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Occurrence of three different binding sites for Bacillus thuringiensis ╬┤-endotoxins in the midgut brush border membrane of the potato tuber moth phthorimaea operculella (zeller).

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Archives of Insect Biochemistry and Physiology 26:315-327 (1 994)
Occurrence of Three Different Binding Sites
for Bacillus thuringiensis &Endotoxins in the
Midgut Brush Border Membrane of the
Potato Tuber Moth, Phthorimaea operculella
(ZeIIer)
Baltasar Escriche, Amparo C. Martinez-Ramirez, M. Dolores Real, Francisco J.
Silva, and Juan F e d
Departamento de Genktica, Facultad de CC. Bioldgicas, Univevsitat de Valencia, Valencia, Spain
The potato tuber moth is susceptible to at least three insecticidal crystal proteins
(ICPs) from Bacillus thuringiensis: CrylA(b), CrylB, and CrylC. To design useful
combinations of toxin genes either in transgenic plants or in new genetically
modified 5. thuringiensis strains, it is necessary to determine the binding characteristics of the different lCPs so as not to combine a pair sharing the same binding
site. This has been accomplished using two different techniques: '251-labeling of
the ICPs with further measurement of the radioactivity bound to brush border
membrane vesicles, and microscopic visualization of the bound lCPs by enzymelinked reagents such as antibodies or streptavidin using biotinylated ICPs. Our
results show that CrylA(b), CrylB, and CrylC bind to different sites in the brush
border membrane of midgut epithelial cells. Also, the affinity of the binding sites
for the lCPs and their concentration in brush border membrane vesicles has been
determined in a laboratory strain and a storage collected population. No significant
ss,
differences were found between these two strains. o 1994 ~ i ~ e y - ~ i Inc.
Key words : Bacillus thuringiensis, Ph thorimaea operculella, insect icida I crysta I protein,
receptors
Acknowledgments: We are indebted to M. Peferoen, J.Van Rie, and P. Denolf from Plant Genetic
Systems (PGS) (Gent, Belgium) for their technical support and for suggesting the use of biotin-labeled
lCPs in tissue sections. We particularly thank S. Jansensfrom PCS for the communication of toxicity
data. This work was supported by a grant of the E.C., under the ECLAIR program (projectAGRE-0003),
and a grant from the Spanish Ministerio de Agricultura, Pesca y Aiimentacion (project AGR91-0238CE). B. Escriche and A.C. Martinez-Kamirezwere supported by grants from the Spanish Ministerio de
Educacion y Ciencia and the Conselleria Valenciana de Educacio y Cikncia, respectively.
Received July 7, 1993; accepted November 1, 1993.
Address reprint requests to Baltasar Escriche, Departamento de Genktica, Facultadde CC. Biologicas,
Universitat de Valencia, 461 00-Burjassot, Valencia, Spain.
0 1994 Wiley-Liss, Inc.
31 6
Escriche et al.
INTRODUCTION
Many strains of the bacterium Bacillus thuringiensis synthesize, at the time of
sporulation, proteinaceous crystals containing one or several types of proteins.
In most cases, these proteins possess insecticidal activity, for which they are
called insecticidal crystal proteins (ICPs) or &endotoxins (Adang, 1991; Hofte
and Whiteley, 1989; Lereclus et al., 1989).Formulations based on B. thuringiensis
spore-crystal mixtures are widely used to control agricultural insect pests,
mainly several species of Lepidoptera that feed on crops, and also some disease
vectors such as several species of mosquitoes. B. thuringiensis products are
environmentally friendly insecticides in that they do not affect species other
than insects, and they are very specific in their insecticidal action (i.e., they d o
not affect beneficial insects).
