A study on the mechanism of resistance to streptomycin in Xanthomonas oryzae pv. oryzae
11 streptomycin-resistant mutants of Xanthomonas oryzae pv. oryzae were obtained by streptomycin selection. These mutants could grow at 100 μg ml-1 of streptomycin while the wild-type strain (PXO99) could not grow at 2 μg ml-1. Specific primers based on the conserved region of X. oryzae pv. oryzae were designed and used to amplify the gene encoding ribosomal protein S12 (rpsL). Sequencing indicated that the rpsL gene has 375 bp encoding 125 amino acid residues. One pair of specific primers with restriction enzyme site of EcoR I enzyme was designed and used to amplify the rpsL gene from streptomycin-resistant strains and the wild-type sensitive strain of X. oryzae pv. oryzae. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. The mutations situated at codon 43 or 88 in rpsL gene of streptomycin-resistant X. oryzae pv. oryzae strains could be detected by PCR-RFLP. Two plasmids, pUFRPS and pUFRPX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRPS and pUFRPX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance were transformed into the sensitive wild-type strain by electroporation. Both transformants, PS1 and PS2, could grow at 50 μg ml-1 of streptomycin. The results strongly suggest that the mutation in rpsL could result in resistance of X. oryzae pv. oryzae to streptomycin.
Key words: Xanthomonas oryzae pv. oryzae, streptomycin, ribosomal protein S12 (rpsL), resistance, functional complementation, molecular diagnosis by PCR-RFLP.