MOLECULAR ANALYSIS OF GENES MEDIATING T-DNA TRAFFICKING INSIDE YEAST CELLS ALAN JOHN LOWTON (B. Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements Gratitude goes to my supervisor, Associate Professor Pan Shen Quan, for giving me the opportunity to undertake this project and The National University of Singapore for financing it. A special mention should go to A/P Leung Ka Yin, Professor Wong Sek Man, A/P Sanjay Swarup for their support and contributions in committees. I would like to thank (in no particular order) the following colleagues and friends past and present who I have shared my time with at NUS: Tan Lu Wee, Guo Minliang, Li Xiaobo, Zhang Li, Tu Haitao, Chang Limei, Hou Qingming, Tang Hock Chun, Seng Eng Khuan, Sheng Donglai, Yu Hongbing, Li Mo, Tung Siew Lai, Joelle Lai, Laurance Gwee, Reena, Joan, Shuba and Yan Tie. My personal everlasting gratitude to Vik and Brenda for reasons that will remain known only unto them. Kudos to all Six Packers (buya, 1, 2…), the wider SRC family, Lara, Jenni, Ellen, Indri, and Maki, for their friendship, support and great times. Last but by no means least I thank my friends and family back home for their long distance support. Over and above all else my heartfelt thanks goes to Lynsey. Table of Contents Acknowledgements Table of Contents List of Figures and Tables List of Abbreviations Summary Chapter 1, Literature review 1.1 Background to Agrobacterium tumefaciens 1.2 Agrobacterium tumefaciens mediated transformation 1.2.1 Agrobacterium chemotaxis 1.2.2 Induction of Agrobacterium 1.2.3 Processing the T-DNA from the Ti-plasmid 1.2.4 T-complex formation 1.2.5 T-strand complex translocation into the host 1.2.6 VirB/D4 channel assembly 1.2.7 Mechanism of T-DNA Export 1.3 The function of the translocated virulence protein in the host 1.3.1 T-DNA nuclear targeting 1.3.2 Inside the host nucleus 1.4 Host factors involved in Agrobacterium-mediated transformation 1.4.1 Host factors involved in Agrobacterium to host attachment 1.4.2 Host factors involved in the intracellular transport of the Tcomplex 1.4.3 Host factors involved in T-complex entry to the nucleus 1.4.4 Host factors involved in intranuclear T-DNA processing 1.4.5 Host factors involved in T-DNA integration 1.4.6 Host factors involved in gene expression 1.5 Plant immunity 1.6 Agrobacterium’s host range 1.7 Aims of this study Chapter 2, Materials and Methods 2.1 Strains, storage and culturing techniques 2.2 DNA preparation 2.2.1 Plasmid DNA preparation from E. coli 2.2.2 Plasmid DNA preparation from A. tumefaciens 2.2.3 Recovering plasmids from S. pombe 2.2.4 Preparation of A. tumefaciens genomic DNA 2.2.5 Preparation of S. pombe genomic DNA 2.2.6 Preperation of S. cerevisiae genomic DNA 2.3 Analysis of potential integrant into the S. pombe genome 2.4 Total RNA Isolation (cDNA synthesis) 2.5 Reverse transcription 2.6 DNA digestion and ligation 2.7 Agarose gel electrophoresis 2.8 DNA Ampflication 2.8.1 Polymerase Chain Reaction (PCR) i ii v vii viii 3 10 13 14 19 22 23 27 32 34 37 42 44 45 49 50 53 57 58 59 59 59 60 61 61 62 63 63 64 64 65 66 66 2.8.2 Long PCR 2.8.3 Whole cell PCR from yeast 2.9 DNA sequencing PCR and sample preparation 2.10 Preparation of E. coli competent cells 2.11 Transformation 2.11.1 Transformation of E. coli by “heat shock” 2.11.2 Transformation of E. coli by electrotransformation 2.11.3 Transformation of A. tumefaciens by electroporation 2.11.4 Transformation of S. pombe by lithium acetate 2.11.5 Transformation of by Agrobacterium-mediated transformation 2.12 Protein preparation and analysis 2.12.1 Native protein extraction from S. pombe 2.12.2 SDS-PAGE gel electrophoresis 2.12.3 Western blot analysis 2.13 Cell Imaging 2.13.1 Confocal Microscopy 2.13.2 DSred-VirD2 nuclear import assay 67 68 69 70 70 70 71 72 72 73 74 74 74 75 76 76 76 Chapter 3, Schizosaccharomyces pombe; a novel host for Agrobacterium tumefaciens-mediated transformation 3.1 Introduction 3.2 Constructing S. pombe binary vectors 3.3 Developing a method for Agrobacterium-mediated transformation of S. pombe 3.4 S. pombe transformation by A. tumefaciens is dependent upon vir gene expression 3.5 Fate of the T-DNA post transformation 3.5.1 Plasmid T-DNA border analysis 3.6 Comparison of S. pombe transformation vectors 3.6.1 Effect of T-DNA orientation between the left and right borders 3.7 Optimising transformation of S. pombe by Agrobacterium mediation 3.8 A novel fate of transformed T-DNA 3.9 Discussion 100 102 106 108 111 113 118 120 Chapter 4, T-complex recognition in the eukaryotic host 4.1 Introduction 4.2 Assessing Vir protein localisation in S. pombe 4.2.1 Construction of vir gene tracking vectors 4.2.2 Localization of VirD2 in S. pombe 4.2.3 Localization of VirE2 and VirE3 in S. pombe 4.2.4 Coexpression of DSred-VirE2 with VirE3 or VIP 4.3 Discussion 123 125 125 127 130 133 137 90 93 98 Chapter 5, Function of importin-
