The potential role and mechanism of an unconventional GTPase and its interacting partner in rice defense response.

Xue, Yan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.Includes bibliographical references (leaves 95-102).Abstract also in Chinese.Thesis committe --- p.2Statement --- p.3Abstract --- p.4Acknowledgement --- p.8General abbreviations --- p.10Abbreviations of chemicals --- p.13List of figures --- p.15List of tables --- p.16Table of contents --- p.17Chapter Chapter 1 --- General IntroductionChapter 1.1 --- Impact of bacterial blight on rice production --- p.25Chapter 1.2 --- The plant immune system --- p.25Chapter 1.2.1 --- Preformed resistance --- p.25Chapter 1.2.2 --- PAMP triggered immunity (PTI) --- p.26Chapter 1.2.3 --- Effecter triggered immunity (ETI) --- p.27Chapter 1.2.3.1 --- R genes --- p.27Chapter 1.2.3.2 --- Hypersensitive responses (HR) --- p.27Chapter 1.2.3.3 --- Systemic acquired resistance (SAR) --- p.28Chapter 1.2.3.3.1 --- Salicylic acid is required for SAR establishment --- p.28Chapter 1.2.3.3.2 --- Involvement of lipid-based molecules in SAR signaling --- p.28Chapter 1.2.3.3.3 --- NPR1: the master regulator of SAR --- p.29Chapter 1.2.3.3.4 --- Expression of pathogenesis related (PR) genes --- p.29Chapter 1.2.4 --- Interaction between SA and JA --- p.29Chapter 1.2.5 --- Other important signaling components in plant defense responses --- p.30Chapter 1.2.5.1 --- G proteins --- p.30Chapter 1.2.5.2 --- G proteins in defense responses --- p.30Chapter 1.3 --- OsGAPl is a C2 (protein kinase C conserved region 2) domain harboring GTPase activating protein --- p.32Chapter 1.4 --- OsYchFl is a GTPase and an interacting partner of OsGAPl --- p.32Chapter 1.5 --- Hypothesis and objectives of this research --- p.33Chapter Chapter 2 --- materials and methodsChapter 2.1 --- Materials --- p.35Chapter 2.1.1 --- Chemicals and reagents --- p.39Chapter 2.1.2 --- Commercial kits --- p.40Chapter 2.1.3 --- Primers used --- p.41Chapter 2.1.4 --- Equipment and facilities used: --- p.47Chapter 2.1.5 --- "Buffer, solution, gel and medium:" --- p.47Chapter 2.2 --- Methods: --- p.51Chapter 2.2.1 --- Culture of bacterial strains --- p.51Chapter 2.2.2 --- Composition of medium used in this work for cultivating bacterial strains: --- p.51Chapter 2.2.3 --- Plant growth and treatment --- p.52Chapter 2.2.3.1 --- Surface sterilization of Arabidopsis thaliana seeds --- p.52Chapter 2.2.3.2 --- Seed germination and Arabidopsis plant growth --- p.52Chapter 2.2.4 --- Generation of transgenic Arabidopsis --- p.53Chapter 2.2.4.1 --- Agrobacterium-mediated Arabidopsis transformation --- p.53Chapter 2.2.5 --- Pathogen inoculation test --- p.54Chapter 2.2.6 --- Molecular cloning --- p.54Chapter 2.2.6.1 --- DNA sequencing: --- p.55Chapter 2.2.6.2 --- Transformation of E. coli strains: --- p.55Chapter 2.2.6.3 --- Transformation of Agrobacteria by electroporation --- p.55Chapter 2.2.7 --- DNA and RNA extraction --- p.56Chapter 2.2.7.1 --- Plasmid DNA extraction from bacterial cells --- p.56Chapter 2.2.7.2 --- Genomic DNA extraction from plant tissues --- p.56Chapter 2.2.7.3 --- RNA extraction from plant tissues --- p.56Chapter 2.2.8 --- Northern blot --- p.57Chapter 2.2.9 --- Subcellular localization studies --- p.58Chapter 2.2.9.1 --- Transformation of tobacco BY-2 cells --- p.58Chapter 2.2.9.2 --- Maintenance of transgenic tobacco BY-2 cells --- p.59Chapter 2.2.9.3 --- Confocal microscopy --- p.59Chapter 2.2.9.4 --- Electron microscopy --- p.59Chapter 2.2.10 --- Bimolecular fluorescence complementation studies (BiFC) --- p.60Chapter 2.2.10.1 --- Construct making --- p.61Chapter 2.2.10.2 --- Preparation of rice protoplasts --- p.61Chapter 2.2.10.3 --- PEG-mediated transfection --- p.62Chapter 2.2.10.4 --- Detection of protein-protein interaction --- p.62Chapter Chapter 3 --- ResultsChapter 3.1 --- OsGAPl interacts with OsYchFl in vivo --- p.63Chapter 3.1.1 --- Construction of vectors for BiFC transient assay in rice protoplasts --- p.64Chapter 3.1.2 --- BiFC assay in rice protoplasts revealed in vivo interaction between the OsGAPl and the OsYchFl proteins --- p.66Chapter 3.2.1 --- Subcellular localization of OsGAPl --- p.68Chapter 3.2.2 --- Localization of OsGAPl and OsYchFl in rice leaves revealed by electron microscopy --- p.70Chapter 3.3 --- Functional characterization of OsYchFlChapter 3.3.1 --- Characterization of Arabidopsis YchF1 knockdown mutant --- p.75Chapter 3.3.2 --- Complementation of AtYchF1 knockdown Arabidopsis --- p.77Chapter 3.3.3.1 --- Pathogen inoculation test --- p.80Chapter Chapter 4 --- DiscussionChapter 4.1 --- Significance of the project --- p.85Chapter 4.2 --- In vivo interaction between OsGAPl and OsYchFl --- p.86Chapter 4.3 --- OsGAPl is located either inside the cytosol or on the plasma membrane in transgenic tobacco BY-2 cells --- p.87Chapter 4.4 --- Study of wounding effect on the subcellular localization of OsGAPl and OsYchFl at whole plant level by EM --- p.88Chapter 4.5 --- OsYchFl functions as a negative regulator of defense responses in A.thaliana --- p.90Chapter 4.6 --- Conclusion --- p.92References --- p.95Appendix --- p.10