| Rice bacterial blight(BB),caused by Xanthomonas oryzae pv.Oryzae(Xoo),is a destructive disease in most rice-growing countries and caused much damage to rice yield. The most efficient and economical mean of controlling BB is the use of host resistance by growing resistant varieties.Thirty bacterial blight resistance genes have been identified so far from cultivated rice or wild rice species Xa23 is a complete dominant,host whole life effective BB resistance gene identified oringinally from wild rice Oryzae rufipogon. To date,no natural Xoo strain that can infect the near-isogenic rice line CBB23 harboring Xa23 has been found.In order to generate a pathogenic Xoo strain for study on the interaction between Xoo and CBB23,the Xoo strain PXO99 was used to construct a random insertional mutant library using the Tn5-derived transposon tagging system.The pathogenicity of each Xoo mutants of the library was assayed by inoculation methods of leaf-cutting or infiltration with a needleless syringe on CBB23.Results showed that the rice line CBB23 is susceptible to 14 among the 24,192 PXO99 mutants.Polymerase chain reaction analysis showed that all the 14 mutants were inserted with Tn5 transposon DNA.Southern blot analysis revealed that there was only one Tn5-insertion event in the genomes of eight mutants.The flanking DNA sequences of the Tn5-insertion sites in two mutants, designated as XMS-16 and XMS-86,were isolated by PCR walking and sequenced. Bioinfomatic analysis of the flanking sequences,by aligning them with the whole genome sequences of Xoo strain KACC10331 in NCBI,revealed that the Tn5-transposon was inserted at 798957th nucleotide located within the Xoo0774 gene that encodes an outer membrane protein in XMS-16.Another copy of the transposon was inserted at 3254316th nucleotide located within the Xoo3031 gene that encodes a cytochrome C peroxidasc,CCPs) in XMS-86.The possible molecular mechanisms on the pathogenicity of the PXO99 mutants XMS-16 and XMs-g6 were discussed. |
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