Environmental factors such as sunlight, heat, and rain inactivate insecticides
based on B. thuringiensis in 2 4 4 8 h (Navon, 1993).However, insecticide persistence is considerably improved when used in situations other than open field
applications. Thus, control of the potato tuber moth (Phthorimaea operculella), a
major pest of potato storage and in field potatoes in warm climates, has been
successful in storage houses (Raman et al., 1987).This insect pest is susceptible,
in the laboratory, to at least three ICPs: CryIA(b), CryIB, and CryIC. CryIB is
the most effective, followed by CryIA(b) (six times less), and CryIC (30 times
less than CryIB) (S. Jansens, personal communication).
The specificity of B. thuringiensis ICPs is linked to its mode of action (Adang,
1991; Gill et al., 1992).After being ingested by the insect, the crystal is dissolved
in the midgut; in most cases activated by partial proteolysis, ICPs bind to
specific sites in the brush border membrane of epithelial cells in the midgut. It
is thought that a hydrophobic domain of the ICP penetrates into the lipid
bilayer, forming a pore that eventually produces the lysis of the cell (Li et al.,
1991). The binding of ICPs to specific binding sites has been demonstrated to
be a key factor of B. thuvingiensis specificity (Hofmann et al., 198813; Van Rie et
al., 1989,1990a).
It has been demonstrated, by field studies and laboratory selection experiments, that the use of B. thuringiensis based products can provoke the development of resistance in the target insects (Could et al., 1992; Kirsch and
Schmutterer, 1988; McGaughey, 1985; Stone et al., 1989; Tabashnik et al., 1990).
In some cases the mechanism of resistance is due to the modification of a
midgut membrane receptor for an ICP (Ferrk et al., 1991; Van Rie et al., 1990b).
Therefore, characterization of binding sites for the ICPs and determination of
their relationship in binding different ICPs will be of great value in helping to
prevent or delay the development of resistance to B. fhzrvingietzsis bioinsecticides. In the present work we investigated the ICP binding sites in P. operculella.
MATERIALS AND METHODS
Colonies and Rearing
Two populations of potato tuber moths were established. One, a laboratory
population originally established in the 1940s,was obtained from the University
Binding Sites for B. thuringiensis Endotoxins
31 7
of California, Berkeley (Etzel, 1985). The other was collected in 1989 from a
potato storage in the Amazonian part of Peru. Insects were reared at 25°C and
60-70% relative humidity with a 16 h/8 h (L/D) photoperiod, which is a slight
modification of published procedure (Etzel, 1985).
Chemicals and Biological Reagents
All chemicals were reagent grade and were obtained from commercial suppliers. B. thuringiensis ICPs were obtained from Plant Genetic Systems (Gent,
Belgium). CryIA(b), CryIB, and CryIC are recombinant proteins expressed in
Escherichia coli (Ferr6 et al., 1991) and were supplied as activated trypsin-digested toxins. CryIIIA, an ICP toxic to Coleoptera but not to Lepidoptera, was
treated with chymotrypsin before use according to Carroll (1990).Monoclonal
antibodies against CryIA(b) (4D6), CryIB (22A2F1), CryIC (5B10 and 1AlO),and
CryIIIA (14A3), and polyclonal antibodies against CryIA(b1 (RaBt21, CryIB
(RaBtl4), and CryIC (RcrBtl5),were also obtained from Plant Genetic Systems.
The biotinylation kit was obtained from Amersham (Buckinghamshire,UK).
Preparation of Brush Border Membrane Vesicles
Last instar larvae were chilled on ice for 10 min to reduce their mobility.
Dissection of larvae and isolation of midguts were carried out in cold 0.3 M
mannitol, 5 mM EGTA, and 17 mM Tris-HC1, pH 7.5. The dissected midguts
were washed in the same buffer, frozen by immersing the vial in liquid nitrogen,
and stored at -80°C until used.
Brush border membrane vesicles (BBMV)from larval midguts of P. opevculella
were prepared by the differential magnesium precipitation method described
by Wolfersberger et al. (1987).The amount of BBMV proteins was measured
using Bradford’s procedure with a Bio-Rad (Richmond, CA) kit (Bradford,1976)
and bovine serum albumin (BSA)as standard.
