Ruiz de Escudero Fuentemilla, Íñigo

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https://academica-e.unavarra.es/handle/2454/49921

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Ruiz de Escudero Fuentemilla

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Íñigo

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Agronomía, Biotecnología y Alimentación

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IMAB. Research Institute for Multidisciplinary Applied Biology

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0000-0002-8698-1481

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88564

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2331

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2006 - 200912010 - 20112
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  • Thumbnail Image
    PublicationOpen Access
    The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles on toxin oligomerization and pore formation
    (American Chemical Society, 2011) Rodríguez Almazán, Claudia; Ruiz de Escudero Fuentemilla, Íñigo; Cantón, Pablo Emiliano; Muñoz Garay, Carlos; Pérez, Claudia; Gill, Sarjeet S.; Soberón, Mario; Bravo, Alejandra; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. Regarding to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects in Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, N-terminal region is able to induce aggregation of Cyt1Aa toxin in solution. Finally, Cterminal domain composed mainly of β-strands, is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation while the C-terminal domain in the interaction of the toxin with the lipid membrane.
  • Thumbnail Image
    PublicationOpen Access
    Potential of the Bacillus thuringiensis toxin reservoir for the control of Lobesia botrana (Lepidoptera: tortricidae), a major pest of grape plants
    (American Society for Microbiology, 2006) Ruiz de Escudero Fuentemilla, Íñigo; Estela, Anna; Escriche, Baltasar; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua, FP2000-5497
    The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.
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    PublicationOpen Access
    Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism
    (Elsevier, 2011) Cantón, Pablo Emiliano; Zanicthe Reyes, Esmeralda; Ruiz de Escudero Fuentemilla, Íñigo; Bravo, Alejandra; Soberón, Mario; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop 6–E K198A, E204A and 7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop -8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.