Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins.

Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.     

Role of Bacillus thuringiensis Toxin Domains in Toxicity and Receptor Binding in the Diamondback Moth

The toxic fragment of Bacillus thuringiensis crystal proteins consists of three distinct structural domains. There is evidence that domain I is involved in pore formation and that domain II is involved in receptor binding and specificity. It has been found that, in some cases, domain III is also important in determining specificity. Furthermore, involvement of domain III in binding has also been reported recently. To investigate the role of toxin domains in the diamondback moth (Plutella xylostella), we used hybrid toxins with domain III substitutions among Cry1C, Cry1E, and Cry1Ab. Neither Cry1E nor G27 (a hybrid with domains I and II from Cry1E and domain III from Cry1C) was toxic, whereas Cry1C and F26 (the reciprocal hybrid) were equally toxic. H04 (a hybrid with domains I and II from Cry1Ab and domain III from Cry1C) showed toxicity that was of a similar level as that of Cry1Ab and significantly higher than that of Cry1C. Binding assays with ¹²⁵I-Cry1C showed that Cry1C and F26 competed for the same binding sites on midgut membrane vesicles, whereas Cry1E, G27, and H04 did not bind to these sites. Our results show that, in contrast to findings in other insects for the toxins and hybrids used here, toxin specificity as well as specificity of binding to membrane vesicles in the diamondback moth is mediated by domain II (and/or I) and not by domain III.     

Enhancement of insecticidal activity of Bacillus thuringiensis Cry1A toxins by fragments of a toxin-binding cadherin correlates with oligomer formation

Cry1A toxins produced by Bacillus thuringiensis bind a cadherin receptor that mediates toxicity in different lepidopteran insect larvae. Insect cadherin receptors are modular proteins composed of three domains, the ectodomain formed by 9-12 cadherin repeats (CR), the transmembrane domain and the intracellular domain. Cry1A toxins interact with three regions of the Manduca sexta cadherin receptor that are located in CR7, CR11 and CR12 cadherin repeats. Binding of Cry1A toxin to cadherin induces oligomerization of the toxin, which is essential for membrane insertion. Also, it has been reported that cadherin fragments containing the CR12 region enhanced the insecticidal activity of Cry1Ab toxin to M. sexta and other lepidopteran larvae. Here we report that cadherin fragments corresponding to CR7 and CR11 regions also enhanced the activity of Cry1Ac and Cry1Ab toxin to M. sexta larvae, although not as efficient as the CR12 fragment. A single point mutation in the CR12 region (I1422R) affected Cry1Ac and Cry1Ab binding to the cadherin fragments and did not enhance the activity of Cry1Ab or Cry1Ac toxin in bioassays. Analysis of Cry1Ab in vitro oligomer formation in the presence of wild type and mutated cadherin fragments showed a correlation between enhancement of Cry1A toxin activity in bioassays and in vitro Cry1Ab-oligomer formation. Our data shows that formation of Cry1A toxin oligomer is in part responsible for the enhancement of Cry1A toxicity by cadherin fragments that is observed in vivo.     

Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids

In order to test our hypothesis that Bacillus thuringiensis delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for Spodoptera exigua, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against S. exigua when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of S. exigua specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against S. exigua or Manduca sexta, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for M. sexta.     

Bacillus thuringiensis Delta-Endotoxin Cry1C Domain III Can Function as a Specificity Determinant for Spodoptera exigua in Different, but Not All, Cry1-Cry1C Hybrids

In order to test our hypothesis that Bacillus thuringiensis delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for Spodoptera exigua, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against S. exigua when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of S. exigua specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against S. exigua or Manduca sexta, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for M. sexta.     

Importance of Cry1 delta-endotoxin domain II loops for binding specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with (125)I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K(com)] = 1.1 nM) for (125)I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for (125)I-Cry1Ab binding sites, though the K(com) values ranged from 179 to 304 nM. Cry1Ab competed for (125)I-Cry1Ac binding sites (K(com) = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the (125)I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.     

Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed to the solvent after insertion of part of domain I into the membrane

Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.     

