Abstract Purinergic signaling appears to be an ancient, conserved trait; for example, P2X receptors trace back one billion years in evolution. However, although virtually all organisms respond to extracellular ATP (eATP), canonical P2X and P2Y receptors appear absent in insects, roundworm and higher plants. Recently, the first plant, purinergic receptor, DORN1, was identified, which defines a new receptor kinase family of purinoreceptors (P2K). This receptor mediates the primary signaling response to eATP, eliciting many responses that are comparable to an animal inflammatory response. Similar to some mammalian, purinergic receptors, DORN1 appears to monitor plasma membrane association with the extracellular matrix, as well as recognition of eATP. Convergent evolution has resulted in different receptors for eATP in plants and animals but the question remains whether other components of the purinergic signaling pathway may be conserved. Preliminary results identify interesting parallels between the animal and plant eATP responses. In Aim 1, we will seek to determine the mechanisms by which DORN1 mediates responses to eATP. For example, in both animals and plants, eATP addition triggers the production of reactive oxygen species. Recent data indicates that DORN1 directly phosphorylates NADPH oxidase (RBOHD), which is the enzyme producing ROS. Aim 1, study 1 will focus on further defining the importance of DORN1-RBOHD interaction. Study 2 describes experiments to define the role of the DORN1 RGD binding sites, which appear to mediate interaction between the plasma membrane and extracellular matrix, analogous to the situation in mammals. DORN1 functions as part of a membrane protein complex, whose components will be identified (Study 3). Animals have multiple P2X and P2Y receptors and this is also the case in plants. Indeed, a second, putative eATP receptor, DORN2, has been identified and Specific Aim 2, Study 4 will focus on characterizing the biochemical features and functional role of this receptor. There is a significant overlap in the plant transcriptional response to eATP and a variety of biotic and abiotic stresses. Hence, it seems likely that purinergic signaling plays a variety of important roles in plants; again mirroring the diverse roles that eATP signaling plays in mammals. Experiments are described to identify additional components of the plant purinergic response pathway by characterization of mutants defective in their response to nucleotide treatment, as well as by identification of specific mutations that result in suppression of the dorn1 mutant phenotype (Study 5). The ultimate goal will be a holistic and integrated view of plant purinergic signaling, beginning with the novel P2K receptors, with definition of plant- specific and, more importantly, plant and animal conserved components. This comparative approach should broaden our knowledge of purinergic signaling in higher organisms, hopefully aiding the design of therapeutic treat...