1. Introduction
Bacillus thuringiensis (Bt) is a Gram-positive, soil-dwelling bacterium that produces δ-endotoxin proteins known as Bt toxins or Cry toxins (crystalline toxins). Bt toxins efficiently kill lepidopteran, dipteran, and coleopteran pests, but are harmless to humans and other vertebrate animals (Bravo et al 2011). The Bt toxins belong to a class of bacterial pore-forming toxins. Once ingested by insects, Bt protoxins are solubilized in the insect midgut, and are then cleaved by proteases to produce activated toxins (Bravo et al 2007). These activated toxins penetrate the insect midgut protrophic membrane and bind to specific target sites, called primary receptors (such as cadherin), of the brush border membrane vesicles (BBMV) (Bravo et al 2008; Bravo et al 2011). Interactions between Bt toxins and cadherin facilitate protease cleavage of the helix α-1 of the toxins, promoting toxin oligomerization (Soberon et al 2007). These toxin oligomers are thought to have increased binding affinity to secondary receptors including glycosylphosphatidylinositol (GPI)-anchored proteins, aminopeptidase N (APN) (Bravo et al 2004), and alkaline phosphatases (ALP) (Jurat-Fuentes et al 2004). Binding of the toxin oligomers to these secondary receptors creates pores in the midgut membranes, thus causing osmotic shock, breakdown of the midgut cells, and insect death (Bravo et al 2004, Soberon et al 2007, Jurat-Fuentes et al 2004, ). However, others have proposed that binding of the activated Bt toxin monomers to cadherin initiates a magnesium-dependent signalling pathway, causing cell disruption (Zhang et al 2006). In either model, the binding of Bt toxins to various midgut receptors is essential for disrupting the midgut membranes, which leads to cell lysis and insect death.
The application of Bt toxins in agriculture is threatened by evolved resistance of insects (Tabashnik and Carrière 2017). The diamondback moth (DBM) Plutella xylostella (Lepidoptera: Plutellidae), caused US $4–5 billion in management costs annually (Furlong et al 2013), is the first insect reported in the fields to have evolved resistance to Bt toxins (Tabashnik 1994). The DBM resistance phenotype involves the reduced binding of toxins to the brush border membranes, a trait that is inherited in a recessive manner but which achieves high resistance levels (Tabashnik et al 1996, Tabashnik et al 1997). Worriedly, our understanding of the molecular basis in DBM midguts involved in Bt toxin binding is scant. So far, all known Bt receptors identified from other lepidopteran insects have been conclusively eliminated as factors conferring resistance to Cry1A in DBM (Heckel et al 2007). Although, the resistance mechanisms in DBM are reported to involve alterations in the expression levels of Bt toxin receptor genes like adenosine triphosphate (ATP)-binding cassette transporter subfamily C (ABCC) gene, no obvious mutations in sequences of these genes between susceptible and resistant DBM strains, casting doubt on the role of these genes in Cry1Ac resistance phenotype of DBM (Guo et al 2015). It is an urgent need in identifying Bt toxin receptors in insects regarding the long-term and sustainable use of Bt and their Cry toxins as insecticides.
Here, we report a novel technique, on-membrane capture, that identified numbers of previously unknown candidates, in addition to well-known receptors cadherin and APN2, from DBM midgut as binding partners of Bt toxins. Among these candidates, we discovered new molecular components, glucosinolate sulfatases, that contribute to the action of Bt toxin Cry1Bd in the DBM midgut. Reduction in GSSs expression remarkably increased tolerance of DBM Bt toxins. Expressing DBM GSSs in silkworms dramatically decreased the tolerance of the transgenic silkworms to Cry1Bd. Therefore, the on-membrane capture provides a new solution to identify Bt toxin receptors in insects.