Insect Control: Biological and Synthetic Agents - Index of
Insect Control: Biological and Synthetic Agents - Index of
Insect Control: Biological and Synthetic Agents - Index of
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<strong>and</strong> Cry1Ac proteins bind to a 120 kDa aminopeptidase-N<br />
(APN) (Knight et al., 1994; Garczynski<br />
<strong>and</strong> Adang, 1995; Denolf et al., 1997) <strong>and</strong> to a<br />
210 kDa cadherin-like protein (Bt-R1) (Belfiore<br />
et al., 1994; Vadlamudi et al., 1995). Cadherins<br />
represent a large family <strong>of</strong> glycoproteins that are<br />
classically responsible for intercellular contacts.<br />
These proteins are transmembrane proteins with a<br />
cytoplasmic domain <strong>and</strong> an extracellular ectodomain<br />
with several cadherin repeats (12 in the case<br />
<strong>of</strong> Bt-R1). The ectodomain contain calcium binding<br />
sites, integrin interaction sequences, <strong>and</strong> cadherin<br />
binding sequences. In Bombyx mori, Cry1Aa binds<br />
to a 175 kDa cadherin-like protein (Bt-R 175)<br />
(Nagamatsu et al., 1998, 1999) <strong>and</strong> to a 120 kDa<br />
APN (Yaoi et al., 1997). In H. virescens Cry1Ac<br />
binds to two proteins <strong>of</strong> 120 kDa <strong>and</strong> 170 kDa<br />
both identified as APN (Gill et al., 1995; Oltean<br />
et al., 1999). Also, a cadherin-like protein is<br />
involved in the mode <strong>of</strong> action <strong>of</strong> Cry1Ac toxin in<br />
H. virescens (Gahan et al., 2001). In P. xylostella <strong>and</strong><br />
Lymantria dispar APNs were identified as Cry1Ac<br />
receptors (Valaitis et al., 1995; Lee et al., 1996;<br />
Denolf et al., 1997; Luo et al., 1997). In L. dispar,<br />
besides APN <strong>and</strong> cadherin-like receptors, a high<br />
molecular weight anionic protein (Bt-R270) that<br />
binds Cry1A toxins with high affinity was identified<br />
(Valaitis et al., 2001). For Cry1C toxin an APN<br />
receptor molecule was identified in Spodoptera<br />
litura (Agrawal et al., 2002). Sequence analysis <strong>of</strong><br />
various APN from lepidopteran insects suggests that<br />
APN’s group into at least four classes (Oltean et al.,<br />
1999). Plutella xylostella <strong>and</strong> B. mori produce the<br />
four classes <strong>of</strong> APN <strong>and</strong> it is anticipated that several<br />
other lepidopteran insects may also produce the four<br />
is<strong>of</strong>orms (Nakanishi et al., 2002). Cry1A binding to<br />
the different B. mori APN is<strong>of</strong>orms revealed that this<br />
toxin only binds the 115 kDa is<strong>of</strong>orm in lig<strong>and</strong> blot<br />
binding analysis (Nakanishi et al., 2002).<br />
Surface plasmon resonance experiments showed<br />
that the binding affinity <strong>of</strong> Cry1A toxins to the<br />
M. sexta APN is in the range <strong>of</strong> 100 nM (Jenkins<br />
<strong>and</strong> Dean, 2000), while that <strong>of</strong> cadherin-like receptors<br />
(Bt-R1) is in the range <strong>of</strong> 1 nM (Vadlamudi et al.,<br />
1995). This difference in the binding affinities between<br />
APN <strong>and</strong> Bt-R 1 suggest that binding to Bt-R 1<br />
might be the first event on the interaction <strong>of</strong> Cry1A<br />
toxins with microvilli membranes <strong>and</strong>, therefore,<br />
the primary determinant <strong>of</strong> insect specificity.<br />
Different experimental evidence supports the involvement<br />
<strong>of</strong> both Cry1A toxins receptors (APN<br />
<strong>and</strong> cadherin-like) in toxicity. Expression <strong>of</strong> the<br />
M. sexta <strong>and</strong> B. mori cadherin-like proteins, Bt-R 1<br />
