Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.     

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC₅₀ = 0.84 mg l^?1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.     

Evaluating the Insecticidal Genes and Their Expressed Products in Bacillus thuringiensis Strains by Combining PCR with Mass Spectrometry

By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels.     

Characterization of Chimeric Bacillus thuringiensis Vip3 Toxins

Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.     

Disruption of a Cadherin Gene Associated with Resistance to Cry1Ac δ-Endotoxin of Bacillus thuringiensis in Helicoverpa armigera

A laboratory strain (GY) of Helicoverpa armigera (Hübner) was established from surviving larvae collected from transgenic cotton expressing a Bacillus thuringiensis var. kurstaki insecticidal protein (Bt cotton) in Gaoyang County, Hebei Province, People's Republic of China, in 2001. The GYBT strain was derived from the GY strain through 28 generations of selection with activated Cry1Ac delivered by diet surface contamination. When resistance to Cry1Ac in the GYBT strain increased to 564-fold after selection, we detected high levels of cross-resistance to Cry1Aa (103-fold) and Cry1Ab (>46-fold) in the GYBT strain with reference to those in the GY strain. The GYBT strain had a low level of cross-resistance to B. thuringiensis var. kurstaki formulation (Btk) (5-fold) and no cross-resistance to Cry2Aa (1.4-fold). Genetic analysis showed that Cry1Ac resistance in the GYBT strain was controlled by one autosomal and incompletely recessive gene. The cross-resistance pattern and inheritance mode suggest that the Cry1Ac resistance in the GYBT strain of H. armigera belongs to “mode 1,” the most common type of lepidopteran resistance to B. thuringiensis toxins. A cadherin gene was cloned and sequenced from both the GY and GYBT strains. Disruption of the cadherin gene by a premature stop codon was associated with a high level of Cry1Ac resistance in H. armigera. Tight linkage between Cry1Ac resistance and the cadherin locus was observed in a backcross analysis. Together with previous evidence found with Heliothis virescens and Pectinophora gossypiella, our results confirmed that the cadherin gene is a preferred target for developing DNA-based monitoring of B. thuringiensis resistance in field populations of lepidopteran pests.     

Dual Insecticidal Activity of Spodoptera-Toxic Bacillus thuringiensis Strain Transformed with Lepidopteran-Specific Cry Toxin

The E. coli-B. thuringiensis shuttle vector for expression of cry1Ac, pHT1K-1Ac plasmid was introduced into acrystalliferous B. thuringiensis Cry⁻B and Spodoptera toxic STB-3 strain. The presence of a recombinant plasmid in transformants after electroporation was confirmed by PCR. The 1K-1Ac/Cry⁻B(Cry⁻B transformant) and 1K-1Ac/STB-3 (STB-3 transformant) produced bipyramidal-shaped parasporal inclusion that was 130 kDa in size as like B. thuringiensis subsp. kurstaki HD-73. In P. xylostella bioassay, these transformants showed significantly high toxicity than the wild-type recipients and further, in case of B. thuringiensis STB-3 transformant still had original Spodoptera toxicity. These results suggested that the pHT1K could be successfully applied for generating individual B. thuringiensis strains that produce various combinations of insecticidal proteins to expand their host spectrum and enhance insecticidal activity.     

Variation in Susceptibility to Bacillus thuringiensis Toxins among Unselected Strains of Plutella xylostella

So far, the only insect that has evolved resistance in the field to Bacillus thuringiensis toxins is the diamondback moth (Plutella xylostella). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.     

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Integrative Model for Binding of Bacillus thuringiensis Toxins in Susceptible and Resistant Larvae of the Diamondback Moth (Plutella xylostella)

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.     

Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris)

Hemipteran pests including aphids are not particularly susceptible to the effects of insecticidal Cry toxins derived from the bacterium Bacillus thuringiensis. We examined the physiological basis for the relatively low toxicity of Cry1Ac and Cry3Aa against the pea aphid, Acyrthosiphon pisum (Harris). Cry1Ac was efficiently hydrolyzed by aphid stomach membrane associated cysteine proteases (CP) producing a 60 kDa mature toxin, whereas Cry3Aa was incompletely processed and partially degraded. Cry1Ac bound to the aphid gut epithelium but showed low aphid toxicity in bioassays. Feeding of aphids on Cry1Ac in the presence or absence of GalNAc, suggested that Cry1Ac gut binding was glycan mediated. In vitro binding of biotinylated-Cry1Ac to gut BBMVs and competition assays using unlabeled Cry1Ac and GalNAc confirmed binding specificity as well as glycan mediation of Cry1Ac binding. Although Cry3Aa binding to the aphid gut membrane was not detected, Cry3Aa bound 25 and 37 kDa proteins in aphid gut BBMV in ligand blot analysis and competition assays confirmed the binding specificity of Cry3Aa. This, combined with low toxicity in feeding assays, suggests that Cry3Aa does bind the gut epithelium to some extent. This is the first systematic examination of the physiological basis for the low efficacy of Cry toxins against aphids, and analysis of Cry toxin-aphid gut interaction.     

Cross-Resistance and Stability of Resistance to Bacillus thuringiensis Toxin Cry1C in Diamondback Moth

We tested toxins of Bacillus thuringiensis against larvae from susceptible, Cry1C-resistant, and Cry1A-resistant strains of diamondback moth (Plutella xylostella). The Cry1C-resistant strain, which was derived from a field population that had evolved resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai, was selected repeatedly with Cry1C in the laboratory. The Cry1C-resistant strain had strong cross-resistance to Cry1Ab, Cry1Ac, and Cry1F, low to moderate cross-resistance to Cry1Aa and Cry9Ca, and no cross-resistance to Cry1Bb, Cry1Ja, and Cry2A. Resistance to Cry1C declined when selection was relaxed. Together with previously reported data, the new data on the cross-resistance of a Cry1C-resistant strain reported here suggest that resistance to Cry1A and Cry1C toxins confers little or no cross-resistance to Cry1Bb, Cry2Aa, or Cry9Ca. Therefore, these toxins might be useful in rotations or combinations with Cry1A and Cry1C toxins. Cry9Ca was much more potent than Cry1Bb or Cry2Aa and thus might be especially useful against diamondback moth.     

Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC₅₀ of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.     

A Bacillus thuringiensis host strain with high melanin production for preparation of light-stable biopesticides

The bacterium Bacillus thuringiensis produces a crystal protein with insecticidal properties; however, crystal proteins can be damaged by ultraviolet (UV) radiation. The aim of this study was to improve the stability of the insecticidal crystal protein (ICP) by constructing a mutant line that expresses high levels of the UV light-protecting pigment, melanin. BMB181, a B. thuringiensis mutant with high melanin production, was obtained after sub-culturing BMB171 for several generations at 42 °C. The melanin yield by BMB181 (without tyrosine supplementation) reached 8.55 mg/ml. The electroporation efficiency of BMB181 reached 10⁶ CFU/μg when a 6.7-kb foreign plasmid was used. Microscopic and SDS-PAGE analyses revealed that ICP (CryIAc10; GenBank: AAA73077.1), which is highly toxic to Lepidoptera, was synthesized efficiently by strain BMB181. The insecticidal properties of a recombinant line derived from strain BMB181, designated BMB32 (cry1Ac10/BMB181), was tested against the cotton bollworm, Helicoverpa armigera. After UV irradiation for 4 h, BMB32 had a half maximal inhibitory concentration value of 1.37 μg/ml, whereas the control line BMB31 (cry1Ac10/BMB171) had a median lethal dose value of 25.85 μg/ml. These results indicate that the B. thuringiensis mutant is a candidate for industrial scale production of light-stable insecticides.     

Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China

A field population (SZ) of Plutella xylostella, collected from the cabbage field in Shenzhen, Guangdong Province of China in 2002, showed 2.3-fold resistance to Cry1Aa, 110-fold to Cry1Ab, 30-fold to Cry1Ac, 2.1-fold to Cry1F, 5.3-fold to Cry2Aa and 6-fold resistance to Bacillus thuringiensis var. kurstaki (Btk) compared with a susceptible strain (ROTH). The SZBT strain was derived from the SZ population through 20 generations of selection with activated Cry1Ac in the laboratory. While the SZBT strain developed 1200-fold resistance to Cry1Ac after selection, resistance to Cry1Aa, Cry1Ab, Cry1F, and Btk increased to 31-, 1900-,>33- and 17-fold compared with the ROTH strain. However, little or no cross-resistance was detected to Cry1B, Cry1C and Cry2Aa in the SZBT strain. Genetic cross analyses between the SZBT and ROTH strains revealed that Cry1Ac-resistance in the SZBT strain was controlled by a single, autosomal, incompletely recessive gene. Binding studies with ¹²⁵I-labeled Cry1Ac showed that the brush border membrane vesicles (BBMVs) of midguts from the resistant SZBT insects had lost binding to Cry1Ac. Allelic complementation tests demonstrated that the major Bt resistance locus in the SZBT strain was same as that in the Cry1Ac-R strain which has “mode 1” resistance to Bt. An F₁ screen of 120 single-pair families between the SZBT strain and three field populations collected in 2008 was carried out. Based on this approach, the estimated frequencies of Cry1Ac-resistance alleles were 0.156 in the Yuxi population from Yunnan province, and 0.375 and 0.472 respectively in the Guangzhou and Huizhou populations from Guangdong province.     

Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag,which facilitates formation of protein inclusion bodies in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A Cry46Ab toxin derived from Bacillus thuringiensis strain TK-E6 shows mosquitocidal activity against Culex pipiens pallens Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity against human cancer cells. In B. thuringiensis cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa, and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. In the present study, the potency of the 4AaCter-tag was investigated for the production of alkali-soluble inclusion bodies of recombinant Cry46Ab in Escherichia coli. The 4AaCter-tag is a polypeptide derived from the C-terminal region of the B. thuringiensis Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in E. coli. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab–4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in E. coli.     

Construction and characterization of the interdomain chimeras using Cry11Aa and Cry11Ba from Bacillus thuringiensis and identification of a possible novel toxic chimera

Three structural domains of mosquitocidal Cry11Aa and Cry11Ba from Bacillus thuringiensis were exchanged to produce interdomain chimeras [BAA (11Ba/11Aa/11Aa), ABA (11Aa/11Ba/11Aa), AAB (11Aa/11Aa/11Ba), ABB (11Aa/11Ba/11Ba), BAB (11Ba/11Aa/11Ba), BBA (11Ba/11Ba/11Aa]. Chimeras BAB, BAA, BBA, and AAB formed inclusion bodies in the crystal-negative B. thuringiensis host and produced expected protein bands on SDS-PAGE gel. However, no inclusion body or target protein could be found for chimeras ABA and ABB. In bioassays using the fourth-instar larvae of Culex quinquefasciatus and Aedes aegypti, AAB had ~50 % lethal concentrations of 4.8 and 2.2 μg ml^?1, respectively; however, the rest of chimeras were not toxic. This study thus helps to understand the domain-function relationships of the Cry11Aa and Cry11Ba toxins. The toxic chimera, AAB, might be a candidate for mosquito control as its amino acid sequence is different from the two parental toxins.     

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22–25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9–11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87–Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.     

Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.     

Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F₄ to F₈) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F₉ found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F₉. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F₉ to F₁₃) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with ¹²⁵I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F₁₅). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F₂ progeny from a backcross of F₁ progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.