Identification of differences in genome content between Xanthomonas oryzae pathovar oryzae and Xanthomoas oryzae pathovar oryzicola by suppressive subtractive hybridization and further analysis

Xanthomonas oryzae pathovar (pv.) oryzae (Xoo) causes bacterial leaf blight of rice. X. o. pv. oryzicola (Xoc) causes bacterial leaf streak of rice. Xoo invades the vascular system, while Xoc colonizes the mesophyll parenchyma. To identify key factors that distinguish vascular from non-vascular pathogens, Xoo and Xoc offer a good model. The work presented here provides specific candidate genes and tools for further study. Suppressive subtractive hybridization (SSH) was used to identify sequences unique to one pathovar or the other. Eight clones specific to the Xoo strain used were detected. Four of these were putative insertion sequences (IS). Two encoded a methyltransferase and a putative O-antigen acetylase, respectively. One matched the upstream sequences of some genes in the avrBs3 family. No clones specific to the Xoc strain used were found. The putative O-antigen acetylase gene, wxoD, is localized in a lipopolysaccharide biosynthesis locus in Xoo. A partial clone of the corresponding locus in Xoc BLS256 was found to be highly conserved with the corresponding region in X. axonopodis pv. citri, a non-vascular pathogen, but different from the loci in Xoo and X. campestris pv. campestris, a vascular pathogen, consistent with a role in tissue specificity determination. Preliminary work to mutagenize this gene for functional characterization is presented. In a related study, tools to identify and compare type III effectors in Xoo and Xoc were assessed. Type III effectors are proteins involved in interactions of pathogenic bacteria and their hosts that may play a role in host and tissue specificity. The feasibility of screening Xoo and Xoc genomic libraries with a reporter of type III secretion based on the avrBs2 gene from X. c. pv. vesicatoria was examined. A strain carrying the avrBs2 gene was demonstrated to elicit a visible hypersensitive response in resistant plants in the presence of four times more cells of a strain not carrying the gene, indicating that a pooling approach would be effective to screen a large number of clones. Xoo and Xoc genomic libraries were constructed for screening