Contents - Faperta

Genetic Engineering of Entomopathogenic Microbes for Pest Management 265
of the Cry3A has revealed the presence of four intermolecular salt bridges, which might
participate in the formation of the crystal inclusion (Li, Carroll, and Ellar, 1991).
Crystallization of Cry2A (71 kDa) and Cyt1A (27 kDa) requires the presence of accessory
proteins (Agaisse and Lereclus, 1995; Baum and Malvar, 1995). These proteins may act at
the post-translational level to stabilize the nascent protoxin molecule and facilitate crystallization.
Cry1Ia toxin has been found in supernatant of B. thuringiensis cultures as a processed
polypeptide of 60 kDa (Kostichka et al., 1996).
Because of the crystalline nature of these proteins, the term Cry is used in gene and
protein nomenclature. Several cry gene promoters have been identifi ed, and their sequences
determined (Brizzard, Schnepf, and Kronstad, 1991; Brown, 1993; Yoshisue et al., 1993;
Dervyn et al., 1995). The toxin genes earlier were classifi ed into four types, based on insect
specifi city and sequence homology (Hofte and Whiteley, 1989). CryI-type genes encode
proteins of 130 kDa, and are usually specifi c to lepidopteran larvae. Type II genes encode
for 70 kDa proteins that are specifi c to lepidopteran and dipteran larvae, while type III
genes encode for 70 kDa proteins specifi c to coleopteran larvae. Type IV genes are specifi c
to the dipteran larvae. The system was further extended to include type V genes that
encode for proteins effective against lepidopteran and coleopteran larvae (Tailor et al.,
1992). The Bt d-endotoxins are now known to constitute a family of related proteins for
which over 140 genes have been described (Crickmore et al., 1998), with specifi cities for
Lepidoptera, Coleoptera, and Diptera. The crystalline proteins get solubilized in the insect
midgut at high pH, releasing proteins called d-endotoxins. The toxin portion is derived
from the N-terminal half of the protoxin, while the C-terminal portion is involved in the
formation of parasporal inclusion bodies and is usually hydrolyzed into small peptides
(Choma et al., 1990).
Mode of Action
The mode of action of the B. thuringiensis Cry toxins involves solubilization of the crystal
protein in the insect midgut, proteolytic processing of the protoxin by proteases to toxin,
binding of the toxin to midgut receptors, and insertion of the toxin into the apical membrane
to create ion channels or pores. For most lepidopterans, protoxins are solubilized
under the alkaline conditions of the insect midgut (Hofmann et al., 1988). Differences in
the extent of solubilization often are associated with differences in toxicity of Cry proteins
to various insect species (Aronson et al., 1991; Du, Martin, and Nickerson, 1994;
Meenakshisundaram and Gujar, 1998). Decreased solubility could be one potential mechanism
for insect resistance to Bt proteins (McGaughey and Whalon, 1992). In cotton bollworm,
H. zea, CryIIA is less soluble than Cry1Ac, and fails to bind to a saturable binding
component in the midgut brush border membrane (English et al., 1994). The unique mode
of action of CryIIA may provide a useful tool for management of resistance to Bt toxins.
Although binding of the Cry toxins to the receptors determines the species sensitivity to
various toxins, there are distinct exceptions, for example, Cry1Ac binds to the ligand bands
of beet armyworm, S. exigua brush border membrane proteins, but there is very little toxicity
to the insect (Garczynski, Crim, and Adang, 1991; Garczynski and Adang, 1995). Cry1Ab
is more toxic to the gypsy moth, Lymantria dispar (L.), than Cry1Ac, but does not bind well
with the receptors in the brush border membrane (Wolfersberger, 1990).
After solubilization, many protoxins need to be processed by midgut proteases to become
activated toxins (Lecadet and Dedonder, 1967; Tojo and Aizawa, 1983). The major proteases
of the lepidopteran insect midgut are of trypsin (Milne and Kaplan, 1993; Lecadet
and Dedonder, 1966) or chymotrypsin type (Johnston et al., 1995; Peterson, Fernando, and