ABSTRACT
The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.
La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios.
Subject(s)
Bacillus thuringiensis/classification , Bacillus thuringiensis/physiology , Bacillus thuringiensis/immunology , Bacillus thuringiensis/metabolism , Bacillus thuringiensis/chemistry , Mutagenesis, Site-Directed/statistics & numerical data , Mutagenesis, Site-Directed/instrumentation , Mutagenesis, Site-Directed/methods , Mutagenesis, Site-Directed/trends , Mutagenesis, Site-DirectedABSTRACT
La polilla del tomate (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) es una de las plagas más devastadorasdel tomate en Colombia y países suramericanos, produciendo pérdidas de hasta el 100% en cultivos sin protección.En 2009, T. absoluta se detectó en España, Portugal y países del mediterráneo, además de Inglaterra,Bulgaria y Alemania. Para su control se utilizan insecticidas químicos que generan resistencia e impactoambiental y de salud. La alternativa de utilizar biopesticidas contra esta plaga es de importancia creciente. Eneste estudio se evaluaron cinco métodos de bioensayo para medir adecuadamente la toxicidad sobre larvasde T. absoluta de tres productos comerciales: Dipel®, XenTary® y Turilav®, formulaciones a base de Bacillusthuringiensis (Bt). El método de Inmersión del folíolo, con el producto Dipel®, causó el 100% de mortalidadde larvas y 96% de supervivencia del testigo; este método presentó diferencias significativas al segundo(F=0,025, p>0,05) y cuarto (F=0,0018, p>0,05) día después de la aplicación (DDA). El método de Aspersiónfoliar por aerógrafo produjo 100% de mortalidad de larvas con Dipel® al segundo DDA (F=7,94x10-10,p> 0,05), y produjo diferencias significativas también al cuarto DDA (F=3,45x10-6, p>0,05). Los métodosFoliolos sumergidos y Medio de cultivo provocaron una alta mortalidad en el control por lo que fueron rechazados.El uso de Dipel®, XenTari® y Turilav® en concentración de 1,25 g/L causó entre 80-100% demortalidad entre el segundo y octavo DDA en tres métodos evaluados válidos (1, 2, 3), además corrobora laactividad biológica de B. thuringiensis sobre este insecto plaga.
The tomato moth (Tuta absoluta Meyrick; Lepidoptera: Gelechiidae) is one of the most devastating tomatopests in Colombia and South-American countries, producing losses of up to 100% in unprotected crops. T.absoluta was detected in Spain, Portugal and Mediterranean countries in 2009, as well as England, Bulgaria andGermany. Chemical insecticides are used for controlling it; however, they produce resistance and an environmentaland human health impact. Finding an alternative to using biopesticides against this pest is becomingincreasingly important. This study evaluated five bioassay methods measuring three commercial productstoxicity on T. absoluta larvae: Dipel, XenTary and Turilav Bacillus thuringiensis (Bt) -based formulations. The leafdipping bioassay method caused 100% larvae mortality with Dipel, the control group having 95% survivalrate. The other products showed significant differences on the 2nd (F=0.025, p>0.05) and 4th (F=0.0018,p>0.05) days after application (DAA). The leaf spray airbrush method produced 100% larvae mortality withDipel on the 2nd DAA, having significant differences from the other products tested on 2nd (DAA F=7.94 x10-10, p>0.05 ), 4th (F=3.45x10-6, p>0.05 ) and 8th (F=1.07x10-5, p>0.05 ) DAA. Submerged leaflet and culturemedium methods caused high mortality in controls and were thus rejected. A variation of the leaflet immersionmethod was standardised. The three commercial products produced high mortality in Lab conditionsregarding T. absolute larvae control at 1.25 g/L concentration, thereby corroborating the biological activity ofB. thuringiensis against this insect pest.
Subject(s)
Lepidoptera/physiology , Lepidoptera/genetics , Lepidoptera/microbiology , Lepidoptera/parasitology , Lepidoptera/chemistry , Bacillus thuringiensis/physiology , Bacillus thuringiensis/genetics , Bacillus thuringiensis/immunology , Bacillus thuringiensis/chemistryABSTRACT
The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins