The goal of this project is to pool the expertise of the PIs in nanotechnology, spectroscopic biosensing, mechanical engineering, physics, plant molecular biology and biochemistry and develop a novel enzyme-enabled Raman imaging (E2RSI) technology to molecularly image for the first time the extracellular structural components of plants at a spatial resolution of ~15 nm. The technology will generate highly resolved 3-D maps of the molecular organization of the polysaccharide and lipid components of the extracellular matrix. Specifically, the plant cell wall, cutin and the interface between these two critical polymeric components of the extracellular matrix will be assessed. We will apply this new imaging technique to further the understanding of mechanisms that integrate plant growth and development with cell shaping processes and how microbes interact with plant cells to enzymatically breach the integrity of the extracellular matrix. Our key approach is to develop a nano-scale tipped-enhanced Raman spectroscopic (TERS) imaging technique with accurate tip positioning to track the in situ enzymatic hydrolysis of cell wall polysaccharides and cutin lipids within the extracellular matrix. A broad set of Arabidopsis transgenic plants with well-characterized cell wall modifications will be utilized for the acquisition of reference Raman spectra that will validate the new datasets relative to cell wall polysaccharide and lipid organization. The knowledge gained through this project will assist in creating better understanding of the molecular organization of the plant extracellular matrix that determine cell morphology and stress resistance. These data will ultimately be used to generate crops with better resistance to pathogens and other stresses, positively impacting crop productivity.