Preparation of Histological Sections
Histological sections were prepared as described by Bravo et al. (1992).
Midgut from last instar larvae were dissected and fixed in Bouin Hollande 10%
sublimate (Brandtzaeg, 1988).After 30 min in the fixative, midguts were transferred to fresh fixative solution and kept for 24 h. Fixed tissue was washed 12
h in distilled water, dehydrated in ethanol, infiltrated with xylol, and embedded
in Paraplast (Monoject Scientific Inc., Kildare, Ireland).
Transverse sections (5 bm thick) were made with a microtome and placed on
mounting glasses coated with 5% glycerol, 1 % ovoalbumin, and 77 pM NaN3.
After stretching at 45°C for 1 min and 40°C for 3 days, tissue sections were
deparaffinated and hydrated by successive incubations in xylol, ethanol, and
distilled water. Excess sublimate was removed with I2/KI and sodium thiosulphate. Finally, the sections were washed with distilled water and equilibrated
with 10 mM Tris-HC1, pH 7.6,150 mM NaC1,l mM thimerosal, and 0.1% triton
X-100 (buffer Ts-T).
Labeling of ICPs
Iodination of ICPs was carried out using carrier free [12511-NaI
(Amersham).
Chloramine-T was used for labeling CryIA(b) (Van Rie et al., 1989) and Iodo-
31 8
Escriche et al.
Gen (Pierce, Rockford, IL) for labeling CryIC (Hofmann et al., 1988a).Specific
activity of iodinated ICPs was determined using a "sandwich ELISA technique
as described by Van Rie et al. (1989).Specific activities on labeling day ranged
between 0.25 and 2.15 pCi/pg for CryIA(b) and between 0.5 and 2.15 pCi/pg
for CryIC. Labeled ICPs were used within a month after labeling.
Biotinylation of CryIA(b) and CryIB was performed following the method
of Denolf et al. (1993). Biotinyl-N-hydroxysuccinimideester (0.2 mg) was
incubated with 1 mg of ICP in 1 ml of borate buffer (pH 8.6) for 1 h. Free
reagent was separated from biotinylated ICP using a Sephadex G-25
column. Biotinylation was confirmed by a dot-blot test: an aliquot (1 pl) of
each fraction was spotted on a nitrocellulose membrane and incubated with
streptavidin-alkaline phosphatase conjugate and then with 5-bromo-4-chloro3-indolyl-phosphate/nitroblue tetrazolium salt (BCIP/NBT) solution. The
concentration of biotinylated ICPs was determined with the Bio-Rad reagent
(Bradford, 1976).
Binding of ICPs to BBMV
To find the optimal assay conditions, experiments were performed to determine the appropriate concentrations of labeled ICPs, incubation time, and
BBMV concentration.According to them, the following conditions were chosen
for competition experiments: BBMV (16 bg protein) were incubated for 90 min
at room temperature, with 0.3 nM lZ5I-labeledCryIA(b)or with4.13 nM 1251-labeled CryIC, in the presence of different concentrations of cold competitor, in
0.1 ml of PBS/O.l% BSA (8mM Na2HPQ, 2 mM KHzP04,150 mM NaC1, pH 7.4,
0.1% BSA. In order to separate bound from free ICP, samples were filtered
through Whatman GF/F glass fiber filters (Whatman Scientific Limited, Maidstone, UK) and washed with 5 ml of PBS/O.l% BSA. The radioactivity retained
in the filters was measured in a 1282 Compugamma CS gamma-counter (LKB,
Uppsala, Sweden).
Data were analyzed using the LIGAND computer program (Mundson and
Rodbard, 1980),which calculates the bound concentration of ligand as a function of the total concentration of ligand and which gives estimates of the affinity
constant (Kd) and the total binding site concentration (RJ.
