ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth

Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix alpha-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.National Research Initiative [2008-03980]; DGAPA/UNAM [IN201515]; CONACYT [179977]Open access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Bacillus thuringiensis Cry1Ab Domain III β-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith)

Spodoptera frugiperdais an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest.Bacillus thuringiensis(Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against differentS. frugiperdapopulations. Thus, the identification of Cry1A mutants that gain toxicity toS. frugiperdaand retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III β-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against differentS. frugiperdapopulations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.</jats:p

Summary of binding and oligomerization of Cry1Ac and Cry1AcMod incubated with BBMV from susceptible and resistant <i>P</i>. <i>gossypiella</i>.

<p>Summary of binding and oligomerization of Cry1Ac and Cry1AcMod incubated with BBMV from susceptible and resistant <i>P</i>. <i>gossypiella</i>.</p

Binding and Oligomerization of Modified and Native Bt Toxins in Resistant and Susceptible Pink Bollworm

<div><p>Insecticidal proteins from <i>Bacillus thuringiensis</i> (Bt) are used extensively in sprays and transgenic crops for pest control, but their efficacy is reduced when pests evolve resistance. Better understanding of the mode of action of Bt toxins and the mechanisms of insect resistance is needed to enhance the durability of these important alternatives to conventional insecticides. Mode of action models agree that binding of Bt toxins to midgut proteins such as cadherin is essential for toxicity, but some details remain unresolved, such as the role of toxin oligomers. In this study, we evaluated how Bt toxin Cry1Ac and its genetically engineered counterpart Cry1AcMod interact with brush border membrane vesicles (BBMV) from resistant and susceptible larvae of <i>Pectinophora gossypiella</i> (pink bollworm), a global pest of cotton. Compared with Cry1Ac, Cry1AcMod lacks 56 amino acids at the amino-terminus including helix α-1; previous work showed that Cry1AcMod formed oligomers <i>in vitro</i> without cadherin and killed <i>P</i>. <i>gossypiella</i> larvae harboring cadherin mutations linked with >1000-fold resistance to Cry1Ac. Here we found that resistance to Cry1Ac was associated with reduced oligomer formation and insertion. In contrast, Cry1AcMod formed oligomers in BBMV from resistant larvae. These results confirm the role of cadherin in oligomerization of Cry1Ac in susceptible larvae and imply that forming oligomers without cadherin promotes toxicity of Cry1AcMod against resistant <i>P</i>. <i>gossypiella</i> larvae that have cadherin mutations.</p></div

Boletín de Segovia: Número 52 - 1849 mayo 2

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Specific binding of Cry1Ac and Cry1AcMod toxins to BBMV from resistant (AZP-R) and susceptible (APHIS-S) larvae of <i>P</i>. <i>gossypiella</i>.

<p>The left lane of each pair shows total binding with no competitor (-). The right lane shows non-specific binding performed in the presence of 1000-fold excess of unlabeled competitor (Cry1Ac or Cry1AcMod, respectively) (+). The numbers under the bands with competitor (+) represent the optical density of the bands relative to the corresponding bands on the left without competitor (-) (set to 100). Total binding minus non-specific binding equals specific binding.</p

Oligomerization of Cry1Ac in the presence of BBMV from resistant and susceptible larvae of <i>P</i>. <i>gossypiella</i>.

<p>Cry1Ac activated toxin (0.5 μg) was incubated with BBMV (15 μg) from <i>P</i>. <i>gossypiella</i> susceptible APHIS-S (lane 5), resistant AZP-R (lane 6) separated by SDS-PAGE after three min heating at 50°C and revealed in western blot using anti-Cry1Ac antibody. Lane 1, shows MW markers; lane 2, Cry1Ac activated toxin; lane 3, BBMV from APHIS-S; lane 4, BBMV from AZP-R. Numbers under the 200 kDa bands represent the percentage of pixels relative to the 200 kDa band of APHIS-S BBMV with Cry1Ac toxin that corresponds to 100, calculated after scanning densitometry of the bands.</p

Cadherin detection in resistant and susceptible strains of <i>Pectinophora gossypiella</i>.

<p>BBMV from susceptible APHIS-S larvae (lanes 1 and 4) and resistant AZP-R larvae (lanes 2 and 5) were separated by SDS-PAGE. We detected cadherin using anti-cadherin antibodies anti-CAD229 (lanes 1 and 2) or anti-CAD230 (lanes 4 and 5). Lane 3 shows the biotinylated molecular weight markers.</p