Dissection and Characterization of the Ax21 Regulatory Network

Xanthomonas oryzae pv. oryzae (Xoo) is a bacterial pathogen of rice, the worldwide staple and grass model. Xoo invades rice vascular tissues to cause bacterial leaf blight, a serious disease of rice in many parts of the world. An effective method to control the disease is by using resistant rice cultivars. The rice Xa21 gene confers immunity to most strains of Xoo. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) of biologically active fractions from Xoo supernatants leads to the identification of a 194-amino acid protein designated Ax21 (Activator of XA21-mediated immunity). A sulfated, 17-amino acid synthetic peptide (axYS̳̳22) derived from the N-terminal region of Ax21 is sufficient for activity, whereas peptides lacking tyrosine sulfation are biologically inactive. axYS̳22 is 100% conserved in all sequenced Xanthomonas species. Ax21 is a quorum sensing factor that controls density-dependent expression of genes involved in motility, c-di-GMP turnover, cell aggregation, and biofilm formation. Additionally, two response regulators RaxR and PhoP control ax21 expression in opposite manners. The putative cognate sensor kinase for RaxR is called RaxH. The reduction of biofilm production in PXO99[delta]raxH, and the failure of this strain respond to addition of exogenous Ax21 protein suggests that RaxH could be the Ax21 receptor. This work reveals the regulatory mechanisms governing peptide-mediated quorum sensing in the Gram-negative bacterium, Xoo, a process previously thought to be restricted to Gram-positive bacteria.