Mechanisms Of Bismerthiazol And Streptomycin Resistance In Xanthomonas Oryzae Pv. Oryzae And Xanthomonas Oryzae Pv. Oryzicola

Bacterial blight of rice (BB) and bacterial leaf streak of rice (BLS) were caused by Xanthomonas oryzae pv. oryzae(Xoo) and X. oryzae pv. oryzicola (Xooc), respectively. BB is one of the most serious diseases of rice, and BLS is emerging in importance. Application of bactericides is an indispensable complementary tool, especially in regions where susceptible cultivars are frequently planted. Bismerthiazol and streptomycin are mainly bactericides which have been used for the control of bacterial plant disease. But bismerthiazol and streptomycin have been used for more than thirty years. Thus, to study the resistance mechanism becomes very important.In this paper, there are five points about resisitance mechanism that were studied:Construction of cosmid gene library of bismerthiazol-resistant Xoo.In vivo selection of the cosmid library of bismerthiazol-resistant Xoo and gene analysis.Study on the molecule mechanism of the streptomycin resistance in Xoo and Xooc.Detection of the resistance to streptomycin in Xoo、Xooc、Erwinia carotovora (Ec) and Xanthomonas axonopodis pv.citri (Xac) by PCR-RFLP within the mutation at codon 43 of the rpsL gene.1. Construction of the cosmid library of bismerthiazol-resistant XooIn order to explore the resistance mechanism of bismerthiazol, the cosmid genome library of the bismerthiazol-resistant Xoo mutant 2-1-1 was constructed. Through incomplete digestion of the genome of 2-1-1, the>23.13 kb gene fragments were gathered. Then, the packaging protein was extracted with the lysogen BHB2688、BHB2690, and the potency came up to 1×108pfu/μgDNA. The gathered gene fragments were connected, packed with packaging protein, and infected with the host S17-1. Then the combined mutants were selected on the LB+Km+Sp plates. A total of 1200 transformants were randomly selected at last and stored. The reservoir storage came up to 2.7 times. Fifteen transformants were randomly selected and digested with enzymes, the results indicated all the cosmids contained heterogenous insertional fragments and the cosmid genome library was successfully constructed, with average 23-40 kb gene fragments inserted.2. In vivo selection of the cosmid library of bismerthiazol-resistant Xoo and gene analysisThe 1200 transformants described above were transformed by conjugation into PXO99 (a bismerthiazol-sensitive strain). Then, the in-vivo selection was done on the treated rice plants (sprayed with 300μg/ml bismerthiazol or water) inoculated with the Xoo which had been transformed with exogenous fragment.The results suggested that transformants numbered 646 and 518 confirmed their resistance to bismerthiazol, and the transformants numbered 5、33、115、152、119、102、172、519、537、611、643、501、694、518、612、654、308、919、670 were obviously stronger than that of the mutants 2-1-land PXO99. The transformants numbered 646 and 518 were subcloned, and the results revealed that two genes mutated. The two genes were raxR2 gene and conserved hypothetical protein(chp) gene. Seven bases mutated in the gene raxR2, but the amino acid sequence remained unchanged. Only one base mutated in the chp gene, resulting in the change of amino acid.3. Study on the molecule mechanism of the streptomycin resistance in Xoo and XoocXoo and Xooc were found to be sensitive to streptomycin, which is an inhibitor of protein synthesis resulting from interference with translational proofreading. Nineteen Xooc and eleven Xoo streptomycin resistant mutants were obtained by UV induction or streptomycin selection. These mutants could grow at 100μg mL-1 of streptomycin while the wild-type strain (RS105 and PXO99) cannot grow at 5μg mL"1 of streptomycin. The mutations at codon 43 and 88 in the rpsL gene might not be related to the reduced pathogenicity of Xoo and Xooc. Sequencing indicated that the rpsL gene has 375 bp encoding 125 amino acid residues. Blasting of the NCBI database indicated that the ORF had 97% similarity in nucleotide sequence and 100% similarity in deduced amino acid sequence with Xoo. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys�'Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS (containing a mutation at codon 43 of rpsL) and pUFRRX (containing a mutation at codon 88 of rpsL), were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys�'Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. The transformants, RS1、RS2、PS1 and PS2, could grow in the medium containing 50μg mL-1 of streptomycin. A mutation at codon 43 or 88 in rpsL could result in resistance of Xoo to streptomycin. Meanwhile, The transformants with point mutation at codon 43 or 88 in the rpsL gene might not be related to the pathogenicity of Xoo and Xooc.4. Detection of mutation at codon 43 of the rpsL gene in Xoo、Xooc、Ec and Xac byPCR-RFLPTwelve Ec、eleven Xac and twelve Ps streptomycin resistant mutants were obtained by UV induction or streptomycin selection. Sequencing indicated that the rpsL gene had a mutation at codon 43 or 88, which AAG was substituted for AGG (Lys�'Arg).To develop a new method for detecting the point mutation of ribosomal S12 protein (rpsL) gene in streptomycin-resistant strains of Xoo, Xooc, Ec and Xac, the PCR-RELP method was developed to detect the point mutation of codon 43 of the rpsL gene in Xoo、Xooc、Ec and Xac. The 304-bp PCR product from the rpsL gene was digested by MboⅡinto two fragments (201 and 103 bp) if there was a mutation at codon 43, or into three fragments (146,103, and 55 bp) if there was no mutation. Compared with the results of nucleotide sequencing, the PCR-RFLP method was feasible in detecting the mutation in codon 43. The PCR-RFLP method could be used in the detection of the point mutation at codon 43 of the rpsL gene in streptomycin-resistant strains of Xoo, Xooc, Ec and Xac. PCR-RFLP is a simple, rapid, and reliable method for detection of the point mutation of codon 43 in the rpsL gene.