in Agrobacterium-mediated transformation 5.1 Introduction 5.2 S. pombe importin-
and VirD2 colocalization 5.3 Importin-
and VirD2 interaction using the yeast two-hybrid system 5.4 Function of importin-
in Agrobacterium-mediated transformation 138 143 147 150 5.5 One S. pombe importin-
mutant reduces the efficiency of Agrobacterium-mediated transformation 5.5.1 The S. pombe cut15-85 mutant displays inefficient DSred-VirD2 nuclear import 5.6 Mutations to S. cerevisiae importin-
reduces efficiency to Agrobacterium-mediated transformation 5.7 Discussion 155 160 162 166 Chapter 6, The T-complex utilises a microtubule based transport pathway to deliver T-DNA to the host nucleus 6.1 Introduction 6.2 -tubulin mutants reduce the efficiency of Agrobacterium-mediated transformation 6.2.1 Mutation to S. pombe -tubulin reduces the efficiency of Agrobacterium-mediated transformation 6.2.2 Mutation at the S. cerevisiae -tubulin gene TUB2 reduces the efficiency of Agrobacterium-mediated transformation 6.3 S. cerevisiae stable microtubules reduce the reduces the efficiency of Agrobacterium-mediated transformation 6.4 Discussion 168 175 175 178 182 185 Chapter 7, T-strand import into the eukaryotic host is independent of endocytosis 7.1 Introduction 7.2 Endocytotic dysfunction increases Agrobacterium-mediated transformation of S. cerevisiae 7.3 Discussion 190 196 Chapter 8, Conclusion and Future work 197 References 203242 187 List of Figures and Tables Fig. 1.1 Overview of Agrobacterium tumefaciens-mediated transformation Fig. 1.2 Agrobacterium type IV secretion system Fig. 1.3 Proposed host factors involved in the intracellular trafficking and processing of the T-complex in planta Fig. 1.4 Species out side of the plant kingdom ameanable to Agrobacteriummediated transformation Table 2.1 Bacteria strains used in this study Table 2.2 Schizosaccharomyces pombe strains used in this study Table 2.3 Saccharomyces cerevisiae strains used in this study Table 2.4 Media used in this study Table 2.5 Antibiotics and other stock solutions Table 2.6 Binary vectors Table 2. S. pombe vir fusion constructs Table 2. S. pombe over expression and deletion cassette constructs Table 2. Yeast two-hybrid constructs Table 2. 10 Primers used in this study Table 2. 11 Proteomics solutions and buffers Fig. 3.1 Construction of pUPT301 Fig. 3.2 Construction of pAPT301 Fig. 3.3 Construction of pLPT19 Fig. 3.4 Development of a method for Agrobacterium-mediated transformation of S. pombe Fig. 3.5 Transformation of S. pombe with and without Agrobacterium induction Fig. 3.6 T-DNA extraction and analysis Fig. 3.7 Border analysis of S. pombe transformants Fig. 3.8 Comparison of S. pombe binary vectors Fig. 3.9 Comparison of S. pombe
and  binary vectors Fig. 3.10 Affect of cocultivation duration on S. pombe transformation Fig. 3.11 Analysis of T-DNA by whole cell PCR Fig. 3.12 Confirming time course S. pombe transformants Fig. 3.13 S. pombe transformants incoorporating endogenous DNA Fig. 3.14 Fate of T-DNA post-transformation Fig. 4.1 Construction of DSred fusion vectors (tracking vectors) Fig. 4.2 Construction of VirD2 tracking vectors Fig. 4.3 DSred-VirD2 fusion proteins localization in S. pombe Fig. 4.4 Construction of VirE2 and VirE3 tracking vectors Fig. 4.5 DSred-VirE2 and VirE3 fusion proteins localization in S. pombe Fig. 4.6 Construction of VIP1 tracking and VIP1 VirE3 over expression vectors Fig. 4.7 Disruption of DSred-VirE2 localization when coexpressed with VirE3 Fig. 4.8 Disruption of DSred-VirE2 localization when coexpressed with VIP1 Fig. 5.1 Conserved homology of importin-
Fig. 5.2 Creating C-terminal Cut15p and Imp1p fusions by homologous integration 15 33 54 77 78 79-80 81-82 83 84 85 86 87 88 89 95 96 97 99 101 105 107 109 112 114 116 117 119 122 126 128 129 131 132 134 135 136 141 144 Fig. 5.3 S. pombe importin-
localization Fig. 5.4 S. pombe importin-
and VirD2 colocalisation Fig. 5.5 Yeast two-hybrid bait and prey vectors Fig. 5.6 X-
gal assay to test for importin-
- VirD2 interaction Fig. 5.7 Cut15p and Srp1p point mutations Fig. 5.8 The effect of S. pombe cut15-85 mutant on Agrobacterium-mediated transformation efficiency Fig. 5.9 Creating imp1
strain using a gene deletion cassette Fig. 5.10 The effect of S. pombe imp1
mutant on Agrobacterium-mediated transformation efficiency Fig. 5. 11 Nuclear localization of DSred-VirD2 in the cut15-85 mutant Fig. 5.12 The effect of S. cerevisiae srp1 mutants on Agrobacteriummediated transformation efficiency Fig. 6.1 S. cerevisiae TUB1, TUB3
