Specific Aims Toxoplasma gondii persists in 25-30% of humans worldwide because there are no vaccines to prevent infections and current therapies are non-curative (1). The first-line therapy for toxoplasmosis consists of pyrimethamine and sulfadiazine, which inhibit intracellular parasite replication but are highly toxic and unable to eliminate the dormant tissue-encysted stage of T. gondii (2). Thus, there is an urgent need for new non-toxic drugs that block T. gondii infectivity in both acute (tachyzoite) and chronic (bradyzoite) life stages. Cyclic guanosine monophosphate (cGMP) has emerged as a master regulator of T. gondii infectivity by coordinating parasite motility (3, 4). Previously, we determined that a T. gondii guanylate cyclase (TgGC) synthesizes cGMP from GTP to stimulate motility for entry and exit of host cells (5). Conditional depletion of TgGC paralyzed T. gondii, rendering parasites incapable of establishing infections and causing disease. Additionally, we determined that a cGMP-dependent protein kinase (TgPKG) at the plasma membrane acts as the central effector of cGMP in T. gondii in both tachyzoites (6) and bradyzoites (7). Chemical inhibition or conditional knockdown of TgPKG phenocopied loss of TgGC, completely blocking parasite motility and infectivity (6, 8). Furthermore, we determined that TgPKG functions by controlling secretion of microneme proteins that are required for parasite motility, host cell invasion, and host cell egress (6). However, the mechanisms by which TgPKG controls microneme secretion and motility remain unclear because its substrates have not been identified or characterized. We hypothesize that TgPKG regulates T. gondii motility and virulence through phosphorylation of proteins that regulate microneme expression and secretion. Here, we will utilize modern genetic, transcriptomic, and proteomic approaches to test our hypothesis with three independent Specific Aims. Specific Aim 1: Identify genes differentially expressed following TgPKG knockdown. TgPKG controls T. gondii infectivity by regulating microneme secretion (6). We speculate that TgPKG directly modifies proteins required for microneme fusion with the plasma membrane. However, TgPKG may also regulate microneme secretion by modulating gene expression since PKGs regulate several transcription factors in other organisms (9). Previously, we generated a T. gondii line that expresses TgPKG fused to an auxin-inducible degron that allows for rapid and robust depletion of TgPKG with auxin treatment (6). Here we will identify genes that are differentially expressed following conditional depletion of TgPKG using RNA-Seq. Specific Aim 2: Identify interactors of TgPKG under basal and activated conditions. As with most kinases, we predict that TgPKG transiently interacts with its protein substrates following activation. We will use liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify TgPKG interactors captur...