A strategy for shuffling numerous Bacillus thuringiensis crystal protein domains

Bacillus thuringiensis that produce Cry1Ba are toxic to Lucilia cuprina Wiedemann blow fly maggots in vivo, and when applied in quantity to sheep fleece, provide up to 6 wk protection against flystrike in the field. These strains also are toxic to Epiphyas postvittana (Walker) light brown apple moth caterpillars. B. thuringiensis expressing Cry1Db are toxic only to E. postvittana. When Cry1Ba and Cry1Db proteins are expressed within Escherichia coli, the recombinant bacteria have the same toxicity profile as the wild-type B. thuringiensis strain. In an effort to develop a Cry protein with improved blow fly toxicity, three different internal regions of Cry1Ba coding DNA, encoding all or part of domains I, II and III respectively were systematically exchanged with the corresponding region from a pool of other Cry protein coding DNAs. The chimeric products were then expressed in recombinant E. coli, and the resulting bacteria assayed for toxicity on L. cuprina and E. postvittana. Clones having insecticide bioactivity were characterized to identify the source of the replacement Cry domain. Despite successfully expressing a large number and variety of chimeric proteins within E. coli, many with measurable insecticidal activity, none of the chimeras had greater potency against L. cuprina than the wild-type Cry1Ba. Chimeric replacements involving domains I and II were rarely active, whereas a much higher proportion of domain III chimeras had some bioactivity. We conclude that shuffling of Cry coding regions through joining at the major conserved sequence motifs is an effective means for the production of a diverse number of chimeric Cry proteins but that such toxins with enhanced bioactive properties will be rare or nonexistent.     

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+²³⁴Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+²³⁴Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.     

The role of Bacillus thuringiensis Cry1C and Cry1E separate structural domains in the interaction with Spodoptera littoralis gut epithelial cells

The Bacillus thuringiensis delta-endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher Bmax and lower Kd than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.     

Bacillus thuringiensis delta-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids

We investigated the role of domain III of Bacillus thuringiensis delta-endotoxin Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this way Cry1Ca, Cry1Fb, and to a lesser extent Cry1Ba were made considerably more toxic.     

Effect of insect larval midgut proteases on the activity of Bacillus thuringiensis Cry toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.     

Differential effects of pH on the pore-forming properties of Bacillus thuringiensis insecticidal crystal toxins

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.     

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins.     

Toxicity and Receptor Binding Properties of Bacillus thuringiensisδ-Endotoxins to the Midgut Brush Border Membrane Vesicles of the Rice Leaf Folders, Cnaphalocrocis medinalis and Marasmia patnalis

Pesticidal activity and receptor-binding properties of Bacillus thuringiensis toxins to rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis, were investigated. Saturation and competition binding experiments were done with iodine (¹²⁵1)-labeled Bt proteins and brush border membrane vesicles prepared from the midgut of C. medinalis and M. patnalis. The results show saturable, specific, and high-affinity binding of all toxins except Cry2A toxin. Cry1Aa and Cry2A toxins were bound with low affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab, and Cry1Ac recognized or shared the same binding site that is different from the binding site for Cry2A toxin. Iodine (¹²⁵I)-labeled Cry1Ac and Cry1Ab toxins were used in ligand blot experiments to detect specific binding proteins in brush border membrane vesicles of C. medinalis and M. patnalis. Cry1Ab toxin protein binds to 205-kDa and 200-kDa proteins respectively in case of C. medinalis and M. patnalis. The apparent molecular mass of the protein bound to labeled Cry1Ac toxins was identified as a 120-kDa protein in both C. medinalis and M. patnalis. Received: 10 April 2000 / Accepted: 23 May 2000     

Effect of Insect Larval Midgut Proteases on the Activity of Bacillus thuringiensis Cry Toxins

