Molecular And Transgenic Dissection Of Harpin_Xoo Signaling In Plant Hypersensitivity And Pathogen Defense

Plants defend themselves against different pathogens by activating distinct signaltransduction pathways. Basal defense pathways leading to non-specific resistance todifferent categories of pathogens are mediated by plant hormones salicylic acid (SA),jasmonates (JA) and ethylene (ET). These pathways often cross-talk one to another and withpathways for insect resistance, plant growth and development in response to stimulations bydifferent exogenous signals. The cross-talk requires modulation by components that canregulate different pathways. Harpins are a group of proteinous elicitors produced by Gram-negative (G-) plantpathogenic bacteria and are required for induction of the hypersensitive response (HR) innon-host of the bacteria. They share some common characteristics such as rich in Glycine,containing no Cysteine, heat-stable and sensitive to protease. Genes encoding harpins havebeen cloned from most G- bacteria and harpins with biological functions are expressed in E.coli. HarpinXoo in this study has been identified recently from a Japanese strain ofXanthomonas oryzae pv. oryzae and expressed in E. coli BL21. It showed some specialproperties such as having small molecular weight, containing Cysteine ect. Application ofharpinXoo to many plants can enhance plant growth, induce resistance to pathogens andinsects. How harpinXoo performes those diverse functions have been unclear. Studies in this Ph. D project was aimed to dissect the signaling pathway leading topathogen defense and hypersensitive cell death (HCD) induced by harpinXoo. Twoapproaches were used to determine signaling pathway in two plant species. The firstapproach is the assays of plants treated with harpinXoo for signaling events. The secondapproach is to test disease resistance and signaling of transgenic tomato plants expressinghrfAXoo. Tobacco and tomato plants sprayed with harpinXoo can enhance disease resistance toTMV and Pseudomonas syringae pv. tomato respectively. Tobacco plants infiltrated withharpinXoo have showed properties of PCD (programmed cell death) but it has not in tomato. 4山东农业大学博士学位论文Transgenic tomato expressing harpinXoo showed increased resistance to P. syringae pv.tomato also. HCD in tobacco induced by harpinXoo showing properties of PCD HR/HCD occurred after infiltration of tobacco leaves with harpinXoo. Histochemical andmolecular changes can be tested during the process of HCD including condensation of nucleicchromatin, cleavage and fragmentation of nucleic DNA, accumulation of H2O2 andexpression of defense genes. In order to determine whether these responses occur in tobaccoinduced by harpinXoo or not, we tested the histochemical and molecular changes in tobaccoleaves after infiltration with harpinXoo. The results are read as follows. 1. Death kinetics study showed that micro-HR began to occur gradually before theoccurrence of macro-HR. HR observed by our naked eyes are results of micro-HR. Results ofRT-PCR showed that the expression of hin1and hsr203 started before the occurrence ofmacro-HR. The expression module of those two genes showed that they involved in tobaccoHCD. 2. To understand the cellular and molecular response in the process of HCD, tobacco leavesinfiltrated with harpinXoo are stained with DAPI and examined under up-right fluorescencemicroscope using UV filters sets. Tobacco cells treated with harpinXoo showed condensationand fragmentation of nucleic chromatin accompanying HCD. DNA were extracted at differenttimes after infiltration and separated by 2% agarose gel electrophoresis. DNA fragmentationbegan to appear 12 hours after induction; Most DNA was degraded to small fragment 24 to 48hours after induction. These results showed that tobacco cells induced by harpinXoo had theproperties of programmed cell death. 3. The results of DAB staining showed that H2O2 accumulated in tobacco leaves duringthe process of HCD, which prior to the condensa...