<strong>and</strong> Bt-R 175 respectively, on the surface <strong>of</strong> different<br />
cell lines render these cells sensitive to Cry1A toxins,<br />
7: Bacillus thuringiensis: Mechanisms <strong>and</strong> Use 257<br />
although the toxicity levels were low (Nagamatsu<br />
et al., 1999; Dorsch et al., 2002; Tsuda et al., 2003).<br />
Cry1Aa toxin was shown to lyse isolated midgut<br />
epithelial cells; this toxic effect was inhibited if the<br />
cells were preincubated with anti-Bt-R 175 antisera in<br />
contrast with the treatment with anti-APN antisera,<br />
suggesting that cadherin-like protein Bt-R175 is a<br />
functional receptor <strong>of</strong> Cry1Aa toxin (Hara et al.,<br />
2003). Also, a single-chain antibody (scFv73) that<br />
inhibits binding <strong>of</strong> Cry1A toxins to cadherin-like<br />
receptor, but not to APN, reduced the toxicity <strong>of</strong><br />
Cry1Ab to M. sexta larvae (Gómez et al., 2001).<br />
Moreover, disruption <strong>of</strong> a cadherin gene by a retrotransposon-mediated<br />
insertion <strong>and</strong> its linkage<br />
to high resistance to Cry1Ac toxin in H. virescens<br />
YHD2 (a laboratory selected line) larvae supports<br />
a role for cadherin as a functional receptor (Gahan<br />
et al., 2001). Overall, these results suggest that<br />
binding to the cadherin-like receptor is an important<br />
step in the mode <strong>of</strong> action Cry1A toxins (Figure 6).<br />
Regarding the APN receptor, several reports<br />
argue against the involvement <strong>of</strong> this protein in<br />
toxin activity. Mutants <strong>of</strong> Cry1Ac toxin affected<br />
on APN binding retained similar toxicity levels to<br />
M. sexta as the wild-type toxin (Burton et al., 1999).<br />
However, there are several reports describing point<br />
mutations <strong>of</strong> Cry toxins located in domain II that<br />
affected both APN binding <strong>and</strong> toxicity (Jenkins <strong>and</strong><br />
Dean, 2000). Expression <strong>of</strong> APN in heterologous<br />
systems did not result in Cry1A sensitivity (Denolf<br />
et al., 1997) <strong>and</strong>, as mentioned above, an APN<br />
antibody did not protect B. mori midgut cells from<br />
Cry1Aa toxic effect in contrast to a cadherin antibody<br />
(Hara et al., 2003). However, two recent<br />
reports clearly demonstrate the importance <strong>of</strong> this<br />
molecule on the mode <strong>of</strong> action <strong>of</strong> Cry1 toxins.<br />
Inhibition <strong>of</strong> APN production in S. litura larvae<br />
by dsRNA interference showed that insects with<br />
low APN levels became resistant to Cry1C toxin<br />
(Rajagopal et al., 2002). Also, heterologous expression<br />
<strong>of</strong> M. sexta APN in midguts <strong>and</strong> mesodermal<br />
tissues <strong>of</strong> transgenic Drosophila melanogaster<br />
caused sensitivity to Cry1Ac toxin (Gill <strong>and</strong> Ellar,<br />
2002). Additionally, previous reports demonstrated<br />
that incorporation <strong>of</strong> M. sexta APN into black lipid<br />
bilayers lowers the concentration <strong>of</strong> toxin needed<br />
for pore formation activity <strong>of</strong> Cry1Aa toxin<br />
(Schwartz et al., 1997b). Finally, all Cry1A APN<br />
receptors are anchored to the membrane by a glycosyl<br />
phosphatidylinositol (GPI) (Knight et al., 1994;<br />
Garczynski <strong>and</strong> Adang, 1995; Denolf et al., 1997;<br />
Oltean et al., 1999; Agrawal et al., 2002; Nakanishi<br />
et al., 2002). And treatment <strong>of</strong> Trichoplusia ni brush<br />
border membrane vesicles with PI-PLC, which<br />
cleaves GPI-anchored proteins from the membrane,