Single Amino Acid Mutations in the Cadherin Receptor from Heliothis virescens Affect Its Toxin Binding Ability to Cry1A Toxins

Xie, Ruiyu; Zhuang, Meibao; Ross, Linda S.; Gómez, Isabel; Oltean, Daniela I.; Bravo, Alejandra; Soberón, Mário; Gill, Sarjeet S.

doi:10.1074/jbc.m408403200

Cited by 122 publications

(156 citation statements)

References 46 publications

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“…Mutants-Previously, we reported that loop 3 of Cry1Ab toxin showed a hydropathic complementary pattern with the binding epitope of H. virescens cadherin, suggesting a putative interaction through a complementary hydropathic profile (17). Here we introduce amino acid substitutions in loop 3 of Cry1Ab toxin designed to disrupt the hydropathic profile of this region.…”

Section: Construction Of Cry1ab Loopmentioning

confidence: 99%

“…Insect cadherins are composed of an ectodomain formed by 11 or 12 cadherin repeats (CRs), a transmembrane domain, and an intracellular domain (14). The exposed loops of Cry1A domain II have been directly involved in binding with cadherin in M. sexta, H. virescens, and B. mori (15)(16)(17)(18). Three Cry1Ab binding sites were mapped in CR7, CR11, and CR12 of the M. sexta Bt-R 1 (15,19).…”

mentioning

confidence: 99%

“…Three Cry1Ab binding sites were mapped in CR7, CR11, and CR12 of the M. sexta Bt-R 1 (15,19). The interaction between Cry1Ab/ Cry1Ac toxins and H. virescens cadherin was analyzed, showing that loop 3 of domain II binds to CR12 of this receptor (17). Accordingly, loop 3 of Cry1Aa binds the corresponding site of B. mori cadherin (Bt-R 175 ) (18).…”

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confidence: 99%

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Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

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Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R 1 ) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R 1 promotes cleavage of the amino-terminal end, including helix ␣-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R 1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.Bacillus thuringiensis is a bacterium that produces crystalline inclusions formed by insecticidal proteins, called Cry toxins, during the sporulation phase of growth. Cry toxins are toxic to different insect orders as well as to other invertebrates, such as nematodes, mites, and protozoa (1). Cry toxins have been used worldwide in the control of insect pests in agriculture, either as transgenic crops or as spray formulations.The molecular mechanism proposed to describe the action of Cry1A toxins, which are active against different lepidopteran insect species, involves several steps. After larval ingestion of the crystalline inclusions, these are solubilized in midgut lumen and activated by proteases releasing a toxic 65-kDa fragment that binds, in a sequential manner, with at least two receptors located in midgut microvilli. The first interaction occurs with cadherin protein (Bt-R 1 2 in the case of Manduca sexta). This interaction promotes further proteolytic processing of the N-terminal end, including helix ␣-1 of the toxin, resulting in the formation of a prepore oligomeric structure (2). The oligomer has higher affinity to secondary receptors, which are anchored by glycosylphosphatidylinositol, such as aminopeptidase-N (APN) or alkaline phosphatase in the case of M. sexta or Heliothis virescens, respectively (3, 4). Glycosylphosphatidylinositol-anchored receptors are located in specific membrane regions called lipid rafts, where the oligomer inserts into the membrane-forming pores, disrupting the osmotic equilibrium and leading to cell death (1, 5). Although this mechanism of action is g...

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Section: Construction Of Cry1ab Loopmentioning

confidence: 99%

mentioning

confidence: 99%

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Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Pacheco

Gómez

Arenas

et al. 2009

Journal of Biological Chemistry

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125

135

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“…Also, a major threat for the use of Bt toxins in transgenic plants is the appearance of insect resistance. It has been demonstrated that a single nucleotide change in the Heliothis virescens cadherin receptor gene produces an amino acid change that abolishes Cry1A toxin binding [35]. Therefore the need of developing an efficient method that allows genetic evolution of Cry toxins to kill novel targets or to recover toxicity to resistant insects, in the case of the appearance of resistance in the field, will be greatly desirable.…”

Section: Displaying Cry Toxins In Phages and Selection Of Novel Variantsmentioning

confidence: 99%

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

et al. 2008

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Phage display is an in vitro method for selecting polypeptides with desired properties from a large collections of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display singlechain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.

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“…Additionally, knockout of a b-1,3 galactosyltransferase from the mutagenized Bt resistant 5 (bre5) line of Caenorhabditis elegans resulted in resistance to Bt toxins (Griffitts et al 2001), which reiterated the role that postranslational glycosylation may play in toxin-binding of receptor proteins (Knowles et al 1991;Jurat-Fuentes et al 2002). Mutations in the receptors, aminopeptidase N and cadherin, were also shown to result in larval Bt toxin-resistance traits among species of Lepidoptera (Gahan et al 2001;Morin et al 2003;Herrero et al 2005;Xie et al 2005;Zhang et al 2009). In contrast, analysis of the Cry1Ac-and Cry2Aa-resistant CP73 strain of Heliothis virescens indicated that neither cadherin nor aminopeptidase N receptor genes contributed to resistance traits Heckel et al 2007).…”

Section: Introductionmentioning

confidence: 99%

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

et al. 2011

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The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance. Abstract The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae

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Single Amino Acid Mutations in the Cadherin Receptor from Heliothis virescens Affect Its Toxin Binding Ability to Cry1A Toxins

Cited by 122 publications

References 46 publications

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Domain II Loop 3 of Bacillus thuringiensis Cry1Ab Toxin Is Involved in a “Ping Pong” Binding Mechanism with Manduca sexta Aminopeptidase-N and Cadherin Receptors

Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

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