-tubulin homology Fig. 6.2 S. pombe and S. cerevisiae -tubulin homology Fig. 6.3 The effect of S. cerevisiae tub3

-tubulin mutant on Agrobacterium-mediated transformation efficiency Fig. 6.4 The effect of S. pombe -tubulin mutant on Agrobacteriummediated transformation efficiency Fig. 6.5 The effect of S. pombe nda3-KM311 mutant on sensitivity to the microtubule depolymerizing drug benomyl Fig. 6.6 The effect of S. cerevisiae -tubulin mutants on Agrobacteriummediated transformation efficiency Fig. 6.7 The varying effect of S. cerevisiae tub2 mutants to the microtubule depolymerizing drug benomyl Fig. 6.8 The effect of S. cerevisiae expressing stable microtubules on Agrobacterium-mediated transformation efficiency Fig. 7.1 Arp2/3 complex involvement in endocytosis Fig. 7.2 The effect of S. cerevisiae myo3
and myo5
mutants on Agrobacterium-mediated transformation efficiency Fig. 7.3 The effect of S. cerevisiae abp1
, vrp1
she4
and arc18
mutants on Agrobacterium-mediated transformation efficiency Fig. 7.4 The effect of S. cerevisiae pan1-101 mutant on Agrobacteriummediated transformation efficiency Fig. 8.1 Overview of host mechanisms involved in Agrobacterium-mediated transformation 145 146 148 149 151 157 158 159 161 163 172 173 174 176 177 179 180 184 188 191 192 193 202 List of Abbreviations aa amino acid(s) mg milligram(s) AS acetosyringone minute(s) bp base pairs μ micro- Ct carboxyl terminal μg microgram(s) Cb carbenicilin μl microliter(s) DMSO dimethylsufoxide μm micrometre DNA deoxyribonucleic acid mM millimole DNase deoxyribonuclease mW molecular weight dNTP deooxyribonucleoside triphosphate nt nucleotides dsDNA double-stranded DNA Nt amino terminal DTT dithiothreitol ORF open reading frame EDTA ethylenediaminetetra acetic acid PAGE Polyacrylamide gel electrophorisis EtBr ethidium bromide r resistant EtOH ethanol RNA ribonucleic acid g grams RNase ribonuclease GFP green fluorescent protein rpm revolutions per minute h hour(s) SAP shrimp alkaline phosphotase HRP horseradish peroxidase SDS sodium dodecyl sulphate kb kilobase(s) or 1000bp sec second kDa kilodalton(s) ssDNA single-stranded DNA km kanamycin UV ultraviolet M molar v/v volume per volume MCS multiple cloning site w/v weight per volume X-
gal 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside Summary Agrobacterium tumefaciens is a natural plant pathogen, capable of transfecting its host via a transferable genetic element termed, T-DNA once induced. The single strand T-DNA molecule is exported from the bacterium and transported to the host cell nucleus as a nucleoprotein termed the T-complex, where it becomes double stranded and in the case of planta incorporates into the genome. Little is known about the posttransfection pathway involved in trafficking the T-DNA to the nucleus. This study introduces the fission yeast Schizosaccharomyces pombe as a novel and powerful host to study factors involved in Agrobacterium-mediated transformation. The pre- established budding yeast (Saccharomyces cerevisiae) model was exploited to corroborate findings and together provide a working model of nucleoprotein trafficking inside eukaryotic cells. By assaying an array of fission and budding yeast mutants mechanisms involved in host cell entry, active transport through the cytoplasm and nuclear import were all examined as a function of Agrobacteriummediated transformation efficiency. Findings from this study suggest T-DNA enters the host via an endocytosis independent pathway. Localization and interaction studies indicate the yeast host recognizes the VirD2 NLSs via importin-
that can associate with microtubules for active transport through the cytoplasm. Fission and budding yeast importin-
and microtubule mutants reduce the efficiency of Agrobacteriummediated transformation. Established links between importin-
and microtubules suggests that importin-
