RESUMEN
Bacillus thuringiensis Cry toxins exert their toxicity by forming membrane pores after binding with larval midgut membrane proteins known as receptors. Spodoptera litura and Spodoptera frugiperda belong to the same genus, but S. litura is tolerant to Cry1Ac, while S. frugiperda is susceptible. The mechanism involved in the differential toxicity of Cry1Ac to these insect species is not understood. Amino acid sequences analysis of ABCC2, a well-recognized Cry1Ac receptor, from both species showed high sequence identity. Hi5 insect cells expressing SfABCC2 from S. frugiperda were 65-fold more susceptible than those expressing the SlABCC2 from S. litura. Substitution of fragments, point mutations and deletions between the ABCC2 of the two species revealed that ABCC2 amino acid Q125 from SfABCC2 or E125 from SlABCC2 was key factor for the differential Cry1Ac toxicity to Hi5 cells expressing these receptors. Consistently with this, cells expressing Helicoverpa armigera HaABCC2Q122-GFP, were more susceptible to Cry1Ac than cells expressing HaABCC2E122-GFP mutant. Q125 or E125 is located in a predicted exposed loop 1 region of ABCC2 indicating that this region could be important for Cry1Ac binding. These findings identified a single amino acid residue located in loop 1 of ABCC2 transporter as responsible for the different levels of susceptibility to Cry1Ac among various lepidopteran species.
Asunto(s)
Proteínas Bacterianas , Endotoxinas , Proteínas Hemolisinas , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Spodoptera/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Línea Celular , Proteínas de Insectos/genética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Polimorfismo de Nucleótido Simple , Especificidad de la EspecieRESUMEN
Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry-SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl-HP cells and partially co-localized with BmATG6-GFP, but it almost has no significant co-localization with GFP-HaATG8. Then, the Western blot analysis demonstrated that GFP-SeATG5 conjugated with ATG12. Moreover, real-time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.
Asunto(s)
Proteína 5 Relacionada con la Autofagia/genética , Regulación de la Expresión Génica , Proteínas de Insectos/genética , Spodoptera/genética , Secuencia de Aminoácidos , Animales , Proteína 5 Relacionada con la Autofagia/química , Proteína 5 Relacionada con la Autofagia/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Perfilación de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Spodoptera/clasificación , Spodoptera/crecimiento & desarrollo , Spodoptera/metabolismoRESUMEN
Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera litura Sl-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the SlABCC3 shared 52-55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of Sl-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SlABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action.