An ABC Transporter Protein and Molecular Diagnoses of Xanthomonas arboricola pv. pruni Causing Bacterial Spot of Stone Fruits

Abstract

The purpose of the research was to develop a detection and identification system for Xanthomonas arboricola pv. pruni using polymerase chain reaction (PCR) analysis and Southern hybridization with a DNA probe. Random amplified polymorphic DNA (RAPD) analysis was used to identify a specific DNA sequence strictly associated and conserved among all X. arboricola pv. pruni strains tested and obtained from various locations and hosts. PCR primers Y17CoF and Y17CoR specific to this DNA fragment were synthesized and evaluated as a diagnostic tool. Primers amplified a 943-bp DNA fragment in all strains previously identified as X. arboricola pv. pruni on the basis of biochemical and physiological tests, and failed to amplify DNA from other xanthomonads and non-xanthomonads including saprophytes and epiphytes associated with Prunus spp. The PCR assay detected between 25 and 50 cells. A digoxigenin-labeled DNA probe, XPRUNI14, was developed and used to detect an extensive collection of this bacterium through dot-blot and Southern analysis. Results indicated that X. arboricola pv. pruni could be accurately detected and identified by PCR analysis and probe hybridizations on symptomatic and asymptomatic plant materials avoiding the need for prior isolation of this phytopathogen. Sequence analysis of this 943-bp DNA fragment revealed the presence of an open reading frame (ORF) predicted to encode a putative protein of 243 amino acids with a calculated molecular mass of 26.8 kDa. In silico analysis predicted this protein to share similarity to the ABC transporter family, with a FtsX conserved domain possibly involved in cell division. In the present study we overexpressed the putative protein and results were confirmed with western and immunoblot analysis. Gene disruption with a streptomycin cassette resulted in small colony phenotype, with no evidence of bacterial mass increase suggestive of impaired division that led them to cell death.