Xylella fastidiosa (Xf) possess a two component regulatory system (TCS) PopP-PopQ which differentially regulates genes in response to environmental stimuli. To elucidate the role of popP in the pathogenicity of X. fastidiosa causing Pierce’s disease (PD) of grapevine, a site-directed deletion method and chromosome-based genetic complementation strategy were employed to create popP deletion mutant XfΔpopP and its complementary strain XfΔpopP-C. In vitro studies showed that while all strains had similar growth curves, XfΔpopP showed significant reduction in cell-cell aggregation and cell-matrix adherence. Biofilm production of XfΔpopP was about 42% less than that of wild type X. fastidiosa and XfΔpopP-C. No symptoms were observed in grapevines inoculated with XfΔpopP, whereas grapevines inoculated with wild type X. fastidiosa and XfΔpopP-C showed typical PD symptoms. Several biofilm-related genes and genes involving protein secretary systems were down regulated in XfΔpopP in compared with wild type X. fastidiosa and XfΔpopP-C. These in vitro and in planta assay results provide strong evidence that the role of PopP is required for pathogenicity of X. fastidiosa on grapevine.