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Applied and Environmental Microbiology, April 1999, p. 1413-1419, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Integrative Model for Binding of Bacillus
thuringiensis Toxins in Susceptible and Resistant Larvae
of the Diamondback Moth (Plutella xylostella)
Victoria
Ballester,1
Francisco
Granero,1
Bruce E.
Tabashnik,2
Thomas
Malvar,3 and
Juan
Ferré1,*
Departament de Genètica, Universitat de
València, 46100 Burjassot, València,
Spain1; Department of Entomology,
University of Arizona, Tucson, Arizona
857212; and Monsanto Co., St. Louis,
Missouri 631983
Received 17 September 1998/Accepted 5 January 1999
 |
ABSTRACT |
Insecticidal crystal proteins from Bacillus
thuringiensis in sprays and transgenic crops are extremely useful
for environmentally sound pest management, but their long-term efficacy
is threatened by evolution of resistance by target pests. The
diamondback moth (Plutella xylostella) is the first insect
to evolve resistance to B. thuringiensis in open-field
populations. The only known mechanism of resistance to B. thuringiensis in the diamondback moth is reduced binding of toxin
to midgut binding sites. In the present work we analyzed
competitive binding of B. thuringiensis toxins Cry1Aa,
Cry1Ab, Cry1Ac, and Cry1F to brush border membrane vesicles from larval
midguts in a susceptible strain and in resistant strains from the
Philippines, Hawaii, and Pennsylvania. Based on the results, we propose
a model for binding of B. thuringiensis crystal
proteins in susceptible larvae with two binding sites for
Cry1Aa, one of which is shared with Cry1Ab, Cry1Ac, and Cry1F. Our
results show that the common binding site is altered in each of the
three resistant strains. In the strain from the Philippines, the
alteration reduced binding of Cry1Ab but did not affect binding of the
other crystal proteins. In the resistant strains from
Hawaii and Pennsylvania, the alteration affected binding of
Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F. Previously
reported evidence that a single mutation can confer resistance to
Cry1Ab, Cry1Ac, and Cry1F corresponds to expectations based on the
binding model. However, the following two other observations do not:
the mutation in the Philippines strain affected binding of only Cry1Ab,
and one mutation was sufficient for resistance to Cry1Aa. The imperfect
correspondence between the model and observations suggests that reduced
binding is not the only mechanism of resistance in the diamondback moth
and that some, but not all, patterns of resistance and cross-resistance can be predicted correctly from the results of competitive binding analyses of susceptible strains.
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INTRODUCTION |
Insecticides derived from the
bacterium Bacillus thuringiensis are the most widely used
biological pesticides (8). During sporulation, B. thuringiensis produces crystals containing toxins that are called
insecticidal crystal proteins (ICPs) or 
-endotoxins (19,
23). The mode of action of B. thuringiensis
includes the following steps in the insect midgut: solubilization of
the crystals, enzymatic activation of protoxins, binding of activated toxins to target sites on midgut membranes, and pore formation (2,
14, 22, 36). B. thuringiensis toxins kill a
specific set of insect pests but do not harm people, wildlife, or even most beneficial insects. The genes encoding ICPs have been incorporated into and expressed by plants so that the plants have become toxic to
some insect pests (11, 43).
Evolution of resistance by pests is the greatest threat to the
continued success of the B. thuringiensis toxins used
in conventional sprays or in transgenic plants (20).
Although laboratory selection has produced resistance to B. thuringiensis in many pests, only the diamondback moth
(Plutella xylostella), a major pest of crucifer crops
worldwide, has evolved resistance to B. thuringiensis in open-field populations (37).
Resistance to B. thuringiensis has been documented in
diamondback moth populations in Hawaii, Asia, the continental United
States, and Central America (35, 37, 41, 48, 49). Reduced
binding of ICPs to midgut target sites is the best-documented
mechanism of resistance in members of the Lepidoptera and the only
known mechanism of resistance in the diamondback moth (10, 41, 42,
48). Nonetheless, some evidence suggests that other mechanisms,
such as reduced activation of protoxins and increased degradation of
toxins, can confer resistance to ICPs (12, 15, 28, 30, 32,
34).
Here we report the results of competitive binding tests performed with
ICPs in a susceptible strain of diamondback moth (strain LAB-V) and in
resistant strains of diamondback moth obtained from the Philippines
(strain PHI), Hawaii (strain NO-QA), and Pennsylvania (strain PEN).
