| Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of rice bacterial blight, is one of the most important phytopathogens of rice. The bacterium could invade into vascular system of host plant through wounds, hydathodes and stomata, and causes serious agricultural yield loss worldwide. Systematic understanding of the molecular mechanism of Xoo pathogenesis will lead us to develop novel strategies to prevent and control this important pathogen. In order to facilitate the genomic-scale studies, firstly we constructed 3 high quality genomic DNA libraries of Xoo strain (PR6) which belongs to Philippine race 6. These libraries have average insert size of 1.6 ~ 3.0 kb, 3.0 ~ 4.0 kb and 8.0 ~ 10.0 kb, respectively. The nucleotide sequences of the inserts from 22,345 clones of the 1.6 ~ 4.0 kb library (pUC18/SmaI/CIP vector) were then determined from both ends using BigDye? terminator by ABI3730 automated sequencers. Atotal of 21,211 sequencing reads have been finished, obtaining 16,459,736 nucleotide basepairs (bp). These sequences (nearly 3.3-fold genome coverage) were assembled using Phred/Phrap/ Consed software package (http://genome.washington.edu) and 529 contigs were obtained, which accomplishes a genome-scaffolds of Xoo PR6 strain. A primary sequence analysis (BlastN search) with the published Xoo genome sequence of KACC 10331 (containing 4,637 protein coding sequences, CDSs) as reference revealed that at least 4,555 (98.23%) CDSs of Xoo KACC 10331 could found homologs in the sequence of PR6, suggesting that the two genomes encode a highly conserved gene content.We screened a previously constructed random Tn5 insertional mutant library of Xoo PR6 against its susceptible rice host (cultivar IR24). From the 17,184 recombinant clones, 112 pathogenicity-deficient mutants were obtained. By Southern blotting analysis, the amount of mutants with single copy transposon insertion accounted to 93.8% (105 mutants) relative to the total. The flanking sequences of these mutants were then dissected by thermal asymmetric interlaced golymerase chains reactions (TAIL-PCR), and were sequenced for bioinformatic analyses. It showed that the Tn5 interrupted ORFs encode 46 genes, with 14 of them being inserted at mobile genetic element (MGE) or promoter or intergenic regions. Among these genes, more than one third are associated to secretion systems (SS), suggesting an important role of secretion systems during disease pathogenesis. Other genes were found to participate diverse of physiological pathways, including biosynthesis of exopolysaccharides, lipopolysaccharides, general metabolic pathways and gene regulations. These results reveal the diversity and complexity of the mechanism of the rice bacterial blight disease.Six non-redundant mutants were found to harbor Tn5 insertio at different xps (for Xanthomonas protein secretion) genes that encode the machinery of general secretion system (also type â…¡ SS). The growth characters on PSA plate of these xps gene mutants are similar to that of the wild-type strain, however, we found that the secretion of extracellular enzymes, including cellulose and xylanase were blocked in these mutants, suggesting that these extracellular enzymes were all secreted by the type â…¡ SS. Primary SDS-PAGE, RT-PCR and Western blot analysis revealed that the transcriptions of these xps genes were under control of a same promoter, with several of them probably possessing of own... |
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