acts as the host adaptor to recognizes the T-complex via NLS interaction and link it to microtubules, thus providing the active transport network for transport to the nucleus. Such a novel model presents a powerful system to offer insights into the trafficking of infecting viruses to the eukaryote host nucleus during the early stage of infection. Chapter Literature review 1.1 Background to Agrobacterium tumefaciens Agrobacterium tumefaciens is a gram-negative soil borne bacteria and a natural phytopathogen of a wide variety of plant species (van Larebeke et al. 1974; Waston et al. 1975). Agrobacterium tumefaciens (or Bacterium tumefaciens as it was then known) has been extensively studied since it was identified as the causative agent of crown gall disease (Smith et al. 1907). Initial research interest focused of a possible link between Agrobacterium-mediated tumour formation and mammalian cancerous growth. However research focus quickly changed when the full potential of using A. tumefaciens as a “natural genetic engineer” became clear. It was Braun’s “tumour inducing principle,” hypothesis that first attempted to link Agrobacterium to tumour induction via a vector (possibly DNA) that was capable of maintaining plant cells in a state of active cell division (Braun 1947; Braun and Mandle, 1948). Evidence that bacterial DNA was indeed present in cultured crown gall tumours was eventually found (Schilperoort et al. 1967) but still debated until latter confirmation and the identification of the tumour inducing (Ti) plasmid (van Larebeke et al. 1974; van Larebeke et al. 1975; Zaenen et al. 1974). Later studies found genetic elements derived from the Ti-plasmid were transferred into the host plant cell (Chilton et al. 1977; Chilton et al. 1978; Depicker et al. 1978). This transfer DNA, or T-DNA, encodes opine biosynthesis genes and oncogenes which are responsible for production of plant growth regulators thus triggering tumorous growths. The A. tumefaciens induced tumors are a source of auxin (Link et al. 1941) and cytokinin (Braun, 1958) both of which are plant growth regulators. The benefit for the Agrobacterium inducing agent is due to the fact that opines (unusual amino acid-like compounds) are Nam, J., Mysore, K.S., Gelvin, S.B. (1998) Agrobacterium tumefaciens transformation of the radiation hypersensitive Arabidopsis thaliana mutants uvh1 and rad5. Mol Plant Microbe Interact. 11, 1136-1141 Nam, J., Mysore, K.S., Zheng, C., Knue, M.K., Matthysse, A.G., Gelvin, S.B. (1999) Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium. Mol Gen Genet. 261, 429-438 Naqvi, S.N., Zahn, R., Mitchell, D.A., Stevenson, B.J., Munn, A.L. (1998) The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Curr Biol. 8, 959-962 Narasimhulu, S.B., Deng, X.B., Sarria, R., Gelvin, S.B. (1996) Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell. 8, 873-886 Neff, N.F., Thomas, J.H., Grisafi, P., Botstein, D. (1983) Isolation of the betatubulin gene from yeast and demonstration of its essential function in vivo. Cell. 33, 211-219 Neff, N.T., Binns, A.N. (1985) Agrobacterium tumefaciens interaction with suspension-cultured tomato cells. Plant Physiol. 77, 35-42 Neff, N.T., Binns, A.N., Brandt, C. (1987) Inhibitory effects of a pectinenriched tomato cell wall fraction on Agrobacterium tumefaciens binding and tumor formation. Plant Physiol. 83, 525-528 Nikolov, D.B., Hu, S-H., Lin, J., Gasch, A., Hoffmann, A., Horikoshi, M., Chua, N-H., Roeder, R.G., Burley, S.K. (1992) Crystal structure of a TFIID TATA-box binding protein. Nature. 360, 40-46 Nishikawa, M., Suzuki, K., Yoshida, K. (1992) DNA integration into recipient yeast chromosomes by trans-kingdom conjugation between Escherichia coli and Saccharomyces cerevisiae. Curr Genet. 21, 101-108 Nurnberger, T., Brunner, F., Kemmerling, B., Piater, L. (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev. 198, 249-266 Nykanen, A., Haley, B., Zamore, P.D. (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 107, 309-321 Offringa, R., De Groot, M.J., Haagsman, H.J., Does, M.P., Van den Elzen, P.J., Hooykaas, P.J. (1990) Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J. 9, 3077-3084 Otten, L., DeGreve, H., Leemans, J., Hain, R., Hooykaas, P. et al. (1984) Restoration of virulence of vir region mutants of Agrobacterium tumefaciens strain B6S3 by coinfection with normal and mutant Agrobacterium strains. Mol Gen Genet. 195, 159-163 Paine, P.L., Moore, L.C., Horowitz, S.B. (1975) permeability. Nature. 254, 109-114 Nuclear envelope Panchal, S.C., Kaiser, D.A., Torres, E., Pollard, T.D., Rosen, M.K. (2003) A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex. Nat Struct Mol Biol. 10, 591-598 Pansegrau, W., Lanka, E. (1991) Common sequence motifs in DNA relaxases and the nick regions from a variety of DNA transfer systems. Nucleic Acids Res. 19, 34-55 Pansergrau, W., Schoumacher, F., Hohn, B. Lanka, E. (1993) Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation. Proc Natl Acad Sci USA. 90, 11538-11542 Pansegrau, W., Schroder, W., Lanka, E. (1993b) Relaxase (TraI) of IncP alpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of singlestranded DNA. Proc Natl Acad Sci USA. 90, 2925-2929 Pansegrau, W., Lanka, E. Barth, P.T., Figurski, D.H., Guiney, D.G., Haas, D., Helinski, D.R., Schwab, H., Stanisich, V.A., Thomas, C.M. (1994a) Complete nucleotide sequence of Birmingham IncP
