Molecular analysis of the roles of yeast microtubule-associated genes in Agrobacterium-mediated transformation Chen Zikai (B Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2012 ACKNOWLEDGEMENTS First of all, my deepest gratitude goes to my supervisor, Associate Professor Pan Shen Quan, not only for providing me the precious opportunity to undertake this promising project but also for his patient encouragement, professional and practical guidance throughout my PhD candidature Secondly, I would like to express my sincere gratitude to Professor Wong Sek Man, A/P Yu Hao and Xu Jian for their valuable instruction and general support to my research program I would also like to thank Ms Tan Lu Wee and Xu Songci for their continuous assistance and support in my experimental manipulation Moreover, I am also indebted to Ms Tong Yan and Mr Yan Tie for their technical help in fluorescent and confocal microscopy I am also grateful to the following friends and laboratory members who have been helping me in different ways: Sun Deying, Tu Haitao, Yang Qinghua, Lim Zijie, Gao Ruimin, Wang Bingqing, Li Xiaoyang, Niu Shengniao, Wen Yi, Wang Yanbin, Gong Ximing, etc Moreover, I must thank my family for their moral support and persistent encouragement during the four years’ PhD study Finally, I gratefully acknowledge National University of Singapore for providing me the research scholarship to conduct this interesting project I TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS II SUMMARY VIII LIST OF MANUSCRIPTS X LIST OF TABLES XI LIST OF FIGURES XII LIST OF ABBREVIATIONS XV CHAPTER LITERATURE REVIEW 1.1 Overview of the structure and functions of microtubules 1.1.1 The properties of tubulins in yeast cells 1.1.2 Motor proteins associated with microtubules 1.1.3 The function of microtubules in mRNA trafficking 1.1.4 The hijacking of microtubules by pathogens 1.1.5 The exploitation of microtubules by Agrobacterium? 1.2 Introduction to Agrobacterium tumefaciens II 1.3 Molecular mechanisms involved in Agrobacterium-mediated gene transfer 1.3.1 The chemotaxis of Agrobacterium 10 1.3.2 Induction of vir genes in Agrobacterium 11 1.3.3 The attachment of Agrobacterium to the host cells 12 1.3.4 The generation of T-DNA and T-complex 13 1.3.5 Translocation of virulence factors through T4SS 16 1.3.6 The functions of virulent proteins imported into the host 17 1.3.7 T-complex transport and nuclear import 19 1.3.8 T-DNA targeting to the chromatin 22 1.3.9 T-DNA uncoating and integration 23 1.4 The response of the host cells to Agrobacterium infection 25 1.5 The Agrobacterium-yeast gene transfer 26 1.6 Aims of this study 28 CHAPTER MATERIALS AND METHODS 31 2.1 Strains, culture media, common solutions plasmid and primers 31 2.2 DNA manipulations technique 39 2.2.1 Plasmid DNA preparation from E coli 40 2.2.2 Total DNA preparation from S cerevisiae 41 2.2.3 Genomic DNA preparation from Agrobacterium 41 2.2.4 DNA digestion and ligation 42 III 2.2.5 Polymerase Chain Reaction (PCR) 42 2.2.6 Real-time PCR 44 2.2.7 DNA gel electrophoresis and purification 44 2.2.8 DNA sequencing 45 2.3 Transformation 46 2.3.1 Transformation of E coli by heat shock 46 2.3.2 Transformation of S cerevisiae by lithium acetate transformation 46 2.3.3 Agrobacterium-mediated transformation in S cerevisiae 47 2.3.4 Transformation of Agrobacterium by electroporation 48 2.4 Protein preparation and analysis 49 2.4.1 Protein extraction from S cerevisiae and protein assays 49 2.4.2 SDS-PAGE gel electrophoresis 52 2.4.3 Coomassie blue staining and Western blot analysis 53 2.5 Sample preparation and microscopy for cell imaging 55 CHAPTER IDENTIFICATION OF YEAST MICROTUBULES-ASSOCIATED GENES INVOLVED IN AGROBACTERIUM-MEDIATED TRANSFORMATION PROCESSES 56 3.1 Introduction 56 3.2 Identification of microtubules-associated genes in AMT 59 3.2.1 Screening of yeast microtubule-related genes 59 IV 3.2.2 Searching for genes consistently affecting AMT efficiency at different input numbers and ratios of yeast to Agrobacterium 62 3.2.3 3.3 Lithium acetate transformation to confirm the effect of genes on AMT 66 Introduction of the H2A-H2A.Z exchanging complex 71 3.3.1 Overview of the histone variant H2A.Z 71 3.3.2 The functions of SWR1 complex and its homologs 72 3.4 The involvement of SWR1 complex in AMT process 74 3.4.1 AMT efficiencies of SWR1 subunit knockout mutants 74 3.4.2 Time course of AMT on WT and arp6∆ 77 3.4.3 Complementation and over-expression assays of ARP6 gene 80 3.5 Conclusion 84 CHAPTER ARP6 REGULATES THE INTERACTION BETWEEN HOST FACTORS AND VIRULENT PROTEINS 86 4.1 Introduction 86 4.2 Localization of Arp6p in S cerevisiae 89 4.3 ARP6 regulates the dynamics of microtubules 91 4.3.1 Comparison of microtubule structures in WT and arp6∆ 92 4.3.2 Microtubules structure was changed during AMT process 94 4.3.3 Colchicine and oryzalin effect on microtubules 97 V 4.4 The regulation of virulence proteins by ARP6 100 4.4.1 Arp6p may prohibit VirD2 from entering the host nucleus 100 4.4.2 VirD2 degradation was accelerated in arp6∆ strain 103 4.4.3 Overexpression of VirD2 decreases transformation efficiency 106 4.4.4 Transport amount of VirE2 is higher in arp6∆ strain 107 4.5 Conclusion 111 CHAPTER T-DNA TRACKING DURING THE AMT PROCESS 113 5.1 Introduction 113 5.2 T-DNA detection in yeast cells by fluorescence in situ hybridization 113 5.2.1 Design of probes for FISH 115 5.2.2 Sample fixation and spheroplast preparation 115 5.2.3 Hybridization with specific probes 117 5.2.4 Results and discussion 118 5.3 T-DNA quantification by semi-quantitative PCR and Real-time PCR 126 5.3.1 Preparation of samples for PCR assay 126 5.3.2 Results and discussion 127 5.4 DNA probes tracing during AMT process 131 5.4.1 Agrobacterium takes in probes through T4SS 131 5.4.2 Probe imported into the yeast cell 134 VI 5.5 DNase activity assay for yeast cells 139 5.6 Conclusion 142 CHAPTER GENERAL CONCLUSIONS AND FUTURE WORK 144 6.1 General conclusions 144 6.2 Future work 147 REFERENCES 149 VII SUMMARY Agrobacterium tumefaciens can genetically transform plants in nature by transferring a piece of DNA (T-DNA) into the host cell Under laboratory conditions, Agrobacterium can also transfer T-DNA into a wide range of other eukaryotic species, including yeast cells A tumefaciens virulence machinery facilitating the transfer of T-DNA has been intensively investigated; however, the trafficking pathway of T-DNA inside eukaryotic cells is not well established It is reasonable to speculate that an active transport mechanism should be involved in the process because macromolecules such as T-complex cannot simply diffuse effectively through the dense cytoplasm This project is using Saccharomyces cerevisiae as a research model to investigate the trafficking pathway of T-DNA inside yeast cells The focus is on yeast microtubule-associated genes, since previous studies have found that in vitro constructed T-complex can utilize a microtubule-based transport pathway to deliver T-DNA to the host nucleus To identify 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The molecular