Dataset Information

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

ABSTRACT: The velvetbean caterpillar Anticarsia gemmatalis is one of the main soybean defoliators in Brazil. Currently, the main biopesticide used to control not only A. gemmatalis but also other insect pests worldwide is the bacteria Bacillus thuringiensis (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 A. gemmatalis is crucial to develop effective strategies to overcome this possible scenario. This study’s goal is to characterize APNs of A. gemmatalis and identify a receptor for Cry1Ac in the midgut. The interaction of Bt spores with the midgut epithelium was observed in situ 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 A. gemmatalis’ transcriptome and subjected to different in silico 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 Cry 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 A. gemmatalis could be involved in the mode of action of Cry toxins in its larval stage.

INSTRUMENT(S):

ORGANISM(S): Anticarsia Gemmatalis

TISSUE(S): Midgut Epithelium

SUBMITTER: Luis Felipe Ramos

LAB HEAD: Danielle Oliveira

PROVIDER: PXD055041 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	APN_AmostrasGel_Marina_Maio2024_DatabaseAgemmatalis.msf	Msf
	QE_000200_Luis_ML2_2.raw	Raw
	QE_000205_Luis_ML6_1.raw	Raw
	QE_000209_Luis_ML4_2.raw	Raw
	QE_000211_Luis_ML1_1.raw	Raw

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Publications

Cry1Ac toxin binding in the velvetbean caterpillar Anticarsia gemmatalis: study of midgut aminopeptidases N.

Lanzaro M D MD Padilha I I Ramos L F C LFC Mendez A P G APG Menezes A A Silva Y M YM Martins M R MR Junqueira M M Nogueira F C S FCS AnoBom C D CD Dias G M GM Gomes F M FM Oliveira D M P DMP

Frontiers in physiology 20241029

The velvetbean caterpillar Anticarsia gemmatalis is one of the main soybean defoliators in Brazil. Currently, the main biopesticide used to control insect pests worldwide is the bacteria Bacillus thuringiensis (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 amino ...[more]

PMID: 39534858

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Project description:Host-pathogen interactions result in complex relationship, many aspects of which are not completely understood. Vip proteins, which are Bacillus thuringensis (Bt) insecticidal toxins produced during the vegetative stage, are selectively effective against specific insect pests. This new group of Bt proteins represents an interesting alternative to the classical Bt Cry toxins because current data suggests that they do not share the same mode of action. We have designed and developed a genome-wide microarray for the beet armyworm Spodoptera exigua, a serious lepidopteran pest of many agricultural crops, and used it to better understand how Lepidopteran larvae respond to the treatment with the insecticidal protein Vip3Aa. With this approach, the goal of our study was to evaluate the changes in gene expression levels caused by treatment with sublethal doses (causing 99% growth inhibition) of Vip3Aa at 8 and 24 h after treatment. Results indicated that the toxin provoked a wide transcriptional response, with 19% of unigenes in the microarray responding significantly to treatment. The number of up- and down-regulated unigenes was very similar.. The number of genes whose expression was regulated at 8 h was similar to the number of genes whose expression was regulated after 24 h of treatment. The up-regulated sequences were enriched for genes involved in innate immune response and in pathogen response such as antimicrobial peptides (AMPs) and repat genes. The down-regulated sequences were mainly unigenes with homology to genes involved in metabolism. Genes related to the mode of action of Bt Cry proteins were found, in general, to be slightly overexpressed. The present study is the first genome-wide analysis of the response of lepidopteran insects to Vip3Aa intoxication. An insight into the molecular mechanisms and components related to Vip intoxication will allow designing of more effective management strategies for pest control. Changes in gene expression levels caused by treatment with sublethal doses (causing 99% growth inhibition) of Vip3Aa were measured at 8 and 24 h after treatment by means of custom Spodoptera exigua microarray. Six to seven larvae were used for each time point and three independent experiments were performed at each time point.

Project description:Summary: Multi-toxins Bt-crops carrying insecticidal toxins with similar host spectrum and different mode of action e.g. Cry and Vip, are expected to improve resistance management in target pests. Control failure has been informed for Cry toxins but not for Vip3A, of which no mechanism of resistance has yet been identified. Here we applied HT-SuperSAGE to analyze the transcriptome profiling in the midgut tissue of a tobacco budworm Heliothis virescens (F.) strain laboratory-selected for Vip3A resistance. A total of 1324252 26-bp tags were sequenced representing 17751 unique transcripts (UniTags) from genetically similar Vip3A-resistant (Vip-Sel) strain and susceptible control (Vip-Unsel) strain. Differential expression was found significant (≥ 2.5-fold or ≤ 0.4) for 1845 unigenes that constitute 10.4% of the total number of UniTags, where 277 represented overexpressed (OE) and 1568 underexpressed (UE) genes in Vip-Sel compared to Vip-Unsel. BLASTN searches mapped 1141 of these UniTags to H. virescens EST sequences, of which, 816 (143 OE and 673 UE) were unambiguously annotated to proteins in NCBI non-redundant protein databases. Gene ontology revealed Vip3A adaptation induced major constitutive transcriptional differences in serine-proteases (SP)-mediated proteolysis, ribosome biogenesis and metabolic processes. Several unigenes homologous to a particular member of the REsponse to PAThogen (REPAT) family were found to be predominantly OE. Since UniTags related to SP and ribosomal proteins (RP) were the most represented in the libraries, they were further analyzed in details. Interestingly, UniTags related to the putative Vip3A-binding protein RpS2 were underexpressed, while, the tumorigenesis suppressor RpL37 accounted for the 35% of total overexpressed RP. A subset of unigenes was chosen to confirm the HT-SuperSAGE data by qRT-PCR. The present study is the first providing a lepidopteran gut transcriptome associated with Vip3A resistance and a foundation for future attempts to elucidate the resistance mechanism. Methods: Midgut transcriptional profiles of third-instar larvae from genetically similar Vip3A-resistant (Vip-Sel) and susceptible control (Vip-Unsel) strains were generated by HT-SuperSAGE. RNA pools of Vip-Sel and Vip-Unsel samples (five RNA preparations each) were prepared and used for the construction of SuperSAGE libraries HvR_GCCT and HvS_GCAC, respectively, according to the procedure described by [Matsumura et al., 2010]. Purified PCR products were mixed and applied to Illumina Genome Analyzer II sequencing with GEX (DpnII) primer in the sequencing reactions as recommended by the manufacturer. Sorting of sequence reads based on index sequences and the subsequent extraction of sequence tags from reads was conducted using a script written in Perl. Fold-change for each tag was calculated as in [Gilardoni et al., 2010]. qRT–PCR validation was performed using TaqMan and SYBR Green assays.

Dataset Information

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

Dataset's files

Publications

Cry1Ac toxin binding in the velvetbean caterpillar <i>Anticarsia gemmatalis</i>: study of midgut aminopeptidases N.

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