plasmids: compilation and comparative analysis. J Mol Biol. 239, 623-663 Pansegrau, W., Schroder, W., Lanka, E. (1994b) Concerted action of three distinct domains in the DNA cleaving-joining reaction catalyzed by relaxase (TraI) of conjugative plasmid RP4. J Biol Chem. 269, 2782-2789 Pansegrau, W., Lanka, E. (1996) Enzymology of DNA transfer by conjugative mechanisms. Prog Nuc Acid Res Mol Biol. 54, 197-251 Pantoja, M., Chen, L., Chen, Y., Nester, E.W. (2002) Agrobacterium type IV secretion is a two-step process in which export substrates associate with the virulence protein VirJ in the periplasm. Mol Microbiol. 45, 1325-1335 Parke, D., Ornston, L.N., Nester, E.W. (1987) Chemotaxis to plant phenolic inducers of virulence genes is constitutively expressed in the absence of the Tiplasmid in Agrobacterium tumefaciens. J. Bacteriol. 169, 5336-5338 Paszkowski, J., Baur, M., Bogucki, A., Potrykus, I. (1998) Gene targeting in plants. EMBO J. 7, 4021-4026 Paulsson, M., Wadstrom, T. (1990) Vitronectin and type-1 collagen binding by Staphylococcus aureus and coagulase-negative streptococci. FEMS Microbiol Immunol. 65, 55-62 Peifer, M. Berg, S., Reynolds, A.B. (1994) A repeating amino acid motif shared by proteins with diverse cellular roles. Cell. 76, 789-791 Pemberton, L.F., Rosenblum, J.S., Blobel, G. (1999) Nuclear import of the TATA-binding protein: mediation by the karyopherin Kap114p and a possible mechanism for intranuclear targeting. J Cell Biol. 145, 1407-1417 Petit, A., Delhaye, S., Tempe, J. and Morel, G. (1970) Researches sur les guanidines des tissues de Crown Gall. Mise en evidence d’une relation biochimiique specific entre les souches d’Agrobacterium tumefaciens et les tumours qu’elles induisent. Physiol Veg. 8, 205-213 Petit, A., and Tourneur, J. (1972) Perte de virulence associee a la perte d’une activite enzymatique chez Agrobacterium tumefaciens. C.R. Hebd Seances Acad Sci Ser D. 275, 137-139 Piers, K.L., Heath, J.D., Liang, X., Stephens, K.M. and Nester, E.W. (1996). Agrobacterium tumefaciens-mediated transformation of yeast. Proc Natl Acad Sci USA. 93, 1613-1618 del Pozo, J.C., Estelle, M. (2000) F-box proteins and protein degradation: an emerging theme in cellular regulation. Plant Mol Biol. 44, 123-128 Preuss, S.B., Jiang, C.Z., Baik, H.K., Kado, C.I., Britt, A.B. (1999) Radiation sensitive Arabidopsis mutants are proficient for T-DNA transformation. Mol Gen Genet. 261, 623-626 Puchta, H. (2003) Towards the ideal GMP: homologous recombination and marker gene excision. J Plant Physiol. 160, 743-754 Puvanesarajah, V., Schell, F.M., Stacey, G., Douglas, C.J., Nester, E.W. (1985) Role for 2-linked-
-D-glucan in the virulence of Agrobacterium tumefaciens. J Bacteriol. 164, 102-106 Radu, A., Blobel, G., Moore, M.S. (1995a) Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci USA. 92, 1769-1773 Radu, A., Moore, M.S., Blobel, G. (1995b) The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell. 81, 215-222. Raponi, M., Arndt, G.M. (2003) Double-stranded RNA-mediated gene silencing in fission yeast. Nucleic Acids Research. 31, 4481-4489 Rashkova, S., Spudich, G.M., Christie, P.J. (1997) Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase. J Bacteriol. 179, 583-591 Relic, B., Andjelkovic, M., Rossi, L., Nagamine, Y., and Hohn, B. (1998). Interaction of DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens: analysis by subcellular localization in mammalian cells. Proc Natl Acad Sci USA. 95, 9105-9110 Rexach, M., Blobel, G. (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell. 83, 683-692 Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C., Citovsky, V. (2000) A genetic system for detection of protein nuclear import and export. Nat Biotechnol. 18, 433-437 Rho, H.S., Kang, S., Lee, Y.H. (2001) Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol Cells. 12, 407-411 Richards, K.L., Anders, K.R., Nogales, E., Schwartz, K., Downing, K.H., Botstein, D. (2000) Structure-function relationship in yeast tubulins. Mol Bio Cell. 11, 1887-1903 Riddick, G., Macara, I.G. (2005) A systems analysis of importin alpha – beta mediated nuclear protein import. J Cell Biol. 168, 1027-1038 Riggleman, R., Wieschaus, E., Schedl, P. (1989) Molecular analysis of the armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene. Genes Dev. 3, 96-113 Risseeuw, E.P., Franke-van Dijk, M.E., Hooykaas, P.J. (1996) Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair. Mol Cells Biol. 16, 59245932 Roberts, R.L., Metz, M., Monks, D.E. Mullaney, M.L., Hall, T., Nester, E.W. (2003) Purine synthesis and increased Agrobacterium tumefaciens transformation of yeast and plants. Proc Natl Acad Sci USA. 100, 6634-6639 Rodal, A.A., Sokolova, O., Robins, D.B., Daugherty, K.M., Hippenmeyer, S., Riezman, H., Grigorieff, N., Goode, B.L. (2005) Conformational changes in the Arp2/3 complex leading to actin nucleation. Nat Struct Mol Biol. 12, 26-31 Rogers, C.W., Challen, M.P., Green, J.R., Whipps, J.M. (2004) Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus Coniothyrium minitans. FEMS Microbiol Lett. 241, 207-214 Rolland, S., Jobic, C., Fevre, M., Bruel, C. (2003) Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences. Curr Genet. 