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A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae
Juan L Jurat-Fuentes1, Michael J Adang
1Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996-4560, USA. jurat@utk.edu
Abstract:
Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.
Insights
Bacillus thuringiensis Cry1Ac toxin resistance in Heliothis virescens involves altered midgut receptors. Proteomics identified membrane-bound alkaline phosphatase (HvALP) as a Cry1Ac binding protein, with reduced HvALP levels correlating to resistance.
Area of Science:
- * Entomology
- * Molecular Biology
- * Biochemistry
Background:
- * Bacillus thuringiensis Cry1Ac toxin requires specific midgut brush border membrane (BBM) receptors for toxicity.
- * Alterations in these receptors are a primary mechanism of insect resistance to Cry1Ac.
- * Heliothis virescens is a significant agricultural pest, and understanding its resistance mechanisms is crucial for pest control.
Purpose of the Study:
- * To identify Cry1Ac binding proteins in the BBM of Heliothis virescens larvae using a proteomic approach.
- * To investigate proteomic alterations in Cry1Ac-resistant Heliothis virescens strains.
- * To validate the utility of proteomic techniques in identifying insect resistance factors.
Main Methods:
- * Preparation of intestinal brush border membrane (BBM) from Heliothis virescens larvae.
- * Two-dimensional (2D) blotting to detect Cry1Ac binding BBM proteins.
- * Peptide mass fingerprinting (PMF) and de novo sequencing for protein identification.
- * Two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare proteomes of resistant and susceptible strains.
Main Results:
- * Membrane-bound alkaline phosphatase (HvALP) and a novel phosphatase were identified as Cry1Ac binding proteins.
- * Reduced HvALP expression levels were directly correlated with Cry1Ac resistance in the YHD2-B strain of H. virescens.
- * Proteomic analysis revealed distinct proteome alterations in resistant H. virescens strains compared to susceptible ones.
Conclusions:
- * Proteomic approaches are effective for identifying insect toxin binding proteins.
- * HvALP is a potential Cry1Ac binding receptor, and its reduced expression contributes to H. virescens resistance.
- * Understanding these molecular mechanisms can inform strategies for managing Cry1Ac resistance in pests.
