Abstract
Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Resistance to B. thuringiensis toxins threatens this technology, and although different resistance mechanisms have been identified, some have not been completely elucidated. To gain new insights into these mechanisms, we performed multiple back-crossing from a 3000-fold Cry1Ac-resistant BtR strain from cotton bollworm (Helicoverpa armigera), isolating a 516-fold Cry1Ac-resistant strain (96CAD). Cry1Ac resistance in 96CAD was tightly linked to a mutant cadherin allele (mHaCad) that contained 35 amino acid substitutions compared with HaCad from a susceptible strain (96S). We observed significantly reduced levels of the mHaCad protein on the surface of the midgut epithelium in 96CAD as compared with 96S. Expression of both cadherin alleles from 96CAD and 96S in insect cells and immunofluorescence localization in insect midgut tissue sections showed that the HaCAD protein from 96S localizes on the cell membrane, whereas the mutant 96CAD-mHaCad was retained in the endoplasmic reticulum (ER). Mapping of the mutations identified a D172G substitution mainly responsible for cadherin mislocalization. Our finding of a mutation affecting membrane receptor trafficking represents an unusual and previously unrecognized B. thuringiensis resistance mechanism.
Highlights
Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests
The observed genotype frequencies of HaCAD on the diet treated with B. thuringiensis toxins differed significantly from the expected genotype frequencies on the untreated diet (2 ϭ 15.5, degree of freedom ϭ 1, p Ͻ 0.001), whereas genotype frequencies of HaABCC2, HaAPN1, and HaALP1 on the diet treated with B. thuringiensis toxins have no difference from the expected genotype frequencies on the untreated diet (2 ϭ 0.5, df ϭ 1, p ϭ 0.48; 2 ϭ 0.1, df ϭ 1, p ϭ 0.8; 2 ϭ 0, df ϭ 1, p ϭ 1, respectively)
To identify the resistance mechanism in the BtR strain, we analyzed single-nucleotide polymorphism (SNP) markers for different insect gut protein genes, such as HaCad, HaApn, HaAbcc, and HaAlp, that have shown to be involved in Cry1Ac toxicity in H. armigera (25, 30 –32)
Summary
Transgenic plants that produce Bacillus thuringiensis (Bt) crystalline (Cry) toxins are cultivated worldwide to control insect pests. Different Cry toxin receptors have been described, such as cadherin (Cad), alkaline phosphatase (ALP), aminopeptidase-N (APN), ATP-binding cassette subfamily C member 2 (ABCC2) transporter, and glycoprotein among others [3,4,5,6,7,8,9,10,11] Their expression in the plasma membrane is a requisite to exert their function as Cry receptors. Glycosylation of cell surface proteins might be critical for localization in the apical membrane [13] Insect pests, such as Helicoverpa armigera, Heliothis virescens, Pectinophora gossypiella, Plodia interpunctella, Ostrinia nubilalis, Plutella xylostella, and Spodoptera exigua, have evolved resistance to Cry toxins in laboratory conditions, whereas P. gossypiella, P. xylostella, Trichoplusia ni, and Spodoptera frugiperda evolved resistance in the field (14 –20). We established a new H. armigera Cry1Ac-resistant strain 96CAD, nearly isogenic to susceptible strain 96S,
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