lopez L, Soberon M, Bravo A . Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiol Rev. 2012; 37(1):3-22. DOI: 10.1111/j.1574-6976.2012.00341.x. View

19.

Guo Z, Kang S, Sun D, Gong L, Zhou J, Qin J . MAPK-dependent hormonal signaling plasticity contributes to overcoming Bacillus thuringiensis toxin action in an insect host. Nat Commun. 2020; 11(1):3003. PMC: 7293236. DOI: 10.1038/s41467-020-16608-8. View

20.

Cantalapiedra C, Hernandez-Plaza A, Letunic I, Bork P, Huerta-Cepas J . eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale. Mol Biol Evol. 2021; 38(12):5825-5829. PMC: 8662613. DOI: 10.1093/molbev/msab293. View