Insect Control: Biological and Synthetic Agents - Index of

144 4: Insect Growth- and Development-Disrupting Insecticides
These studies clearly demonstrate the potential
of utilizing EcR based gene switches that can be
activated by ecdysteroids and nonsteroidal ecdysone
agonists like tebufenozide, methoxyfenozide, and
others. The fact that both tebufenozide and methoxyfenozide
are registered as commercial insecticides,
and have proven reduced risk mammalian and
ecotoxicology profiles, makes them very attractive
as inducers of the EcR based gene switches. The
work of Kumar et al. (2002) clearly demonstrates
the potential of mutating EcR to change its ligand
specificity, thus opening additional possibilities
of extending the use of the EcR gene switch in a
multiplexed manner.
4.2.9.2. Gene switch for trait regulation in
plants The reader is referred to very good recent
reviews on this topic that not only describe the EcRbased
chemically inducible gene regulation systems,
but also other systems that have utility in plants
(Jepson et al., 1998; Zuo and Chua, 2000; Padidam,
2003). This section is restricted to descriptions of
EcR-basedgeneswitchsystems.
In the mid-1990s, a number of agricultural companies
initiated research to exploit the use of the
EcR-based gene switch and nonsteroidal ecdysone
agonists like tebufenozide as chemical inducers for
regulation of traits (for example, fertility, flowering,
etc.) in plants. Initial work was done using DmEcR
and DmUSP as components of the gene switch (Goff
et al., 1996). In this case, the researchers used
chimeric polypeptides (GAL4 DBD fused to LBD
of DmEcR and VP16 activation domain fused
to DmUSP) to activate the luciferase reporter gene
fused to GAL4 response element in maize cells.
In the presence of 10 mM tebufenozide, about 20- to
50-fold activation of luciferase expression was
obtained. Subsequently, a number of researchers
developed variants of EcR gene switches using
different chimeric combinations of heterologous
DBD, LBDs from different lepidopteran EcRs,
and transactivation domains that could interact
with appropriate response elements to transactivate
a reporter gene in a ligand-dependent manner
(Martinez et al., 1999a; Unger et al., 2002;
Padidam et al., 2003). For example, Jepson et al.
(1996) and Martinez et al. (1999b) used chimeric
H. virescens EcR (HvEcR) composed of glucocorticoid
receptor transactivation and DBD fused to
LBD of HvEcR and GUS reporter gene fused to
glucocorticoid receptor response element for transfection
of maize and tobacco protoplast. In both
cases, weak transactivation of the GUS reporter
gene was obtained with tebufenozide and muristerone
A, though the response with the latter was much
lower than with tebufenozide. However, 10 mM
and higher concentrations of the two ligands were
required to transactivate the reporter genes.
Padidam et al. (2003) used a chimeric C. fumiferana
EcR (CfEcR) composed of a CfEcR LBD, GAL4
and LexA DBDs, and VP16 activation domains that
could be activated with methoxyfenozide in a dosedependent
manner from a GAL4- or LexA-response
element to express a reporter gene. These researchers
used Arabidopsis and tobacco plants for transformation
with the gene switch components and
obtained several transgenic plants that had little or
no basal level of expression in the absence of methoxyfenozide.
In the presence of methoxyfenozide,
reporter expression was several fold higher than in
the absence of methoxyfenozide. The above studies
provided ample evidence of the utility of EcR gene
switch, especially those that utilize EcR from a lepidopteran
species, and tebufenozide and methoxyfenozide
as chemical inducers for trait regulation in
plants.
Demonstration of the utility of EcR-based gene
switch for trait regulation in maize was demonstrated
by Unger et al. (2002). A mutation in maize (MS45),
which results in male-sterile phenotype, could be
reversed by complementation to gain fertility using
methoxyfenozide-dependent chimeric receptor gene
switch to express the wild-type MS45 protein in
tapetum and anthers. These researchers used the
EcR LBD from, O. nubialis to generate a chimeric
receptor. The chimeric receptor was introduced into
MS45 maize with the MS45 gene fused to the GAL4
response element, which in the absence of methoxyfenozide
were male sterile. However, application
of methoxyfenozide to plants containing either a
constitutive promoter or anther specific promoter
resulted in the restoration of fertility to MS45 plants
grown in either the greenhouse or the field.
It is interesting to note that in all the above studies,
except those by Goff et al. (1996), reporter
transactivation response to tebufenozide, methoxyfenozide,
or muristerone A via the EcR gene switch
was obtained without the requirement of an exogenous
heterodimeric partner (USP or RXR), suggesting
that there may be other factor(s) in plants that
can substitute for USP as a partner for EcR, or
that EcR can function as a homodimer. However,
as far as is known, there is no evidence of EcR
binding an ecdysteroid or nonsteroid ligand in the
absence of USP or RXR. Irrespective, these studies
provide ample demonstration of the utility of EcRbased
gene switch, which can be regulated with an
ecdysteroid or a nonsteroidal ecdysone agonist. The
use of nonsteroidal ecdysone agonists, like any of
the commercialized products, is attractive because