Contribution of the transcription factor SfGATAe to Bt Cry toxin resistance in Spodoptera frugiperda through reduction of ABCC2 expression.

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.

Helicoverpa armigera GATAe transcriptional factor regulates the expression of Bacillus thuringiensis Cry1Ac receptor gene ABCC2 by its interplay with additional transcription factors.

Helicoverpa armigera is a worldwide pest that has been efficiently controlled by transgenic plants expressing Bt Cry toxins. To exert toxicity, Cry toxins bind to different receptors located in larval midgut cells. Previously, we reported that GATA transcription factor GATAe activates the expression of multiple H. armigera Cry1Ac receptors in different insect cell lines. Here, the mechanism involved in GATAe regulation of HaABCC2 gene expression, a key receptor of Cry1Ac, was analyzed. HaGATAe gene silencing by RNAi in H. armigera larvae confirmed the activation role of HaGATAe on the expression of HaABCC2 in the midgut. The contribution of all potential GATAe-binding sites was analyzed by site-directed mutagenesis using Hi5 cells expressing a reporter gene under regulation of different modified HaABCC2 promoters. DNA pull-down assays revealed that GATAe bound to different predicted GATA-binding sites and mutations of the different GATAe-binding sites identified two binding sites responsible for the promoter activity. The binding site B9, which is located near the transcription initiator site, has a major contribution on HaABCC2 expression. Also, DNA pull-down assays revealed that all other members of GATA TF family in H. armigera, besides GATAe, HaGATAa, HaGATAb, HaGATAc and HaGATAd also bound to the HaABCC2 promoter and decreased the GATAe dependent promoter activity. Finally, the potential participation in the regulation of HaABCC2 promoter of several TFs other than GATA TFs expressed in the midgut cells was analyzed. HaHR3 inhibited the GATAe dependent activity of the HaABCC2 promoter, while two other midgut-related TFs, HaCDX and HaSox21, also bound to the HaABCC2 promoter region and increased the GATAe dependent promoter activity. All these data showed that GATAe induces HaABCC2 expression by binding to HaGATAe binding sites in the promoter region and that additional TFs participate in modulating the HaGATAe-driven expression of HaABCC2.

Novel roles for GATAe in growth, maintenance and proliferation of cell populations in the Drosophila renal tubule.

The GATA family of transcription factors is implicated in numerous developmental and physiological processes in metazoans. In Drosophila melanogaster, five different GATA factor genes (pannier, serpent, grain, GATAd and GATAe) have been reported as essential in the development and identity of multiple tissues, including the midgut, heart and brain. Here, we present a novel role for GATAe in the function and homeostasis of the Drosophila renal (Malpighian) tubule. We demonstrate that reduced levels of GATAe gene expression in tubule principal cells induce uncontrolled cell proliferation, resulting in tumorous growth with associated altered expression of apoptotic and carcinogenic key genes. Furthermore, we uncover the involvement of GATAe in the maintenance of stellate cells and migration of renal and nephritic stem cells into the tubule. Our findings of GATAe as a potential master regulator in the events of growth control and cell survival required for the maintenance of the Drosophila renal tubule could provide new insights into the molecular pathways involved in the formation and maintenance of a functional tissue and kidney disease.

Cell-Specific Imd-NF-κB Responses Enable Simultaneous Antibacterial Immunity and Intestinal Epithelial Cell Shedding upon Bacterial Infection.

Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding.

GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult midgut.

Adult intestinal tissues, exposed to the external environment, play important roles including barrier and nutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adult intestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its first-generated knock-out mutant as well as cell type-specific RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell regulation.