Effect Of Mutagenesis Of Phar Gene Of Xanthomonas Oryzae PV. Oryzae PXO99~A On The Pathogenicity In Rice And Hypersensitive Response On Tobacco

Xanthomonas oryzae pv. oryzae(Xoo) is one of the most important bacterial pathogens, which caused bacterial blight in rice. Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. There are numerous genes playing important roles in polyhydroxyalkanoate metabolism through the sequence analysis. One of the genes encodes the regulatory protein of PHA.The PhaR gene knock out mutant of X. oryzae pv. oryzae strain PXO99A was generated based on double crossover homologous recombination. Functional complementation tests were performed to confirm the function of PhaR gene in Xoo. The biological phenotype, pathogennicity in rice and hypersensitive response in tobacco of the mutant were tested.Firstly, the encoding PhaR gene combined with the expression vector pHMl, and then were electroporated into the cells of the mutant. The functional complementation transformant of the mutant was get. The bacterial growth and motility of the mutant was reduced. The mutant also showed a significant decrease in the secretion of exopolysaccharides. However, the sress tolerance and resistance to the germicide for the mutant was the same as the wild-type strain. And there was no change of the lipases.Expression of synthesis-related genes of PHA was detected by quantitative real time PCR. Compared to the wild type, transcription of these genes was all decreased including PhaC, PhaD, PhaE, PhaF, Phbsub, Phbsyn and Phbdep.We also found that the mutant displayed reduced virulence in the host rice. Number of bacteria population in rice leaves was the same as that. The encoding sequence of PhaR gene restored the changed phenotype of the mutant to the level close to that of the wild type. The PhaR gene mutant enhanced hypersensitive response on the non-host tobacco. We also detected ROX burst and micro-HR in the inoculated tobacco leaves by DAB and trypan blue staining. ROX burst by the mutant enhanced, and the number of cell death increased. Expression of defense-related genes in tobacco was detected by semi-quantitative RT-PCR. When inoculated on tobacco leaf, the mutant induced high level expression of HMGR and MAPK genes than that by the wild type5hours post inoculation.These results indicate that PhaR gene has a direct effect on the biological characteristics in Xoo. The PhaR gene mutant could only synthetize a small quantity of PHA. Furthermore, the PhaR gene plays an important role in determining the pathogenicity of Xoo in rice and negatively regulating the hypersensitive response on tobacco.