To test the possibility that proteolytic cleavage by midgut juice enzymes could enhance or inhibit the activity of Bacillus thuringiensis insecticidal toxins, once activated, the effects of different toxins on the membrane potential of the epithelial cells of isolated Manduca sexta midguts in the presence and absence of midgut juice were measured. While midgut juice had little effect on the activity of Cry1Aa, Cry1Ac, Cry1Ca, Cry1Ea, and R233A, a mutant of Cry1Aa from which one of the four salt bridges linking domains I and II of the toxin was eliminated, it greatly increased the activity of Cry1Ab. In addition, when tested in the presence of a cocktail of protease inhibitors or when boiled, midgut juice retained almost completely its capacity to enhance Cry1Ab activity, suggesting that proteases were not responsible for the stimulation. On the other hand, in the absence of midgut juice, the cocktail of protease inhibitors also enhanced the activity of Cry1Ab, suggesting that proteolytic cleavage by membrane proteases could render the toxin less effective. The lower toxicity of R233A, despite a similar in vitro pore-forming ability, compared with Cry1Aa, cannot be accounted for by an increased susceptibility to midgut proteases. Although these assays were performed under conditions approaching those found in the larval midgut, the depolarizing activities of the toxins correlated only partially with their toxicities.     

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa,Cry1Ab,Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests,Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with ¹²⁵I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.     

Differential Effects of pH on the Pore-Forming Properties of Bacillus thuringiensis Insecticidal Crystal Toxins

The effect of pH on the pore-forming ability of two Bacillus thuringiensis toxins, Cry1Ac and Cry1C, was examined with midgut brush border membrane vesicles isolated from the tobacco hornworm, Manduca sexta, and a light-scattering assay. In the presence of Cry1Ac, membrane permeability remained high over the entire pH range tested (6.5 to 10.5) for KCl and tetramethylammonium chloride, but was much lower at pH 6.5 than at higher pHs for potassium gluconate, sucrose, and raffinose. On the other hand, the Cry1C-induced permeability to all substrates tested was much higher at pH 6.5, 7.5, and 8.5 than at pH 9.5 and 10.5. These results indicate that the pores formed by Cry1Ac are significantly smaller at pH 6.5 than under alkaline conditions, whereas the pore-forming ability of Cry1C decreases sharply above pH 8.5. The reduced activity of Cry1C at high pH correlates well with the fact that its toxicity for M. sexta is considerably weaker than that of Cry1Aa, Cry1Ab, and Cry1Ac. However, Cry1E, despite having a toxicity comparable to that of Cry1C, formed channels as efficiently as the Cry1A toxins at pH 10.5. These results strongly suggest that although pH can influence toxin activity, additional factors also modulate toxin potency in the insect midgut.     

Toxicity of Bacillus thuringiensis delta-endotoxins against bean shoot borer (Epinotia aporema Wals.) larvae, a major soybean pest in Argentina

The toxicity of seven Bacillus thuringiensis Cry protoxins was tested against neonate larvae of Epinotia aporema, a major soybean pest in Argentina and South America. The most active protoxins were Cry1Ab and Cry1Ac, with LC50 values of 0.55 and 1.39 microg/ml, respectively. Cry1Aa, Cry1Ba, Cry1Ca, and Cry9Ca protoxins were equally toxic with LC50 values about 4 microg/ml, whereas Cry1Da was not toxic. The synergistic activity of different protoxin-mixtures was also analyzed, no synergistic effect between the Cry proteins was observed, with the exception of the poorly toxic Cry1Ba/Cry1Da mixture that was slightly synergistic. The binding capacity of individual Cry1 and Cry9Ca toxins to brush border membranes of E. aporema was also determined. The non-toxic Cry1Da toxin was the only toxin unable to bind to E. aporema membranes. In addition the heterologous competition experiments showed that Cry1Ab and Cry1Ac toxins share a common binding site. Based on these data, we propose that Cry1Ab and Cry1Ac toxins could be used in the biological control of E. aporema.     

Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with ¹²⁵I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K_com] = 1.1 nM) for ¹²⁵I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for ¹²⁵I-Cry1Ab binding sites, though the K_com values ranged from 179 to 304 nM. Cry1Ab competed for ¹²⁵I-Cry1Ac binding sites (K_com = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the ¹²⁵I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.