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Endocytosis is essential for eukaryotic cells. However, relatively little is known about the endocytic pathway and its molecular machinery in plant cells. In this research, a highly conserved membrane protein called secretory carrier membrane protein 1 (SCAMP) from rice (Oryza sativa) (OsSCAMP1) was employed as a tool to study the plant endocytosis. Toward this goal, I have generated polyclonal antibodies specific to SCAMP and transgenic tobacco BY-2 cell lines expressing yellow fluorescence protein (YFP)-SCAMP fusion. Confocal microscopy study showed that SCAMP localized to both plasma membrane (PM) and motile organelles. Further drug treatment and uptake studies demonstrated that these organelles are early endosomes distinct from Golgi and prevacuolar compartment (PVC), because they colocalized with the endosomal marker FM4-64. Immunogold electron microscopy study with SCAMP antibodies has identified the early endosome (EE) as a vesicular tubular membrane organelle, which resembles the structure of trans-Golgi network (TGN). These results indicate that the secretory and endocytic pathways are merged at the TGN which may serve as the sorting station for both pathways. / Since brefeldin A (BFA) induced both TGN and Golgi to form similar aggregates or BFA compartments in tobacco BY-2 cells, studies were also performed to sort out these BFA-induced compartments. Here I have demonstrated that the BFA-induced compartments derived from Golgi and TGN are physically distinct where the TGN aggregates were usually found to be surrounded by the Golgi aggregates in the same cells in both confocal immunofluorescent and immunogold EM studies. Furthermore, the internalized endosomal marker FM4-64 was found to colocalize with the TGN-derived BFA compartments but separated from the Golgi aggregates, whereas the endocytosis inhibitor tyrphostin A23 prevented TGN but not Golgi from forming BFA compartments. Therefore, the secretory Golgi organelle is functinally distinct from the endocytic TGN/EE in their responses to BFA treatment in plant cells. / The possible roles of SCAMPs in cytokinesis were also investigated. In transgenic tobacco BY-2 cells expressing the TGN/EE marker SCAMP-YFP, SCAMPs were found to be concentrated in the developing cell plate together with the internalized endosomal marker FM4-64 under confocal microscopy and this was further confirmed by immunogold electron microscopy studies with SCAMP antibodies. These results have demonstrated that SCAMPs, TGN and endocytosis are all involved in the cell plate formation during cytokinesis in plant cells. / Lam, Sheung Kwan. / Adviser: Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3266. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 176-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Xue, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 95-102). / Abstract also in Chinese. / Thesis committe --- p.2 / Statement --- p.3 / Abstract --- p.4 / Acknowledgement --- p.8 / General abbreviations --- p.10 / Abbreviations of chemicals --- p.13 / List of figures --- p.15 / List of tables --- p.16 / Table of contents --- p.17 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Impact of bacterial blight on rice production --- p.25 / Chapter 1.2 --- The plant immune system --- p.25 / Chapter 1.2.1 --- Preformed resistance --- p.25 / Chapter 1.2.2 --- PAMP triggered immunity (PTI) --- p.26 / Chapter 1.2.3 --- Effecter triggered immunity (ETI) --- p.27 / Chapter 1.2.3.1 --- R genes --- p.27 / Chapter 1.2.3.2 --- Hypersensitive responses (HR) --- p.27 / Chapter 1.2.3.3 --- Systemic acquired resistance (SAR) --- p.28 / Chapter 1.2.3.3.1 --- Salicylic acid is required for SAR establishment --- p.28 / Chapter 1.2.3.3.2 --- Involvement of lipid-based molecules in SAR signaling --- p.28 / Chapter 1.2.3.3.3 --- NPR1: the master regulator of SAR --- p.29 / Chapter 1.2.3.3.4 --- Expression of pathogenesis related (PR) genes --- p.29 / Chapter 1.2.4 --- Interaction between SA and JA --- p.29 / Chapter 1.2.5 --- Other important signaling components in plant defense responses --- p.30 / Chapter 1.2.5.1 --- G proteins --- p.30 / Chapter 1.2.5.2 --- G proteins in defense responses --- p.30 / Chapter 1.3 --- OsGAPl is a C2 (protein kinase C conserved region 2) domain harboring GTPase activating protein --- p.32 / Chapter 1.4 --- OsYchFl is a GTPase and an interacting partner of OsGAPl --- p.32 / Chapter 1.5 --- Hypothesis and objectives of this research --- p.33 / Chapter Chapter 2 --- materials and methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Chemicals and reagents --- p.39 / Chapter 2.1.2 --- Commercial kits --- p.40 / Chapter 2.1.3 --- Primers used --- p.41 / Chapter 2.1.4 --- Equipment and facilities used: --- p.47 / Chapter 2.1.5 --- "Buffer, solution, gel and medium:" --- p.47 / Chapter 2.2 --- Methods: --- p.51 / Chapter 2.2.1 --- Culture of bacterial strains --- p.51 / Chapter 2.2.2 --- Composition of medium used in this work for cultivating bacterial strains: --- p.51 / Chapter 2.2.3 --- Plant growth and treatment --- p.52 / Chapter 2.2.3.1 --- Surface sterilization of Arabidopsis thaliana seeds --- p.52 / Chapter 2.2.3.2 --- Seed germination and Arabidopsis plant growth --- p.52 / Chapter 2.2.4 --- Generation of transgenic Arabidopsis --- p.53 / Chapter 2.2.4.1 --- Agrobacterium-mediated Arabidopsis transformation --- p.53 / Chapter 2.2.5 --- Pathogen inoculation test --- p.54 / Chapter 2.2.6 --- Molecular cloning --- p.54 / Chapter 2.2.6.1 --- DNA sequencing: --- p.55 / Chapter 2.2.6.2 --- Transformation of E. coli strains: --- p.55 / Chapter 2.2.6.3 --- Transformation of Agrobacteria by electroporation --- p.55 / Chapter 2.2.7 --- DNA and RNA extraction --- p.56 / Chapter 2.2.7.1 --- Plasmid DNA extraction from bacterial cells --- p.56 / Chapter 2.2.7.2 --- Genomic DNA extraction from plant tissues --- p.56 / Chapter 2.2.7.3 --- RNA extraction from plant tissues --- p.56 / Chapter 2.2.8 --- Northern blot --- p.57 / Chapter 2.2.9 --- Subcellular localization studies --- p.58 / Chapter 2.2.9.1 --- Transformation of tobacco BY-2 cells --- p.58 / Chapter 2.2.9.2 --- Maintenance of transgenic tobacco BY-2 cells --- p.59 / Chapter 2.2.9.3 --- Confocal microscopy --- p.59 / Chapter 2.2.9.4 --- Electron microscopy --- p.59 / Chapter 2.2.10 --- Bimolecular fluorescence complementation studies (BiFC) --- p.60 / Chapter 2.2.10.1 --- Construct making --- p.61 / Chapter 2.2.10.2 --- Preparation of rice protoplasts --- p.61 / Chapter 2.2.10.3 --- PEG-mediated transfection --- p.62 / Chapter 2.2.10.4 --- Detection of protein-protein interaction --- p.62 / Chapter Chapter 3 --- Results / Chapter 3.1 --- OsGAPl interacts with OsYchFl in vivo --- p.63 / Chapter 3.1.1 --- Construction of vectors for BiFC transient assay in rice protoplasts --- p.64 / Chapter 3.1.2 --- BiFC assay in rice protoplasts revealed in vivo interaction between the OsGAPl and the OsYchFl proteins --- p.66 / Chapter 3.2.1 --- Subcellular localization of OsGAPl --- p.68 / Chapter 3.2.2 --- Localization of OsGAPl and OsYchFl in rice leaves revealed by electron microscopy --- p.70 / Chapter 3.3 --- Functional characterization of OsYchFl / Chapter 3.3.1 --- Characterization of Arabidopsis YchF1 knockdown mutant --- p.75 / Chapter 3.3.2 --- Complementation of AtYchF1 knockdown Arabidopsis --- p.77 / Chapter 3.3.3.1 --- Pathogen inoculation test --- p.80 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Significance of the project --- p.85 / Chapter 4.2 --- In vivo interaction between OsGAPl and OsYchFl --- p.86 / Chapter 4.3 --- OsGAPl is located either inside the cytosol or on the plasma membrane in transgenic tobacco BY-2 cells --- p.87 / Chapter 4.4 --- Study of wounding effect on the subcellular localization of OsGAPl and OsYchFl at whole plant level by EM --- p.88 / Chapter 4.5 --- OsYchFl functions as a negative regulator of defense responses in A.thaliana --- p.90 / Chapter 4.6 --- Conclusion --- p.92 / References --- p.95 / Appendix --- p.103

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