RESUMO
Studies on the transcriptional response to pathogens in the insect larval gut have shown the regulation of several genes after the infection. Repat (REsponse to PAThogens) genes were first identified in Spodoptera exigua midgut as being up-regulated in response to the exposure to Bacillus thuringiensis toxins and baculovirus. Recently, new members of the REPAT family showed a constitutive up-regulation in a B. thuringiensis-resistant population. Based on a yeast two-hybrid screening, we have detected the interaction of REPAT1 with other members of the REPAT family, leading to the discovery of a new member: REPAT8. The functional role of this interaction was shown by following the changes of the subcellular localization of REPAT1 in the presence of REPAT8. REPAT1 alone was localized exclusively in the cytoplasm, while the presence of REPAT8 led to the migration of REPAT1 to the nucleus. Finally, analysis of the expression pattern of eight REPAT members has shown that B. thuringiensis-related treatments (Cry1Ca toxin, Xentari™ product and an acrystalliferous strain) induced a general up-regulation of repat genes, especially of repat2. In contrast, no significant effect was detected after treatment with Escherichia coli or Enterococcus sp., or by the presence of microbiota in the midgut. The results suggest that the different repat genes play different roles in response to pathogens.
Assuntos
Bacillus thuringiensis/fisiologia , Interações Hospedeiro-Patógeno/genética , Proteínas de Insetos/metabolismo , Família Multigênica , Spodoptera/genética , Spodoptera/microbiologia , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes de Insetos/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/genética , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Frações Subcelulares/metabolismoRESUMO
Repat (REsponse to PAThogens) genes were first identified in the midgut of Spodoptera exigua (Lepidoptera: Noctuidae) in response to Bacillus thuringiensis and baculovirus exposure. Since then, additional repat gene homologs have been identified in different studies. In this study the comprehensive larval transcriptome from S. exigua was analyzed for the presence of novel repat-homolog sequences. These analyses revealed the presence of at least 46 repat genes in S. exigua, establishing a new gene superfamily in this species. Phylogenetic analysis and studies of conserved motifs in these hypothetical proteins have allowed their classification in two main classes, αREPAT and ßREPAT. Studies on the transcriptional response of repat genes have shown that αREPAT and ßREPAT differ in their sequence but also in the pattern of regulation. The αREPAT were mainly regulated in response to the Cry1Ca toxin from B. thuringiensis but not to the increase in the midgut microbiota load. In contrast, ßREPAT were neither responding to Cry1Ca toxin nor to midgut microbiota. Differential expression between midgut stem cells and the whole midgut tissue was studied for the different repat genes revealing changes in the gene expression distribution between midgut stem cells and midgut tissue in response to midgut microbiota. This high diversity found in their sequence and in their expression profile suggests that REPAT proteins may be involved in multiple processes that could be of relevance for the understanding of the insect gut physiology.