Each of the three resistant strains evolved resistance to three ICPs
present in B. thuringiensis sprays (Cry1Aa, Cry1Ab, and
Cry1Ac) while remaining susceptible to Cry1C and some other ICPs not
present in the sprays (39, 41). NO-QA and PEN evolved cross-resistance to two ICPs that were not in the sprays (Cry1F and
Cry1J), whereas PHI did not (39, 41). Previous analyses of
noncompetitive binding provided a preliminary description of ICP-target
site interactions in the four strains of diamondback moth which we used
(41). Previous results also show that the ICPs Cry1Aa,
Cry1Ab, and Cry1Ac bind to a common target site in diamondback moth and
other moths (1, 7, 25, 44) and that Cry1F also binds to this
common target site in diamondback moth (16). In the present
study, we used results from our competitive binding assays along with
previously published data (9, 16) to produce an integrative
model for binding of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, and Cry1F in
a susceptible strain and variations on the model that explain the
patterns observed in the three resistant strains.
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MATERIALS AND METHODS |
Insects.
The three resistant strains of diamondback moth
were started from field populations that had been sprayed repeatedly
with commercial formulations of B. thuringiensis subsp.
kurstaki containing the toxins Cry1Aa, Cry1Ab, and Cry1Ac
(41). Before we compared the three resistant strains, each
was exposed to B. thuringiensis in the laboratory to
eliminate susceptible individuals. Susceptible strain LAB-V originated
from The Netherlands and had been reared in the laboratory for more
than 10 years (9).
Source of ICPs.
Trypsin-activated Cry1Aa, Cry1Ab, Cry1Ac,
and Cry1F were kindly provided by Luke Masson (National Research
Council of Canada, Montreal, Canada). The Cry1A proteins were obtained
from recombinant Escherichia coli HB101, and Cry1F was
obtained from recombinant E. coli EG1945 (from Ecogen Inc.,
Langhorne, Pa.).
Iodination of ICPs.
Cry1A proteins (25 µg each) were
labeled with Na125I by using the chloramine-T method
(44). Cry1Aa was labeled twice, once with 0.5 mCi of
Na125I (1 Ci = 37 GBq) and once with 1 mCi of
Na125I, and the two preparations were used in independent
experiments. Cry1Ab and Cry1Ac were labeled with 1 mCi of
Na125I. Labeled ICPs were separated from free iodine by
using a Bio-Gel P30 (Bio-Rad) column. The specific activities were 0.63 mCi/mg for Cry1Aa labeled with 0.5 mCi of Na125I, 5.2 mCi/mg for CryAa labeled with 1 mCi of Na125I, 1.9 mCi/mg
for Cry1Ab, and 2.1 mCi/mg for Cry1Ac.
Preparation of BBMV.
Brush border membrane vesicles (BBMV)
were prepared from whole last-instar larvae by the differential
magnesium precipitation method (5, 46). BBMV were frozen in
liquid nitrogen and kept at 
80°C until they were used. The
concentration of proteins in BBMV preparations was determined with the
Bio-Rad reagent (3) by using bovine serum albumin as the standard.
Binding of 125I-ICPs to BBMV.
Binding
experiments were performed essentially as described previously
(9). BBMV (5 to 10 µg of vesicle protein per assay) were
incubated in 0.1 ml (final volume) of binding buffer (8 mM Na2HPO4, 2 mM KH2PO4,
150 mM NaCl [pH 7.4], 0.1% bovine serum albumin) containing 1.1 nM
125I-labeled Cry1Aa, 1.5 nM 125I-labeled
Cry1Ab, 1.0 nM 125I-labeled Cry1Ac, and various
concentrations of unlabeled competitor. Incubations were carried out at
room temperature for 30 min. Bound ICPs were separated from free ICPs
by filtration with glass fiber filters (type GF/F; Whatman). The
filters were washed with 5 ml of cold binding buffer, and the
radioactivity retained in the filters was measured with a model 1282 Compugamma CS gamma-counter (LKB). A 150- to 1,000-fold excess of
unlabeled toxin was used to determine the extent of nonspecific
binding. The maximum specific binding was 4 to 6% for Cry1Aa, 3% for
Cry1Ab in strain LAB-V, 9% for Cry1Ac in strain LAB-V, and 3% for
Cry1Ac in strain PHI (41).
