Reactive Oxygen Species And Factors Related To The Hypersensitive Cell Death In Rice-Xanthomonas Oryzae Pv.oryzae Interaction

Plant hypersensitive response (HR) is a type of disease resistance induced by the incompatible pathogen, showing a rapid death of the infected cells and their surrounding ones in association with the restriction of the pathogen growth. Hypersensitive cell death is not caused directly by the destructive potential of the pathogen but rather is under an intrinsic genetic control, i.e., a type of programmed cell death (PCD). Up to now, the mechanisms modulating hypersensitive cell death is still unclear. Using the compatible and incompatible combinations of rice (Oryza sativa L.) and Xanthomonas oryzae pv. oryzae (Xoo) as a model, this study focused on the roles of oxidative burst, nitric oxide (NO) generation, mitochondrial oxidative burst, nucleases, and the apoptosis-related homologs of genes or proteins in rice hypersensitive cell death elicited by Xoo infection. In addition, we studied the possible enzyme systems implicated in the generation of reactive oxygen species (ROS), and characterized a gene encoded a subunit of NADPH oxidase which is related to ROS production. The purpose of the study is to elucidate the possible regulatory mechanisms for hypersensitive cell death in rice- Xoo interaction.Rice cultivar IR-BB10 with resistance gene Xa-10 was used. The corresponding incompatible and compatible strains of Xoo were strain PXO99A(pBUavrXa10) with avrXa10 and PXO99A(pBU) without avrXa10, respectively. Suspension-cultured rice cells were stained with Evans blue within 24 h after inoculation with Xoo at intervals. The results showed hypersensitive cell death occurred at 8 h after inoculation in the incompatible interaction, whereas no cell death was detected within 24 h after inoculation in the compatible interaction.The apoplastic generation of O2- and H2O2 was analysed by cytochrome c and xylenol orange after inoculation with Xoo, respectively. The results indicated that two transient increases in apoplastic ROS (O2- and H2O2) production were detected in the incompatibleIVinteraction. The first transient increase occurred at 0.5 h after inoculation, and the second transient increase within 1.5-4 h after inoculation followed. However, only the first transient increase occurred in the compatible interaction. Moreover, the production of cellular O2-and intracellular H2O2 was measured by XTT and H2DCF-DA after inoculation, respectively. The results showed the dynamics of the cellular O2- production were similar to those of the apoplastic, whereas the two peaks of intracellular H2O2 production lagged behind those of the apoplastic. Treatment of rice with superoxide dismuatse (SOD) and catalase (CAT) inhibited partially the production of cellular O2- and intracellular H2O2, respectively. These observations suggested that there was a biphasic oxidative burst in development of the hypersensitive cell death of rice elicited by Xoo, and the ROS generation occurred both in extracellular and in the intracellular matrix. In the incompatible cells, the increased activity of cytosolic SOD was detected 2 h after Xoo inoculation, and the decreased activities of cytosolic CAT, peroxidase (POD) and ascorbate POD, respectively, to varying degrees were also monitored. The addition of N-acetylcysteine or flavone inhibited mostly rice hypersensitive cell death, implicating the ROS production in hypersensitive cell death of rice inoculation with Xoo.A biphasic NO burst was observed in the incompatible interaction. The first increase in NO production occurred at 1 h after inoculation. The second increase occurred within 3-4 h after inoculation. In the compatible interaction, however, only the first NO peak was detected. The treatment of NO scavenger (PTIO) or NO synthase inhibitors (NMMA, L-NAA and 1,3-PBIT) suppressed markedly hypersensitive cell death in the incompatible interaction, whereas NO donor (SNP) induced cell death in the control and compatible interaction. Therefore, these results suggested the involvement of NO production in the induction of rice hypersensitive cell death by Xoo.The enzyme systems...