Specific aims Transposons are major genome constituents with the potential to trigger genome instability and disease, or generate beneficial genetic diversity that can drive evolution. The interplay between mobile elements and the ?immobile? genome is controlled by the piRNA pathway, which has a conserved role in transposon silencing during germline development. This pathway must respond to new genome invaders, distinguish piRNA source loci from genes, and modulate transposition in response to stress. The three aims address these critical, but poorly understood, processes. Aim 1. How does the germline respond to genome invasion? piRNA control of established transposon families has been extensively studies, but the response to new genome invaders is poorly understood. The KoRV-A gammaretrovirus is currently invading the koala germline, and progressing in a north to south sweep through the wild population. Gammaretroviruses produce spliced Env mRNAs and unspliced transcript which encode Gag, Pol, and the viral genome. Our initial analysis this process indicates that these unspliced transcripts, which are essential to retroviral replication, are directed to the piRNA biogenesis machinery and processed into sense strand piRNAs. Significantly, we find that selective processing of unspliced proviral transcripts is conserved from flies to placental mammals. These findings suggerst that inefficient proviral transcript splicing generates molecular ?pattern? that triggers an innate genome defense response, which is followed by adaptive genome immunity, mediated by anti-sense piRNAs. These model will be tested through extended analysis of theKoRV-A/koala system, and mechanistic studies in the tractable fly model. Aim 2. How are piRNA source loci defined? piRNAs are sequence specific guides in the germline transposon silencing system, and the specificity of this genome immune system is defined by the genomic loci that produce piRNA precursors. From flies to mice, transposon mapping piRNAs are produced from both long non-coding ?clusters? containing transposon fragments, and a subset of euchromatic transposon insertions. In flies, these loci are marked by the Rhino-Deadlock-Cuff (RDC) complex, but how the RDC is specifically localized to these sites is not understood. Studies under this aim will define the genetic and epigenetic mechanisms that localize the RDC, and specify where piRNA precursors are made. Aim 3. How does the piRNA pathway respond to stress? McClintock discovered transposition as a response to chromosome breaks, and transposon mobilization is a driving force in evolution, suggesting a critical role in adaption to stress. To generate heritable benefits, stress induced transposon activation must occur in the germline, but how the piRNA pathway responds to stress is not understood. Our preliminary studies, and work from several other labs, point to a critical role for Checkpoint kinase 2 (Chk2) in DNA damage control of the piRNA biogenesis machinery, and we find that heat shock induces rapid, reversible disassembly of chromatin organization at piRNA source loci. This aim focuses on characterization of these stress responses, and uses reversible disruption of the biogenesis machinery by heat shock to probe the mechanisms controlling piRNA cluster chromatin assembly and de novo initiation of piRNA production.