Statistical analyses.
Data from the competition experiments
were analyzed by using the LIGAND (33) and PRISM
(17) computer programs. Both programs were used to analyze
homologous competition (competition of a labeled ligand and its
unlabeled analogue for binding to the receptor) curves to see if they
fit a one-binding-site model or a two-binding-site model. In both
programs, the null hypothesis assumes that the two populations of
binding sites really are the same. If the P value obtained
from the analysis is less than 0.05, then the null hypothesis is
rejected, which indicates that the experimental curve does not fit a
one-binding-site model. The results obtained with the two programs were
always in agreement. Binding parameters (dissociation constant
[Kd] and concentration of receptors
[Rt]) were estimated from homologous
competition curves, as well as heterologous competition curves (when
the unlabeled ligand was not an analogue of the labeled ligand), by
using the LIGAND program. Heterologous competition also revealed
whether different ICPs bound to the same binding site.
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RESULTS |
Binding assays performed with the susceptible strain (strain
LAB-V).
Binding of 125I-labeled Cry1Aa was
determined at different concentrations of unlabeled Cry1Aa,
Cry1Ab, Cry1Ac, or Cry1F (Fig. 1).
Results obtained from both the LIGAND and PRISM program analyses showed
that the data for the homologous competition curve (labeled Cry1Aa
versus unlabeled Cry1Aa) fit a two-binding-site model better than they
fit a one-binding-site model. The Kd values for
Cry1Aa indicated that a high-affinity binding site
(Kd1, 0.1 ± 0.1 nM) and a
low-affinity binding site (Kd2, 17.7 ± 1.0 nM) were present (Table 1). Considerable binding of labeled Cry1Aa
was detected in the presence of the homologous competitor at a
concentration of 200 nM (Fig. 1), which indicated that a
substantial portion of the labeled Cry1Aa bound nonspecifically to
the BBMV. Cry1Ab and Cry1Ac competed with labeled Cry1Aa but did
not completely impede specific binding of labeled Cry1Aa.
These results suggest that the three ICPs compete for binding to one of
the Cry1Aa binding sites but not the other. Cry1F apparently did not
compete for binding with labeled Cry1Aa.
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FIG. 1.
Binding of 125I-labeled Cry1Aa to BBMV of
the susceptible strain (LAB-V) at different concentrations of unlabeled
competitor. Symbols:  , Cry1Aa;  , Cry1Ab;  , Cry1Ac;  ,
Cry1F.
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The results obtained for binding of
125I-labeled Cry1Ab and
125I-labeled Cry1Ac at different concentrations of
unlabeled Cry1Aa,
Cry1Ab, and Cry1Ac are shown in Fig.
2 and
3.
Analysis of the
homologous competition data indicated that the data fit
a one-binding-site
model better than they fit a two-site model. In both
cases, the
heterologous competitor inhibited specific binding of the
labeled
ligand almost completely, suggesting that the three ICPs bound
to the single receptor recognized by the labeled ICPs. Although
different values were obtained depending on the labeled ICP used,
Cry1Ab was the ICP that showed the highest affinity for the binding
site, followed by Cry1Ac and Cry1Aa (Table
1). These results
suggest that the shared
binding site is the low-affinity binding
site for Cry1Aa.
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FIG. 2.
Binding of 125I-labeled Cry1Ab to BBMV of
the susceptible strain (LAB-V) at different concentrations of
unlabeled competitor. Symbols: , Cry1Aa; , Cry1Ab; ,
Cry1Ac.
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FIG. 3.
Binding of 125I-labeled Cry1Ac to BBMV of
the susceptible strain (LAB-V) at different concentrations of
unlabeled competitor. Symbols: , Cry1Aa; , Cry1Ab; ,
Cry1Ac; , Cry1F.
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TABLE 1.
Equilibrium Kd and
Rt values for B. thuringiensis
crystal proteins for BBMV of susceptible strain LAB-V of P. xylostella obtained with different 125I-labeled ICPs
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Competition of Cry1F with labeled Cry1Ab for the same binding site has
been observed previously in LAB-V (
16). Here we found
that
Cry1F also competes with labeled Cry1Ac (Fig.
3), with an
affinity
similar to the affinity observed when Cry1F was competing
with labeled
Cry1Ab (Tables
1 and
2), which is
consistent with
the observation that Cry1Ab and Cry1Ac share the same
binding
site.
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TABLE 2.
Previously reported values for Kd
and Rt of B. thuringiensis
crystal proteins for BBMV from susceptible P. xylostella
strains (strains LAB-V, LAB-P, and ROTH) and resistant P. xylostella strains (strains Philippines, NO-QA, SERD3, and
Bta-Sel)
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Binding assays performed with three resistant strains
(strains PHI, NO-QA, and PEN).
In a previous study, we
demonstrated that specific binding of 125I-labeled Cry1Aa
occurs in resistant strains PHI, NO-QA, and PEN (41). Here,
we expanded the study with competition analyses. With PHI, the
homologous competition data for Cry1Aa fit a two-binding-site model
better than they fit a one-binding-site model (Fig.
4A), and the two
Kd values were essentially the same as the
Kd values obtained with the susceptible strain
(Table 3). This indicates that binding of
Cry1Aa is not affected in this resistant strain. In contrast, with
strains NO-QA and PEN, the homologous competition data fit a
one-binding-site model better than they fit a two-binding-site model (Fig. 4B). The Kd and
Rt values of Cry1Aa for NO-QA (4.8 ± 2.7 nM and 1.9 ± 0.8 pmol/mg of protein, respectively) and PEN (4.0 ± 2.8 nM and 1.9 ± 0.8 pmol/mg of protein,
respectively) are similar to each other and are intermediate between
the values obtained for the two binding sites of LAB-V and PHI (Tables
1 and 3).
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FIG. 4.
(A) Binding of 125I-labeled Cry1Aa to BBMV
of resistant strain PHI at different concentrations of unlabeled
competitor. Symbols: , Cry1Aa; , Cry1Ab; , Cry1Ac;  , Cry1F.
(B) Binding of 125I-labeled Cry1Aa to BBMV of resistant
strains NO-QA ( ) and PEN () at different concentrations of
unlabeled Cry1Aa.
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TABLE 3.
Equilibrium Kd and
Rt values for B. thuringiensis
crystal proteins for BBMV from resistant strain PHI of P. xylostella obtained with different 125I-labeled ICPs
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Binding was low or nil in all three resistant strains for
Cry1Ab (
41) and in NO-QA and PEN for Cry1Ac (
41).
However, Cry1Ac
exhibits specific binding to BBMV from strain PHI
(
41). Analysis
of the homologous competition data (Fig.
5) gave a better fit
with a
one-binding-site model than with a two-binding-site model.
Kd and
Rt values were
calculated from both the homologous and
heterologous competition data
(Table
3). The values of
Kd and
Rt depended on which ICP was labeled. However,
for a particular
labeled ICP, the
Kd and
Rt values for Cry1Ac did not differ
between
strain PHI and strain LAB-V, which indicates that binding
of Cry1Ac
in strain PHI was not altered compared to binding of
Cry1Ac in
strain LAB-V.
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FIG. 5.
Binding of 125I-labeled Cry1Ac to BBMV of
resistant strain PHI at different concentrations of unlabeled
competitor. Symbols: , Cry1Aa; , Cry1Ab; , Cry1Ac; ,
Cry1F.
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Heterologous competition experiments confirmed the homologous
competition results showing that binding of Cry1Aa and Cry1Ac
is not
altered in strain PHI. The results of competition of Cry1Ab,
Cry1Ac,
and Cry1F with labeled Cry1Aa for binding sites in BBMV
of PHI larvae
are shown in Fig.
4A. As in susceptible strain LAB-V,
Cry1F did not
compete with labeled Cry1Aa for binding, and Cry1Ab
did not
compete either (as expected, since this ICP does not bind
to BBMV
of this strain), but there was some competition with Cry1Ac
(as a
consequence of the shared binding site). The results of
competition of
Cry1Aa, Cry1Ab, and Cry1F with labeled Cry1Ac are
shown in Fig.
5,
which shows that the main difference compared
with strain LAB-V is
the lack of competition by Cry1Ab. Cry1F
competed with labeled Cry1Ac
for binding (Table
3) with essentially
the same affinity as the
affinity in strain LAB-V (Table
1).
| |
DISCUSSION |
If we considered the results of competition binding experiments
performed with strain LAB-V in this work along with the results of
other studies in which binding of Cry1B and binding of Cry1C were also
determined (9, 16), an integrative model for the sites
involved in binding to ICPs in the diamondback moth could be developed.