Binding of ICPs to Tissue Sections
Immunocytochemical detection of ICP binding sites was carried out according to the procedure of Bravo et al. (1992). Mounting glasses with
rehydrated tissue sections were covered with 0.3 ml of ICP (5 yg/ml) and
incubated for 2 h. Then samples were incubated overnight with 0.3 ml of
primary antibody (1 pm/ml) and finally for 2 h with 0.3 ml of rabbit
antimouse antibody coupled with horseradish peroxidase (Sigma Chemical
Co., St. Louis, MO) diluted 1:200. Peroxidase activity was detected by incubating with 0.35 mM diaminobenzidine solution and 0.03% H202 in 50 mM
Tris-HC1 (pH 7.6). The reaction was stopped by immersing the mounting
glasses for 1 min in a 50 mM Tris-HC1 (pH 7.6) solution.
For detection of binding sites with biotinylated ICPs, tissue sections were
covered with blocking solution (0.5 ml of 100 mM maleic acid, pH 7.5,150 mM
NaCl, 1%dry milk) for 30 min, incubated with 0.3 ml of biotinylated ICP
Binding Sites for B. fhuringiensis Endotoxins
31 9
(5 pg/ml) for 45 min, and then incubated for 1 h with 0.3 ml of streptavidin
conjugated with alkaline phosphatase (Amersham) diluted 1:lOO. Color development was obtained by incubation with 0.5 ml of BCIP/NBT solution for 10
min. The reaction was stopped by immersing the mounting glasses in 50 mM
Tris-HC1 (pH 7.6). Binding competition between nonlabeled and biotin-labeled
ICPs was performed by preincubating the tissue sections with 0.3 ml of nonlabeled competitor during 45 min before adding the biotinylated ICP.
Incubations were done at room temperature (22°C). All reagents and dilutions were done in Ts-T buffer. Every step described above was followed by a
1min washing in this buffer, and negative controls for each step were included
in each experiment. After the color reaction, slides were dehydrated by successive incubations in ethanol and xylol. Finally, tissue sections were mounted
with Entellan (Merck, Darmstadt, Germany).
RESULTS
Binding of Iodinated ICPs to BBMV
CryIAb) and CryIC were iodinated with 1251and used for binding to BBMV
from the two different colonies of potato tuber moth. Saturable binding was
obtained, with maximum binding at 400 pg/ml for CryIA(b) and 500 pg/ml for
CryIC (Fig. 1). Quantitative estimates of the binding characteristics to BBMV
binding sites were obtained from homologous competition experiments (i.e.,
competition of a labeled ligand with its nonlabeled analogue for binding to the
receptor) (Fig. 2). Dissociation constants and receptor concentrations, given in
Table 1, were essentially the same for the two colonies ( P > 0.05 t-test).
Heterologous competition experiments were carried out in order to check for
binding of more than one type of ICP to the same receptor. Figure 2 shows that
CryIB and CryIC do not bind to the receptor for CryIA(b) and that CryIA(b)and
CryIB do not bind to the receptor for CryIC.
.- I
"
n
CrylA(b)
8
0
-
rn
"
6
c
0
.-m
c
zT
4
$ 2
n
0
0,i
0,2
0,3
0.4
0.5
0,6
0,7
BBMV (mglml)
Fig. 1. Specific binding (solid symbols) of '251-labeled CrylA(b) and CrylC to increasing amounts of
BBMV of the laboratory strain. Nonspecific binding (open symbols) is shown for each ICP and was
subtracted from the total binding for cach data point.
320
Escriche et al.
-e
120
A
0;001
0,Ol
0,l
10
1
100
1000
10000
100
1000
10000
Competitor (nM)
0 ~ 0 0 1 0.01
0.1
1
10
Competitor
(nM)
.,
Fig. 2. Binding of '251-labeled CrylA(b) (A) and CrylC (B) as afunction of increasing concentrations of
nonlabeled competitor. BBMVs of the laboratory strain were used. Each point represents the mean
of a duplicate sample. e,CrylA(b); A,CrylB;
CrylC.
