UC Riverside

Pantoea stewartii subspecies stewartii (Pnss) causes Stewart’s Wilt in sweet corn and maize, which is characterized by wilting, caused by the formation of exopolysaccharide (EPS)-based biofilms that block the xylem, and the formation of water-soaked lesions. These lesions can form via Pnss-induced expression of a repeat-in-toxin (RTX)-like protein called RTX2. Deleting rtx2 prevents water-soaked lesion formation and effective plant colonization. The rtx2 gene is in an operon with a second gene that encodes another putative rtx-like toxin (rtx1), and two genes that encode the phosphotransferase and the response regulator of the Regulator of Capsular Synthesis (Rcs) phosphorelay, which regulates gene expression via external stimuli. This operon also has a predicted upstream OxyR transcription factor binding site. Water-soaked lesions contain lethal Reactive Oxygen Species (ROS), and previous work by the Roper lab demonstrated that OxyR protects Pnss against ROS while deletion of oxyR caused increased H2O2 sensitivity and negated EPS production. As the operon contains genes for the Rcs phosphorelay, it is hypothesized that ROS promotes activity of OxyR, thereby inducing gene expression of this operon. Indeed, results of this study show OxyR does bind to the predicted binding site of the rtx1/rtx2/rcsD/rcsB operon, and induces its activity. Furthermore, sublethal concentrations of ROS induce gene expression of certain components of the operon in wild type Pnss, but not in an oxyR deletion mutant. Therefore, ROS appears to be a signal that induces expression of key components of the Rcs phosphorelay via OxyR. The Rcs phosphorelay also plays a key role in cell shape and membrane integrity, and results of this study show that RTX2 affects these cellular characteristics as well, wherein the deletion of rtx2 causes decreased cell length, increased sensitivity to Polymyxin B, and increased overall cellular hydrophobicity when compared to wild type. RTX2-dependent alteration of the cell envelope also causes increased biofilm formation activity as Pnss transitions between the apoplastic and xylem phases of Stewart’s Wilt. Furthermore, RTX2 is required for both adhesion and biofilm formation in-planta, and impacts biofilm height in a non-EPS producing background (mimicking the initial stages of biofilm formation) in-vitro.