44, 164-171 Rossi, L., Hohn, B., Tinland, B. (1993) The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants. Mol Gen Genet. 239, 345-53 Rossi, L., Hohn, B., Tinland, B. (1996) Integration of the complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens. Proc Natl Acad Sci USA. 93, 126-130 Rosso, M.G., Li, Y., Strizhov, N., Reiss, B., Dekker, K., Weisshaar, B. (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequencing tag-based reverse genetics. Plant Mol Biol. 53, 247-259 Saito, T., Ishiguro, S., Ashida, H., Kawamukai, M., Matsuda, H., Ochiai, H., Nakagawa, T. (1995) Cloning and sequencing analysis of genes for cyclophilin from Arabidopsis thaliana. Plant Cell Physiol. 36, 377-382 Salman, H., Abu-Arish, A., Oliel, S., Loyter, A., Klafter, J., Granek, R., Elbaum, M. (2005) Nuclear localization signal peptides induce molecular delivery along microtubules. Biophysical J. 89, 2134-2145 Sambrook, J.F., Fritsch, E.F., Maniatis, T. (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. Sambrook, J.F., Russell, T. (2001) Molecular cloning: A laboratory manual (3rd edition). Cold Spring Harbor Laboratory Press. Cold Spring Harbor N.Y. Samejima, I., Matsumoto, T., Nakaseko, Y., Beach, D., Yanagida, M. (1993) Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis. J Cell Sci. 105, 135-143 Sauter, C. (1995) Is Hodgkin’s disease a human counterpart of bacterially induced crown-gall tumours? Lancet. 346, 1433 Savvides, S., Yeo, H.-J., Beck, M.R., Blaesing, F., Lurz, R., Lanka, E., Buhrdorf, R., Fischer, W., Haas, R., Waksman, G. (2003) VirB11 ATPases are dynampic hexameric assemblies: new insights into bacterial type IV secretion. EMBO J. 22, 1969-1980 Sawasaki, Y., Inomata, K., Yoshida, K. (1996) Trans-kingdom conjugation between Agrobacterium tumefaciens and Saccharomyces cerevisiae, a bacterium and a yeast. Plant Cell Physiol. 37, 103-106 Scheifflele, P., Pansegrau, W. Lanka, E. (1995) Initiation of Agrobacterium tumefaciens T-DNA processing. Purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro. J Biol Chem. 270, 1269-1276 Schell, J., Van Montagu, M., De Beuckeleer, M., De Block, M. Depicker, A., De Wilde, M., Engler, G., Genetello, C., Hernalsteens, J.P., Holsters, M., Seurinck, J., Silva, B., Van Vliet, F., Villarroel, R. (1979) Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host. Proc R Soc Lond. B204, 251-266 Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P.C., Baron, C. (1999) Vir proteins stabilize VirB5 and mediate its association with the T-pilus of Agrobacterium tumefaciens. J Bacteriol. 181, 7485-7492 Schatz, P.J., Pillus, L., Grisafi, P., Solomon, F., Botstein, D. (1986a) Two functional
-tubulin genes of the Saccharomyces cerevisiae encode divergent proteins. Mol Cell Biol. 6, 3711-3721 Schatz, P.J., Solomon, F., Botstein, D. (1986b) Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins. Mol Cell Biol. 6, 3722-3733 Schrammeijer, B., Risseeuw, E., Pansegrau, W., Regensburg-Tuink, T.J.G., Crosby, W.L., Hooykaas, P.J.J. (2001) Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein. Curr Biol. 11, 258-262 Schrammeijer, B., den Dulk-Ras, A., Vergunst, A.C., Jacome, E.J., Hooykaas, P.J.J. (2003) Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3. Nucleic Acids Res. 31, 860-868 Schulein, R., Guye, P., Rhomberg, T.A., Schmid, M.C., Schroder, G., Verhunst, A.C., Carena, I., Dehio, C. (2005) A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc Natl Acad Sci USA. 102, 856-861 Sciaky, D., Montoya, A.L., Chilton, M.D. (1978) Agrobacterium Ti plasmids. Plasmid. 1, 238-253 Fingerprints of Seisenberger, G., Ried, M.U., Endress, T., Buning, H., Hallek, M., Brauchle, C. (2001) Real-time single-molecule imagining of the infection pathway of an adeno-associated virus. Science. 294, 1929-1932 Sen. P., Pazour, J., Anderson, D., Das, A. (1989) Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA. J Bacteriol. 