ABSTRACT Just as the function of metazoan tissues and organs is mediated by the patterned assembly of phenotypically distinct cells during development, the functions of oral microbes in promoting health and disease arise from the emergent properties of their patterned structure: the biofilm. For example, the accumulation of supragingival dental plaque into stable, long-range biofilms at the gingival margin is implicated in reversible gingivitis, which may progress to chronic periodontitis. Recent studies have called into question the relative contribution of two conspicuous, filamentous organisms in structuring supragingival plaque over long distances: Fusobacterium nucleatum and Corynebacterium matruchotii. There exists a critical need to understand the role that filamentous organisms play in biofilm assembly because it is well understood that changes in taxonomic abundance, ecological succession and biofilm structure mediate the transition from health to inflammatory periodontal diseases in the mouth. To address this knowledge gap, we have developed an in vitro oral biofilm model, amenable to systems imaging in which to study the molecular basis of long-range community structure and biochemical interaction. We will elucidate the role of micron-scale spatial structure in oral biofilm assembly, growth and function, through combined metagenomic sequencing and systems imaging of biofilms inoculated with dental plaque from healthy donors. We will further map novel Corynebacterium genes involved in long range spatial structure of biofilms, through co-culture RNA-seq and mRNA FISH imaging. We will next decipher the specific F. nucleatum molecular components that underly long-range biofilm structure, through targeted inhibition of F. nucleatum outer membrane proteins with neutralizing antibodies and through genetic complementation of these proteins in E. coli cells co-cultured with dental plaque inocula. The knowledge gained from the research proposed here will comprise a holistic understanding of oral community assembly processes and physical structure across spatial scales from molecule to microbiome and may identify new paradigms for intervening in the progression of dental plaque-mediated diseases.