! PROJECT SUMMARY The eukaryotic actin cytoskeleton is required for the function of numerous cellular processes, including response to stress, injury, and disease. At present, we know almost nothing about the link(s) between changes in actin cytoskeletal dynamics and the regulation of gene expression. Our long-term goals are to define how pathogens disrupt the stochastic behavior of the host actin cytoskeleton to cause disease. To achieve this, we are using pathogens, and their associated virulence factors, to probe and define the mechanisms associated with the cellular organization of the actin cytoskeleton, including the immune signaling processes linked to actin cytoskeletal dynamics. Using this approach, we will not only gain insight into the cellular processes required for immunity, but the mechanisms by which perturbations in cytoskeletal function give rise to disease. Previous work from my research group has uncovered the actin-based mechanisms used by plants and pathogens to resist infection and to cause disease, respectively. Based on our published and preliminary data, we can propose the following central hypothesis: Pathogens directly target the host actin cytoskeleton to disrupt the nucleocytoplasmic movement of actin and members of the ADF/cofilin family of proteins. In support of this hypothesis, we have identified two key events that underpin the transition from the naïve homeostatic function of the cytoskeleton to an immune-activated signaling platform. First, we have identified a bacterial pathogen effector protein that cleaves and inactivates the ADF4 kinase, CPK3. Second, we have identified an interaction within the nucleus between ADF4 and 4 members of a stress-associated transcription factor family. To define the immunity and pathogen virulence mechanisms associated with these events, we propose to pursue three Specific Aims to characterize the nucleocytoplasmic regulation of actin: (1) Characterize ADF4/CPK3 phosphorylation dynamics during immune signaling; (2) Define the organization dynamics of the actin cytoskeleton; and (3) Characterize the function and activity of actin and ADF4 immune-associated regulons. In total, our proposed research will impact the field by identifying and characterizing the processes required for immunity and disease, including uncovering the mechanisms required for actin cytoskeletal function. We posit that the work described herein will lead to the discovery of novel mechanisms underpinning pathogen virulence and innate immunity in both plants and animals. !