Insect Control: Biological and Synthetic Agents - Index of

een subject to mutagenesis studies to determine
their role in receptor recognition. Domain II was
first recognized as an insect toxicity determinant
domain based on hybrid toxin construction using
cry genes with different selectivities (Ge et al.,
1989). Site-directed mutagenesis studies of Cry1A
toxins showed that some exposed loop regions, loop
a-8, loop 2, and loop 3, of domain II are involved in
receptor recognition (Rajamohan et al., 1996a,
1996b; Jenkins et al., 2000; Lee et al., 2000,
2001). Specifically, the loop 2 region of Cry1Ab
toxin was shown to be important in the interaction
with M. sexta brush border membrane vesicles.
Characterization of mutants affected at Arg368-
Arg369 in loop 2 indicated that these residues
have an important role in the reversible binding of
the toxin to brush border membrane vesicles, while
Phe371 is involved in the irreversible binding of the
toxin (Rajamohan et al., 1995, 1996b; Jenkins and
Dean, 2000). Reversible binding is related to the
initial interaction of the toxin to the receptor while
irreversible binding is related to the insertion of
the toxin into the membrane (Rajamohan et al.,
1995). This result was interpreted as suggesting
that Phe371 might be involved in membrane
insertion (Rajamohan et al., 1995, 1996b). Also,
evidence has been provided showing that Arg368-
Arg369 in loop 2 of Cry1Ab are involved in APN
binding since this mutant showed no binding to
purified APN in surface plasmon resonance experiments
(Jenkins and Dean, 2000; Lee et al., 2000). In
this regard it is interesting to note that a truncated
derivative of Cry1Ab toxin containing only domains
II and III was still capable of receptor interaction but
was affected in irreversible binding (Flores et al.,
1997). Therefore, it is likely that mutations in
Phe371 affect membrane insertion probably by
interfering with conformational change, necessary
for membrane insertion, after initial recognition of
the receptor or as originally proposed this residue
could be directly involved in membrane interaction
(Rajamohan et al., 1995). Phylogenetic relationship
studies of different Cry1 loop 2 sequences show that
there is a correlation between the loop 2 amino acid
sequences and cross-resistance with several Cry1
toxins in resistant insect populations, implying that
this loop region is an important determinant of
receptor recognition (Tabashnik et al., 1994; Jurat-
Fuentes and Adang, 2001). Overall these results
suggest that loop 2 of Cry1A toxins plays an important
role in receptor recognition. Regarding loop a-8
and loop 3, mutations of some residues in these
regions of Cry1A toxins affected reversible binding
to brush border membrane vesicles or the purified
APN receptor (Rajamohan et al., 1996a; Lee et al.,
7: Bacillus thuringiensis: Mechanisms and Use 259
2001). The closely related toxins Cry1Ab and
Cry1Aa have different loop 3 amino acid sequences
even though they interact with the same receptor
molecules (Rajamohan et al., 1996a). Analysis of
Cry1Ab and Cry1Aa binding to the cadherin-like
Bt-R1 receptor in ligand blot experiments and
competition with synthetic peptides corresponding
to the toxin exposed loop regions, showed that,
besides loop 2, loop 3 of Cry1Aa toxin was important
for Bt-R1 recognition (Gómez et al., 2002a).
Although the Cry1A loop 1 region seems not to
play an important role in receptor recognition this
is certainly not the case for other Cry toxins. Mutagenesis
of Cry3A loop 1 residues showed that this
region, besides loop 3, is important for receptor
interaction in coleopteran insects (Wu and Dean,
1996). Loop 1 residues of two Cry toxins with dual
insecticidal activity (Cry1Ca and Cry2Aa, active
against dipteran and lepidopteran insects) are
important for toxicity against mosquitoes but not
against lepidopterous insects (Widner and Whiteley,
1990; Smith and Ellar, 1994; Morse et al., 2001).
Besides loop1 of Cry1C toxin, loops 2 and 3 are
important for toxicity to dipteran and lepidopteran
insects (Abdul-Rauf and Ellar, 1999). In the case
of Cry2Aa toxin, an amino acid region involved in
lepidopteran activity was not located in loops 1, 2,
or 3 regions but was located to a different part of
domain II in loops formed by b-5 and b-6, and by b-7
and b-8 (Morse et al., 2001). These results show that
the loop regions of domain II are important determinants
for receptor interaction, although other
regions of this domain, in certain toxins, could
also participate in receptor recognition.
As mentioned previously (see Section 7.3.3),
domain III swapping indicated that this domain is
involved in receptor recognition (Bosch et al., 1994;
de Maagd et al., 2000), and it has been proposed
that domain swap has been used as an evolutionary
mechanism of these toxins (Bravo, 1997; de Maagd
et al., 2001). The swapping of domain III between
Cry1Ac and Cry1Ab toxins showed that the Cry1Ac
domain III was involved in APN recognition (Lee
et al., 1995; de Maagd et al., 1999a). The interaction
of Cry1Ac domain III and APN was dependent
on N-acetylgalactosamine (Gal-Nac) residues
(Burton et al., 1999; Lee et al., 1999). Mutagenesis
studies of Cry1Ac domain III identified Gln509-
Asn510-Arg511 Asn506, and Tyr513 as the epitope
for sugar recognition (Burton et al., 1999; Lee et al.,
1999). The three-dimensional structure of Cry1Ac
in the presence of the sugar confirmed that these
residues are important for Gal-Nac recognition
and that sugar binding has a conformational effect
on the pore forming domain I (Li et al., 2001).