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Applied and Environmental Microbiology, September 2001, p. 4372-4373, Vol. 67, No. 9
Department of Biological Sciences, Imperial
College of Science, Technology, and Medicine, Silwood Park, Ascot,
Berkshire SL5 7PY,1 and School of
Biological Sciences, University of Sussex, Falmer, Brighton, East
Sussex BN1 9QG,2 United Kingdom
Received 7 May 2001/Accepted 19 June 2001
Resistant and susceptible populations of the diamondback moth
(Plutella xylostella) were tested with crystalline,
solubilized, and partially and fully activated forms of the
Bacillus thuringiensis Cry1Ac Bacillus thuringiensis
crystalline (Cry) protoxins are solubilized and cleaved by
proteinases in the insect midgut to form active toxins which then bind
to specific sites on midgut brush border membranes, leading to pore
formation and cell lysis (8). Changes that cause
disruption of any of these steps could confer resistance. The
diamondback moth (Plutella xylostella) is the only insect in
which resistance to B. thuringiensis has been selected in
the field, and the only significant resistance mechanism described for
P. xylostella involves the loss of toxin binding, although there is indirect evidence for alternative mechanisms of resistance in
some field populations (3, 10, 11). One possible
alternative mechanism has been described in a B. thuringiensis-resistant strain of Plodia
interpunctella, where the loss of a specific proteinase was
believed to be responsible for the observed resistance
(6). Reduced production of active toxin has also been
proposed as a mechanism of resistance in Heliothis virescens
(1). Proteolytic activation of Cry1 protoxins involves the
removal of peptide sequences from both N and C termini. The
observations that a mutant toxin in which N-terminal cleavage is
prevented has reduced toxicity (4) and that N-terminally
truncated toxins are toxic to Escherichia coli
expression hosts (4, 9) suggest that N-terminal cleavage may be an important part of the toxic mechanism.
An N-terminally activated Cry1Ac was constructed by deleting the DNA
encoding amino acids 2 to 29. This and full-length Cry1Ac were
expressed in E. coli or B. thuringiensis and used
to bioassay P. xylostella using a leaf dip assay as
described previously (7). A Cry1Ac-resistant field
population of P. xylostella from Serdang, Malaysia (SERD5;
collected in August 1999), was divided into two subpopulations. One
subpopulation was left unselected (UNSEL); the other (Cry1Ac-SEL) was
reselected with in vitro-activated Cry1Ac from F2
to F9. Compared with the susceptible strain
(ROTH), UNSEL and Cry1Ac-SEL showed resistance ratios (ratio of the
50% lethal concentration for the resistant population to that for the
susceptible population) of 44 and 1,170, respectively, towards crystalline Cry1Ac protoxin synthesized in E. coli (Table
1) . To test whether the
resistant strain might be defective in toxin solubilization or
activation, bioassays were performed using Cry1Ac toxin which had been
either solubilized or both solubilized and activated in vitro (Table
2). For ROTH, the activity of both forms of the toxin was equivalent to that of the crystalline protoxin. For the SERD5 populations, the activated form of toxin was
significantly (P < 0.01) more toxic than the
solubilized, but nonactivated, form. In the case of Cry1Ac-SEL, the
activated toxin was at least 100-fold more toxic than the solubilized
toxin. Activation of Cry1Ac involves removal of peptide fragments from
both N and C termini. To test whether removal of the N-terminal peptide
is an important step, an engineered protein lacking the N-terminal peptide was used in bioassays. No significant difference was found between the N-terminally truncated and full-length protoxins for any of
the insect populations (Table 1), suggesting that N-terminal activation
is not a limiting step.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4372-4373.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Susceptibility of a Field-Derived, Bacillus
thuringiensis-Resistant Strain of Diamondback Moth to In
Vitro-Activated Cry1Ac Toxin
ABSTRACT
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-endotoxin. Fully
activated toxin greatly reduced the resistance ratio (ratio of the 50%
lethal concentration for the resistant population to that for the
susceptible population) of the resistant population, suggesting
that a defect in toxin activation is a major resistance mechanism.
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TABLE 1.
Toxicity of crystalline Cry1Ac for ROTH, UNSEL, and
Cry1Ac-SEL populations of P. xylostella
TABLE 2.
Toxicity of in vitro solubilized and activated Cry1Ac for
ROTH, UNSEL, and Cry1Ac-SEL populations of P. xylostella
The results described above suggest that reduced availability of active toxin is a major factor explaining the increased resistance in the Cry1Ac-SEL subpopulation. The resistance phenotype could be due to a specific defect in the activation step, as has been proposed for a resistant strain of P. interpunctella (6). Alternatively, a mechanism not directly involved in this step could be responsible. Prevention of protoxin activation by degradation or precipitation of the solubilized toxin, for example, could result in the observed phenotype. Both of these possibilities have been observed in other insect larvae (1, 2, 5) and have been put forward as explanations for reduced susceptibility to B. thuringiensis toxins. The SERD5 strain is believed to have initially developed resistance through exposure to B. thuringiensis sprays; the reselected population was maintained through exposure to in vitro-activated Cry1Ac. The high levels of resistance generated through this reselection would argue against a specific defect in the conversion of protoxin to toxin. More likely is a mechanism that reduces the availability of protoxin or toxin for pore formation, the kinetics being such that protoxin is removed more rapidly than it is converted into toxin. The reduced toxicity of the solubilized toxin compared to that of the crystalline form with the SERD5 populations might be an effect of the leaf dip bioassay, with solubilized toxin not being presented to the larvae as efficiently as crystalline toxin is; this effect may not be so significant for the much more susceptible ROTH strain. Alternatively, it might represent the fact that crystalline protoxin is less of a target for resistance. Concomitant solubilization and activation may partially bypass the resistance mechanism.
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ACKNOWLEDGMENTS |
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We thank Dzolkhifli Omar and Angeliki Martinou for the supply of the field population of P. xylostella from Malaysia.
A.H.S. was supported by the Hundred Scholarship Scheme of the Government of Pakistan. This work was conducted under MAFF licenses PHL 17A/3057(5/1999) and PHL 17B/3479(06/2000) to culture and experiment with the insect populations.
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FOOTNOTES |
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* Corresponding author. Mailing address: School of Biological Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9QG, United Kingdom. Phone: 44 1273 678917. Fax: 44 1273 678433. E-mail: n.crickmore{at}sussex.ac.uk.
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Applied and Environmental Microbiology, September 2001, p. 4372-4373, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4372-4373.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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