|Schetelig, Marc Florian|
|Handler, Alfred - Al|
Submitted to: International Congress On Apoptosis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2011
Publication Date: 8/1/2011
Citation: Schetelig, M.A., Xavier, N., Handler, A.M. 2011. Pro-apoptotic cell death genes, hid and reaper, from the tephritid pest species, Anastrepha suspensa. International Congress On Apoptosis. 16(8):759-768. Interpretive Summary: The ability to create transgenic strains of economically and medically important insect species has the potential to greatly improve existing biological control methods, which is a major goal of our laboratory at the Center for Medical, Agricultural and Veterinary Entomology, USDA, Agricultural Research Service, Gainesville, FL. Development of effective and safe transgenic insect strains for biological control will depend on robust and reliable expression of lethal effectors isolated from specific pest insects. Pro-apoptotic genes, which are the primary regulators of apoptosis, are a group of such potential lethal effectors. But thus far these genes have been found only in very few insect species. To both broaden our knowledge of insect cell death, and isolate potential lethal effectors for transgenic biological control systems, hid and reaper genes were isolated from the tephritid species, Anastrepha suspensa, and functionally characterized in insect cell cultures. Functional cell death assays demonstrate the cell death activity of these two genes as well as the importance of two cell death motifs present in their predicted protein sequence. This appears to be the first report of cell death genes from a tephritid fruit fly and will be critical in creating transgenic strains for a safe environment-friendly biological control system.
Technical Abstract: Pro-apoptotic proteins from the reaper, hid, grim (RHG) family are primary regulators of programmed cell death in Drosophila due to their antagonistic effect on inhibitor of apoptosis (IAP) proteins, thereby releasing IAP inhibition of caspases that effect apoptosis. Using a degenerate PCR approach to conserved domains from the 12 Drosophila species genome project, we have identified the first reaper and hid orthologs from a tephritid insect, the Caribbean fruit fly, Anastrepha suspensa. As-hid is the first identified non-drosophilid homolog of hid, and As-rpr is the second non-drosophilid rpr homolog. Both genes share more than 50% amino acid sequence identity with their Drosophila homologs, suggesting that insect pro-apoptotic peptides may be more conserved than previously anticipated. Importantly, both genes encode the conserved IBM and GH3 motifs that are key for IAP-inhibition and mitochondrial localization. Functional verification of both genes as cell death effectors was demonstrated by cell death assays in A. suspensa embryonic cell culture, as well as in heterologous Drosophila melanogaster S2 cells. Notably, heterologous cell death activity was found to be higher for the Anastrepha genes than their Drosophila counterparts. In common with the Drosophila cognates, As-hid and As-rpr negatively regulated the Drosophila inhibitor of apoptosis (DIAP1) gene to promote apoptosis, and both genes were synergistic in effecting increased cell death activity when used in concert versus individually, indicating a cooperative function for As-Hid and As-Rpr. We show that these tephritid cell death genes are functional and potent as cell death effectors, and could be used to design improved transgenic lethality systems for insect population control.