We propose a model which includes at least four binding sites involved
in binding of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, and Cry1F (Fig.
6). In this model Cry1A proteins and
Cry1F compete for binding to a common binding site, and Cry1Aa binds with low affinity to the common binding site and binds with high affinity to a different binding site not shared with any of the other
ICPs. Cry1B and Cry1C each bind to different binding sites. This
model explains the finding that the homologous competition data for
Cry1Aa fit a two-site model, whereas the homologous competition data
for Cry1Ab, Cry1Ac, and Cry1C fit a one-site model. As far as we know,
labeled Cry1B has not been tested in a competition experiment.
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FIG. 6.
Model proposed for binding of B. thuringiensis ICPs to binding sites in the P. xylostella epithelial midgut membrane in susceptible insects (A),
in strain PHI (B), and in strains NO-QA and PEN (C). The wider arrows
indicate greater binding affinity. Dashed arrows indicate that no
binding or extremely reduced binding occurs.
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The model also explains the heterologous competition data. Cry1Ab and
Cry1Ac cannot completely displace specific binding of labeled Cry1Aa.
In the case of Cry1F, we interpret the lack of competition with labeled
Cry1Aa as the result of Cry1F competing with low affinity to the common
binding site that in turn Cry1Aa also binds to with low affinity. The
low level of binding apparently escaped detection in our assay. In
contrast, binding of labeled Cry1Ab or Cry1Ac can be eliminated by
competition with the three Cry1A proteins and Cry1F, which indirectly
suggests that Cry1Aa and Cry1F bind to the same common site.
Alternatively, these two toxins could bind to different determinants in
the binding site in such a way that their binding interferes with
Cry1Ab or Cry1Ac binding but not with each other. The latter
possibility could be tested in competition experiments performed with
Cry1Aa and labeled Cry1F.
Resistant strain PHI apparently has an alteration in the common binding
site that greatly reduces or eliminates binding of Cry1Ab without
affecting binding of Cry1Aa or Cry1Ac. Binding is likely to occur
through multiple interactions between the binding sites and several
loops of domain II of ICPs (18, 26). Binding of Cry1Aa and
Cry1Ac to the common binding site in PHI suggests that binding of
Cry1Ab involves a distinct binding determinant that can change without
affecting binding of the other ICPs. This same phenotype has been
reported in a resistant diamondback moth population from Malaysia, in
which binding was reduced for Cry1Ab but not for Cry1Aa, Cry1Ac, or
Cry1C (48). This very specific change in the binding site is
also consistent with the observation that in one set of bioassays
conducted soon after PHI was established in the laboratory, this strain
showed extremely high resistance to Cry1Ab but apparently not to Cry1Aa
or Cry1Ac (1). Genetic analyses also have shown that in PHI,
resistance to Cry1Aa is not controlled by the same gene that confers
resistance to Cry1Ab (41).
The binding changes in NO-QA are the same as those observed in PEN.
Both strains show very little or no binding of Cry1A proteins to the
putative common binding site. These two strains also have virtually
identical values of Kd and
Rt for binding of Cry1Aa to the site that is not
shared by the other ICPs. According to our model, NO-QA and PEN have an
alteration in the common binding site that impedes binding of any ICP.
The model nicely explains the resistance phenotype; both strains are
resistant to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F (and Cry1J), although
they are susceptible to Cry1C and Cry1B (39, 41). We
observed specific binding of Cry1C in both of these resistant strains
(41). A previous analysis also detected no reduction in
Cry1C binding in NO-QA (38). One gene can confer resistance
to Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F in NO-QA (40), whereas
in a related diamondback moth strain from Hawaii, resistance to Cry1C
segregates independently from resistance to Cry1Ab (27). The
genetics of resistance in NO-QA and PEN supports our model, because it
appears that one gene is responsible for the major alteration in the
common binding site and one or more other genes confer resistance to Cry1C.
