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.