PROJECT SUMMARY/ABSTRACT Plasmodesmata (PD), plasma membrane-lined pores in the plant cell walls, allow trafficking of small molecules, macromolecules, metabolites, signaling molecules and including hormones from one plant cell to another. Several hormones have been found to traffic between cells via PD. Interestingly, salicylic acid (SA), a plant defense hormone, does not traffic via PD but through the apoplast. Nonetheless, SA is a critical hormonal signal that negatively regulates cell-to-cell permeability in response to pathogen infection. Previous studies have shown SA to regulate PD through regulated callose accumulation along the pores. A second effect of PD is to induce the insertion of additional pores at existing PD leading to the formation of complex (branched) pores. Because PD are very important for plant growth, development, and defense, we seek to understand the molecular mechanisms involved in regulating PD function and formation. This study will focus on investigating the cellular mechanisms of how SA regulates intercellular trafficking by adjusting PD permeability. These studies will apply a combination of advanced microscopic techniques and cell and molecular biology approaches to study how SA is connected to PD transport in epidermal leaf tissues of Nicotiana benthamiana. The specific aims of the research will determine the effects of SA alone or in combination with another defense hormone, jasmonic acid, on PD structure and function. We will also investigate how the concentration of SA and/or JA change PD permeability based on various times after exposure to the hormone (Aim 1); determine PD structure and the number of PD in SA-treated tissues relative to untreated controls to examine hormone effects on PD formation (Aim 2); determine the effect of SA on endocytosis and ER-PM contact sites as part of the process inserting new pores into existing cell walls, (Aim 3). The results from this research will advance understanding of how hormones influence intercellular trafficking and PD, and they will be of interest to basic plant scientists and agricultural scientists.