TABLE 1. Binding Characteristics of Bacillus thuringiensis ICPs
to BBMV From Two Strains of P . operailella
ICP
Strain
CrylA(b)
Laboratory
Peruvian
Laboratory
Peruvian
CrylC
Kd(nM)a
Rt(pmol/mg prot)"
2.71 (0.83)
2.47 (0.19)
2.13 (0.98)
1.32 (0.27)
3.90 (1.74)
1.67 (0.47)
2.10 (0.94)
2.04 (0.98)
%tandad deviation is shown in brackets. Values represent the
mean of two independent determinations.
Binding Sites for 6.fhuringiensisEndotoxins
321
Binding of CryIB to the Brush Border Membrane of Midgut Epithelial Cells
Since CryIB does not bind to either the CryIA(b) receptor or the CryIC
receptor, it must bind to a different one. However, because of the poor labeling
obtained with CryIB using 1251,its binding to the brush border membrane was
demonstrated by a different approach. Midgut tissue sections were incubated
with CryIB, and specificbinding of this ICP to the brush border membrane was
revealed with the use of a monoclonal antibody (Fig. 3A,B). No binding was
detected when CryIIIA (Hoffeet al., 1987)(an ICP nontoxic to P. opevculella) was
used as a control (data not shown). These results suggest the presence of specific
binding sites for CryIB, with the caveat that the antibody technique does not
demonstrate whether or not binding is saturable because one cannot use a
homologous competitor.
Accordingly, biotinylated CryIB was bound to the brush border membrane
of midgut tissue sections, as revealed after incubation with streptavidin coupled
to alkaline phosphatase (Fig. 3C,D). Preincubation of tissue sections with a
fiftyfold excess of nonlabeled CryIB before incubation with biotinylated CryIB
inhibited binding of the labeled ICP, resulting in no streptavidin reaction (Fig.
3E). These data demonstrate that binding of CryIB to the midgut epithelium is
saturable.
The specificity of CryIB binding was evaluated by preincubation with
CryIA(b) and CryIC at excess concentrations of 250-fold and 50-fold, respectively. Neither reagent inhibited the binding of biotinylated CryIB (data not
shown). Biotinylated CryIA(b) was used as a positive control in these experiments. Its binding to the midgut epithelium (Fig. 3F) was inhibited by preincubation with a fiftyfold excess of nonlabeled CryIA(b),but not with either CryIB
or CryIC (data not shown). Therefore, competition in the biotin-streptavidin
assay system correlated with competition experiments carried out with 1251labeled ICPs.
DISCUSSION
Binding of CryIA(b) and CryIC to specific sites in BBMV prepared from
midguts of the potato tuber moth was demonstrated using 12JI-labeledtoxins.
and the concentration of binding sites (RJ in BBMV for
The binding affinity (h)
the two ICPs was similar in both colonies (Table 1).This similarity could be
interpreted as a uniform feature of the wild type of this species.
It was formerly suggested that ICP toxicity is positively correlated with the
overall binding capacity, as reflected by the parameter Rt/Kd (Hofmann et al.,
1988a;Van Rie et al., 1989).However, in many cases this does not hold true, and
the correlation between toxicity and Rt/Kd can even be inverse (Wolfersberger,
1990). In P. opevculella, CryIC is five times less toxic than CryIA(b) (S. Jansens,
personal communication),although we found only small differences in binding
parameters. Apparently, the toxicity in each insect, and for each ICP, is influenced by additional factors (e.g., pH of the midgut, solubility of the ICP,
proteolytic activity).