Quantitative differences between Kd and
Rt values obtained by using homologous
competition data and Kd and
Rt values obtained by using heterologous
competition data are common (4, 45), even though both sets
of data should give the same result. Differences may arise because the
labeled proteins are not the same as the unlabeled proteins used as
homologous competitors, since the labeling procedure changes the
proteins at least by adding the 125I residue. Values of
Kd and Rt usually are
calculated from the results of homologous competition experiments,
which are regarded as more indicative of the binding of the ICPs to
their target sites.
The values of Kd and Rt
obtained in the present work agree well with those previously published
for P. xylostella (Table 2). Note that the values reported
previously for Cry1Aa were obtained in heterologous competition with
labeled Cry1Ab (Table 2) and therefore represent the low-affinity
binding site of Cry1Aa observed in the present study (Table 1). The
values obtained here for Cry1Ab in homologous competition experiments
(Table 1) are not much different from the values reported previously
(Table 2). For Cry1Ac, two different sets of values have been obtained
previously for Kd (Table 2). Values of 22.4 and
27.3 nM (48) were obtained with the same batch of labeled
Cry1Ac that was used in the present work; experiments with
strains LAB-V (Table 1), ROTH (48), and SERD (48)
were performed simultaneously in the same laboratory. The value
obtained in heterologous competition with labeled Cry1Ab, 3.7 nM (Table
2), agrees well with the value obtained in the present work with
labeled Cry1Ab (5.5 nM) and is close to the value obtained for
homologous competition in a different laboratory (2.0 nM). Therefore,
for now, we think that there are not enough independent replicates to
choose a Kd value for homologous competition of
Cry1Ac. However, the differences in the values for the binding parameters of Cry1Ac do not invalidate the model which we propose and
do not affect the comparisons among the strains of diamondback moth
studied here. Finally, the Kd values obtained
for Cry1F (Tables 1 and 3) are also close to the value shown in Table
2.
Binding of Cry1A proteins to strain NO-QA has been determined by
using techniques different from the technique used in our study.
Binding of Cry1Aa, Cry1Ab, and Cry1Ac to tissue sections of the midguts
of NO-QA larvae was observed by using histochemical detection
(6), as well as binding of Cry1Ac to BBMV with surface plamon resonance (31). Finally, a Cry1Ac binding protein
with aminopeptidase activity was purified from both susceptible insects and NO-QA (29). Several explanations have been proposed for these discrepancies (6, 29, 31). The hypothesis underlying all of them is that strain NO-QA has binding sites for the ICPs that
have been altered in such a way that they do not bind toxins in vivo
but in vitro bind ICPs under some circumstances but not others.
The results presented in this paper have important implications for
resistance management. On the one hand, they show that binding site
models obtained by using susceptible insects may correctly predict some
aspects of the patterns of resistance. For example, based on the model
derived from our competitive binding assays performed with susceptible
strain LAB-V, we would have predicted that a single mutation could
confer resistance to Cry1Ab, Cry1Ac, and Cry1F because these ICPs each
bind only to a shared target site. However, because Cry1Aa binds to two
sites in LAB-V, we might have made the incorrect prediction that two
mutations were necessary for resistance to Cry1Aa. The finding that
binding to the high-affinity binding site for Cry1Aa occurs in NO-QA
and PEN without toxicity agrees with the results of other reports showing that binding is not sufficient for toxicity (7, 13, 21,
24, 47). The receptor alteration observed in PHI that confers
resistance to Cry1Ab but not Cry1Ac would not have been predicted from
the binding model. Furthermore, the results also show that a binding
site model does not provide any information concerning the mechanisms
of resistance that are not related to binding site alteration. This is
true for strain PHI, and the model does not predict that this strain is
resistant to Cry1Aa and Cry1Ac. However, in cases like this, the fact
that the model cannot explain the resistance pattern clearly shows that
there is more than one mechanism of resistance.
| |
ACKNOWLEDGMENTS |
We thank Luke Masson, who generously provided toxins.
Funding was provided in part by grants TSTAR 95-34135-1771, NRI-CGP
96-35302-3470, and WRPIAP 97RA0304/0305-WR96-16 from the U.S.
Department of Agriculture and by European Community ECLAIR project
AGRE-0003.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departament de
Genètica, Universitat de València, 46100 Burjassot,
València, Spain. Phone: 34-96-386-4506. Fax: 34-96-398-3029. E-mail: juan.ferre{at}uv.es.
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Applied and Environmental Microbiology, April 1999, p. 1413-1419, Vol. 65, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