171, 2573-2580 Shah, J. (2003) The salicylic acid loop in plant defense. Curr Opin Plant Biol. 6, 365-371 Shaw, C.H.J., Ashby, A.M., Brown, A., Royal, C., Loake, G.J. Shaw, C.H. (1989) VirA and G are necessary for acetosyringone chemotaxis in Agrobacterium tumefaciens. Mol Microbiol. 2, 413-417 Sheng, J., Citovsky, V., (1996) Agrobacterium-plant cell interaction: have virulence proteins will travel. Plant Cell. 8, 1699-1710 Sheff, M.A., Thorn, K.S. (2004) Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast. 21, 661-670 Shiozaki, K., Russell, P. (1995) Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature. 378, 739-743 Shirasu, K., Koukolikova-Nicloa, Z., Hohn, B., Kado, C.I. (1994) An innermembrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activitly and similarities to conjugative transfer genes. Mol Microbiol. 11, 581-588 Shulga, N., Roberts, P., Gu, Z., Spitz, L., Tabb, M.M., Nomura, M., Goldfarb, D.S. (1996) In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation. J Cell Biol. 135, 329-339 Shurvinton, C.E., Hodges, L., Ream, W. (1992) A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation. Proc Natl Acad Sci USA. 89, 11837-41 Sigova, A., Rhind, N., Zamore, P.D. (2006) A single argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. Gene and Dev. 18, 2359-2367 Simone, M., McCullen, C.A., Stahl, L.E., Binns, A.N. (2001) The carboxyterminus of VirE2 from Agrobacterium tumefaciens is required for its transport to host cells by the virB-encoded type IV transport system. Mol Microbiol. 41, 1283-1293 Sipiczki, M. (1995) Phylogenesis of fission yeasts. Contradictions surrounding the origin of century old genus. Antonie van Leeuwenhoek. 68, 119-149 Sipiczki, M. (2001) Where does fission yeast sit on the tree of life? Genome Biol. 1. 1011.1-1011.4 Smardon, A., Spoerke, J.M., Stacey, S.C., Klein, M.E., Mackin, N., Maine, E.M. (2000) EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans. Curr Biol. 10, 169-178 Smit, G., Logman, T.J.J., Boerrigter, M., Kijne, J.W., Lugtenberg, B.J.J. (1989) Purification and partial characterization of the Rhizobium leguminosarum biovar Ca2+ -dependent adhesin which mediates the first step in attachment of cells of the family Rhizobiaceae to plant root hair tips. J Bacteriol. 171, 4054-4062 Smith, E.F., Townsend, C.O. (1907) Science, 25, 671-673 A plant tumor of bacterial origin. Smith, H.M., Raikhel, N. (1998) Nuclear localization signal receptor importin
associates with the cytoskeleton. The plant Cell. 10, 1791-1799 Sodeik, B., Ebersold, M.W., Helenius, A. (1997) Microtubule-mediated transport of incoming herpes simplex virus capsids to the nucleus. J Cell Biol. 136, 1007-1021 Solsbacher, J., Maurer, P., Bischoff, F.R., Schlenstedt, G. (1998) Cse1p is involved in export of yeast importin alpha from the nucleus. Mol Cell Biol. 18, 6805-6815 Solsbacher, J., Maurer, P., Vogel, F., Schlenstedt, G. (2000) Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha. Mol Cell Biol. 20, 8468-8479 Sonti, R.V., Chiurazzi, M., Wong, D., Davis, C.S., Harlow, G.R., Mount, D.W., Signer, E.R. (1995) Arabidopsis mutants deficient in T-DNA integration. Proc Natl Acad Sci USA. 92, 11786-11790 Spudich, G.M., Fernandez, D., Zhou, X.-R., Christie, P.J. (1996) Intermolecular disulphide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus. Proc Natl Acad Sci USA. 93, 7512-7515 Stachel, S.E., Timmerman, B. Zambryski, P. (1987) Activation of Agrobacterium tumefaciens vir gene expression generates multiple singlestranded T-strand molecules from the pTiA6 T-region: requirement for 5’ virD gene products. EMBO J. 6, 857-863 Stachel, S.E., Zambryski, P.C. (1986a) virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 46 325-333 The genetic and transcriptional Stachel, S.E., Nester, E.W. (1986b) organisation of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. Nature. 318, 624-629 Stachel, S.E., Nester, E.W., Zambryski, P.C. (1986c) A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci USA. 83, 379-383 Stade, K., Ford, C.S., Guthrie, C., Weis, K. (1997) Exportin (Crm1p) is an essential nuclear export factor. Cell. 90, 1041-1050 Stahl, L.E., Jacobs, A. Binns, A.N. (1998) The conjugal intermediate of plasmid RSF1010 inhibits Agrobacterium tumefaciens virulence and VirBdependent export of VirE2. J Bacteriol. 180, 3933-3939 Steck, T.R., Close, T.J., Kado, C.I. (1989) High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid. Proc Natl Acad Sci USA. 86, 2133-2137 Sugui, J.A., Chang, Y.C., Kwon-Chung, K.J. (2005) Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol. 71, 1798-1802 Suikkanen, S., Aaltonen, T., Nevalainen, M., Valilehto, O., Lindholm, L., Vuento, M., Vihinen-Ranta, M. (2003) Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus. J Virol. 