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We reported previously a direct correlation between reduced soybean agglutinin binding to 63-and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens).In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [ 125 I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F 1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.Keywords: alkaline phosphatase; Cry1Ac; Heliothis virescens; resistance; N-acetylgalactosamine. Specific binding to insect midgut receptors is a key step in the mode of action of insecticidal Cry toxins from the bacterium Bacillus thuringiensis (Bt). Despite exceptions [1], in most cases Cry toxin specificity and potency correlate with the extent of toxin binding to midgut brush border membrane receptors in vitro [2,3]. Effective toxin binding to receptors results in toxin insertion and oligomerization on the midgut cell membrane, leading to pore formation and cell death by osmotic shock [4].In brush border membrane vesicles (BBMV) from Heliothis virescens (tobacco budworm) larvae, three groups of binding sites (A, B, and C) for Cry1A toxins were proposed based on their toxin binding specificities [5,6] Membrane-bound ALP from Bombyx mori and M. sexta are attached to the brush border cell membrane by a glycosylphosphatidylinositol (GPI) anchor [13][14][15]. Specific interactions between Cry1Ac and ALPs under native conditions resulting in inhibition of phosphatase activity have been reported for M. sexta [16] and H. virescens [17]. However, the potential role for alkaline phosphatases in Cry1Ac intoxication has not been addressed directly.The main goals of the present study were to identify the 68-kDa glycoprotein and characterize its oligosaccharide residues as a first step to investigate the specific alteration of this glycoprotein in Cry1Ac-resistant YHD2 larvae. Based on reported molecular sizes of insect alkaline phosphatases, and their interaction with Cry1 toxins, we hypothesized the 68-kDa glycoprotein to be a form of alkaline phosphatase. Immunoblotting and enzymatic activity experiments identified the 68-kDa protein as a GPI-anchored form of alkaline phosphatase we term HvALP (for H. virescens alkaline phosphatase). Ligand blots and glycosidase digestion Correspondence to M. J...

Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

Evolution of resistance threatens sustainability of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The fall armyworm (Spodoptera frugiperda) is a devastating pest of corn in the Western Hemisphere initially controlled by transgenic Bt corn producing the Cry1Fa insecticidal protein (event TC1507). However field-evolved resistance to TC1507 was observed in Puerto Rico in 2007 and has subsequently been reported in a number of locations in North and South America. Early studies on Puerto Rico fall armyworm populations found that the resistance phenotype was associated with reduced expression of alkaline phosphatase. However, in this work we show that field-evolved resistance to Cry1Fa Bt corn in Puerto Rico is closely linked to a mutation in an ATP Binding Cassette subfamily C2 (ABCC2) gene that functions as a Cry1Fa receptor in susceptible insects. Furthermore, we report a DNA-based genotyping test used to demonstrate the presence of the resistant (SfABCC2mut) allele in Puerto Rico populations in 2007, coincident with the first reports of damage to TC1507 corn. These DNA-based field screening data provide strong evidence that resistance to TC1507 in fall armyworm maps to the SfABCC2 gene and provides a useful molecular marker for detecting the SfABCC2mut allele in resistant fall armyworm.

Extensive planting of crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has suppressed some major pests, reduced insecticide sprays, enhanced pest control by natural enemies, and increased grower profits. However, rapid evolution of resistance in pests is reducing these benefits. Better understanding of the genetic basis of resistance to Bt crops is urgently needed to monitor, delay, and counter pest resistance. We discovered that a point mutation in a previously unknown tetraspanin gene in the cotton bollworm (Helicoverpa armigera), a devastating global pest, confers dominant resistance to Cry1Ac, the sole Bt protein produced by transgenic cotton planted in China. We found the mutation using a genome-wide association study, followed by fine-scale genetic mapping and DNA sequence comparisons between resistant and susceptible strains. CRISPR/Cas9 knockout of the tetraspanin gene restored susceptibility to a resistant strain, whereas inserting the mutation conferred 125-fold resistance in a susceptible strain. DNA screening of moths captured from 23 field sites in six provinces of northern China revealed a 100-fold increase in the frequency of this mutation, from 0.001 in 2006 to 0.10 in 2016. The correspondence between the observed trajectory of the mutation and the trajectory predicted from simulation modeling shows that the dominance of the mutation accelerated adaptation. Proactive identification and tracking of the tetraspanin mutation demonstrate the potential for genomic analysis, gene editing, and molecular monitoring to improve management of resistance.