Reduced Membrane-Bound Alkaline Phosphatase Does Not Affect Binding of Vip3Aa in a Heliothis virescens Resistant Colony

Abstract

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

The Rapid Evolution of Resistance to Vip3Aa Insecticidal Protein in Mythimna separata (Walker) Is Not Related to Altered Binding to Midgut Receptors

“…Here, we have tested the binding of 125 I-Vip3Aa to BBMV from susceptible and resistant M. separata insects to see whether we could find differences that could explain resistance. The lack of qualitative or quantitative binding differences between susceptible and resistant insects is in line with previous results with other Vip3Aa-resistant strains from other insect species for which Vip3Aa binding differences were not found [ 33 , 34 ]. Slower activation of Vip3Aa by midgut juice of H. armigera larvae has been shown in Vip3Aa-resistant insects, though because of the small differences with the susceptible insects, it did not seem to be the main reason for resistance [ 33 ].…”

“…Slower activation of Vip3Aa by midgut juice of H. armigera larvae has been shown in Vip3Aa-resistant insects, though because of the small differences with the susceptible insects, it did not seem to be the main reason for resistance [ 33 ]. Additionally, Vip3Aa-resistant H. virescens larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase, but its involvement in resistance could not be demonstrated [ 34 ]. Therefore, unlike for Cry proteins, altered binding to membrane receptors seems not to be the main mechanism of resistance to Vip3Aa proteins, and other mechanisms should be explored [ 35 ].…”

Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers

“…The biochemical basis of resistance could be the down regulation of membrane bound alkaline phosphatase (mALP) isoform HvmALP1, observed in Vip3 resistant insects, which is thought to be the functional receptor of Vip3. In addition, mALP can be used as a marker for the detection of Vip3A resistance [79]. Moreover, Cry1F and Vip3A do not share common binding sites in S. frugiperda [17], and also lack cross resistance [54].…”

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein

“…glycinivorella malp are orthologous to H. virescens HvmALP1 and HvmALP2 that show a strong correlation between reduced transcript and protein levels in Cry1Ac resistant strains of H. virescens [ 101 ]. Additional evidence shows that mALP is a functional receptor for Cry1A and Cry2Ab2 proteins [ 102 ], but not Vip3A [ 103 ], where N-acetylgalactosamine modification is required for Cry1Ac binding [ 78 ]. Furthermore, constitutively reduced mALP or malp levels are documented in Cry1Ac resistant laboratory strains of Helicoverpa armigera and Spodoptera frugiperda [ 101 ], Cry1Ab resistant Ostrinia nubilalis [ 43 ] and Cry1C resistant Chilo suppressalis [ 104 ], but not in Cry1Ab resistant Diatraea saccharalis [ 96 ].…”