Goal: Characterize the composition and functions of the extracellular matrix of listerial biofilms on plants, and elucidate the mechanisms controlling biofilm formation.Objectives1. Decipher the regulatory mechanisms controlling Pss EPS formation in listeria.2. Identify an antibiofilm component in tree extracts and characterize the mechanism of EPS-biofilm inhibition.3. Uncover the origin of EPS-independent appendages and characterize their role in listerial plant biofilms.Listeria monocytogenes is one of the most notorious foodborne pathogens responsible for annual multi-billion dollar economic losses. In the environment, listera grows in plant-associated biofilms, yet our understanding of these biofilms is poor. This knowledge gap may hinder our ability to prevent listerial contamination of fruits and vegetables, which is problematic because the frequency of listeriosis outbreaks caused by contaminated produce has been increasing. Our preliminary data indicate that listerial biofilms on plant matter differ drastically from the biofilms formed on non-plant surfaces. One of the major matrix components of listeria-plant biofilms, the Pss exopolysaccharide, greatly increases tolerance of listeria to desiccation and disinfectants, and may contribute to improved listerial survival on contaminated produce during processing, transportation and storage. The exopolysaccharide-independent extracellular appendages of unknown origin may promote attachment to plant surfaces. In Objective 1 of this project, we will decipher environmental stimuli and regulatory mechanisms controlling exopolysaccharide synthesis and degradation. In Objective 2, we will capitalize on our discovery that some tree species have potent antibiofilm compounds. We will identify these compounds from aqueous wood extracts and elucidate their mechanisms. In Objective 3, we will investigate exopolysaccharide-independent extracellular appendages. We will uncover the origin of these appendages and elucidate their role in plant attachment. As a result, we will gain deeper understanding of the extracellular matrix components that allow listeria to form biofilms on plants, learn how synthesis and degradation of these components are regulated, and how we can prevent listerial biofilm formation on fresh produce.