Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus productionworldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important forthe successful development of a pathogenic relationship between various bacteria and their host(s). To understand themechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposonmutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilmformation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilmformation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X.axonopodis pv. citri strain 306 and 30–100% identity with two-component response regulators in various pathogens andenvironmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leafsurfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to causecanker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicatedthat BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, andlecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium,which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for cankerdevelopment in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR viaregulation of rpfF, which is required for the biosynthesis of a diffusible signal factor
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