Binding of CryIB to the brush border membrane of midgut epithelial cells
was initially demonstrated by immunocytochemical detection using a mono-
Fig. 3. Cytochemical staining showing the binding of lCPs to the brush border membrane
of the midgut epithelium of the laboratory strain. A: lmmunocytochemical staining after
CrylB incubation. B: Control without ICP incubation. C: Streptavidin/CrylB-biotin staining.
D: Detail of streptavidin/CrylB-biotin staining. E: Streptavidin/CrylB-biotin staining after
preincubation with excess nonlabeled CrylB. F: Streptavidin/CrylA(b)-biotin staining. AMV,
apical niicrovilli; BM,basement membrane and connective tissue; L, lumen. Bars = 10 km.
324
Escriche et al.
clonal antibody against CryIB. We also used biotin-labeled CryIB in order to
perform competition experiments in tissue sections. Another advantage of
using biotinylated ICPs is that it does not require the preparation of antibodies
against the ICPs. We have successfully biotinylated CryIB and CryIA(b). Biotinylated ICPs were used to show binding of ICPs to the brush border membrane and to demonstrate, in the case of CryIA(b), CryIB, and CryIC, that
binding was not inhibited by lCPs different from the biotinylated one. Cytochemical techniques for determining ICP binding to epithelial membrane of the
midgut require very few insects as compared with binding to BBMV, which
requires several thousand insects of the size of P. operculella. However, the
main disadvantage of these techniques is that they are not quantitative, and,
therefore, binding parameters for CryIB could not be obtained in the present
study.
Competition among ICPs for the same binding sites has been determined in a number of lepidopteran species (Hofman et al., 198813; Van Rie
et al., 1989, 1990a,b; Wolfersberger, 1990; Ferrk et al., 1991; Denolf et al.,
1993). Although the general pattern is to find competition for the same
binding site only among ICPs belonging to the same subclass-that is,
CryIA(a), CryIA(b) and CryIA(c)-competition has also been found between CryIA(b) and CryIC in Plodia interpunctella (Van Rie et al., 1990b)
and between CryIC and CryIE in Mnnduca sextn and Spodoptera littoralis
(Van Rie et al., 1990a). However, this is not the case in P . opercuIeEEa, where
each of the ICPs tested bind to different sites with no cross-competition
for the sites recognized by the other ICPs.
An advantage of ICPs is that their genes can be genetically manipulated
to improve their insecticidal action (Honke et al., 1990)and, furthermore, can
be incorporated into plants, rendering them in resistant to insect attack
(Fischoff et al., 1987; Vaek et al., 1987). Also, ICP genes can be combined in
bacterial hosts to obtain varieties with new toxicity spectra (Lecadet et al.,
1992). Transformation of potatoes with genes for B. thuringiensis ICPs has
already begun in the biotechnological companies (Peferoen et al., 1990).
Therefore, determination of the different ICP binding sites in this insect may
help resistance management by permitting one to choose the appropriate
combination of ICP genes, either in transgenic plants, in modified bacteria,
or in field applications.
To improve formulation effectiveness, it is very important to know which
ICPs are suitable for combinations (in products, bacteria, or transgenic plants)
or for rotation programs (sequential use of different insecticides). These combinations must take into account that resistance in some insects could involve
changes in binding characteristics (Ferr6 et al., 1991; Van Rie et al., 1990b).This
stresses the importance of determining the number (and affinity) of different
binding sites involved in the toxic action of B. thuringiensis ICPs, as well as the
presence of common binding sites for different toxic ICPs. Results from the
present work allow us to propose a model for the binding of CryIA(b), CryIB,
and CryIC in the potato tuber moth, with three different binding sites, each
binding only one type of ICP. This will be useful to design combinations of these
three ICPs for better resistance management.
Binding Sites for B. thuringiensis Endotoxins
325
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site, endotoxin, midgut, different, three, thuringiensis, membranes, bacillus, phthorimaea, operculella, potato, moth, occurrence, zellen, brush, border, binding, tube
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