77, 10270-10279 Sullivan, T.D., Rooney, P.J., Klein, B.S. (2002) Agrobacterium tumefaciens integrates transfer DNA into single chromosomal sites of dimorphic fungi and yields homokaryotic progeny from multinucleate yeast. Eukaryot Cell. 1, 895905 Sundberg, C., Meek, L., Carroll, K., Das, A., Ream, W. (1996) VirE1 protein mediates export of the single-stranded DNA binding protein VirE2 from Agrobacterium tumefaciens into plant cells. J Bacteriol. 178, 1207-1212 Sundberg, C.D., Ream, W. (1999) The Agrobacterium tumefaciens chaperonelike protein, VirE1, interacts with VirE2 at domains required for single-stranded DNA binding and cooperative interaction. J Bacteriol. 181, 6850-6855 Suomalainen, M., Nakano, M.Y., Keller, S., Boucke, K., Stidwill, R.P., Greber, U.F. (1999) Microtubule-dependent plus- and minus-end directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol. 144, 657-672 Swart, S., Smit, G., Lugtenberg, B.J.J., Kijne, J.W. (1993) Restoration of attachment, virulence and nodulation of Agrobacterium tumefaciens chvB mutants by rhicadhesin. Mol Microbiol. 10, 597-605 Swart, S., Logman, T.J., Smit, G., Lugtenberg, B.J., Kijne, J.W. (1994) Purification and partial charaterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin, an attachment protein of Rhizobiacae. Plant Mol Biol. 24, 171-183 Swart, S., Lugtenberg, B.J.J., Smit, G., Kijne, J.W. (1994b) Rhicadhesinmediated attachment and virulence of an Agrobacterium tumefaciens chvB mutant can be restored by growth in a highly osmotic medium. J Bacteriol. 176, 3816-3819 Szabados, L., Kovacs, I., Oberschall, A., Abraham, E., Kerekes, I., Zsigmond, L., Nagy, R., Alvarado, M., Krasovskaja, I., Gal, M., Berente, A., Redei, G.P., Haim, A.B., Koncz, C. (2002) Distribution of 1000 sequenced TDNA tags in the Arabidopsis genome. Plant J. 32, 233-242 Tabara, H., Yigit, E., Siomi, H., Mello, C.C. (2002) The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, an a DExH-box helicase to direct RNAi in C. elegans. Cell. 109, 861-871 Tanguay, P., Breuil, C. (2003) Transforming the sapstaining fungus Ophiostoma piceae with Agrobacteria tumefaciens. Can J Microbiol. 49, 301304 Tao, Y., Rao, P.K., Bhattacharjee, S., Gelvin, S.B. (2004) Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium Tcomplex protein VirD2. Proc Natl Acad Sci USA. 101, 5164-5169 Telotte, H., Davis, I. (2002) Intracellular mRNA localization: motors move messages. Trends Genet. 18, 636-642 Thomas, J.H., Neff, N.F., Botstein, D. (1985) Isolation and characterisation of mutants in the -tubulin gene of Saccharomyces cerevisiae. Genetics. 112, 715734 Tijsterman, M., Tasseron-de Jong, J.G., Verhage, R.A., Brouwer, J. (1998) Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not genome nucleotide excision repair. Mutat Res. 409, 181-188 Timeny, B.L., Tetenbaum-Novatt, J., Agate, D.S., Williams, R., Zhang, W., Chait, B.T., Rout, M.P. (2006) Simple kinetic relationships and non-specific competition govern nuclear import rates in vivo. J Cell Biol. 175, 579-593 Tinland, B., Koukolikova-Nicola, Z., Hall, M.N., Hohn, B. (1992) The TDNA-linked VirD2 protein contains two distinct functional nuclear localisation signals. Proc Natl Acad Sci USA. 89, 7442-46 Tinland, B., Schoumacher, F., Gloeckler, V., Bravo-Angel, A.M., Hohn, B. (1995) The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome. EMBO J. 14, 3585-3595 Tinland, B. (1996) The integration of T-DNA into plant genomes. Trends Plant Sci. 1, 178-184 Toda, T., Adachi, Y., Hiraoka, Y., Yanagida, M. (1984) Identification of the pleiotropic cell division cycle gene NDA2 as one of two different
-tubulin genes in Schizosaccharomyces pombe. Cell. 37, 233-242 Toi, H., Fujimura-Kamada, K., Irie, K., Takai, Y., Todo, S., Tanaka, K. (2003) She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae. Mol Biol Cell. 14, 2237-2249 Toone, W.M., Kuge, S., Samuels, M., Morgan, B.A., Toda, T., Jones, N. (1998) Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stressactivated MAP kinase styl/Spc1. Genes Dev. 12, 1453-1463 Toro, N., Datta, A., Carmi, O.A., Young, C., Prusti, R.K., Nester, E.W. (1989) The Agrobacterium tumefaciens virC1 gene product binds to overdrive a T-DNA transfer element. J Bacteriol. 171, 6845-6849 Turk, S.C.H.J., Van Lange, R.P., Regensburg-Tuink, Tonny, J.G., Hooykaas, P.J.J. (1994) Localisation of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens. Plant Mol Biol. 25, 899-907 Tzfira, T. Citovsky, V. (2000) From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-plant cell interaction. Mol. Plant Pathol. 1, 201-212 Tzfira, T., Vaidya, M., Citovsky, V. (2001) VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J. 20, 3596-3607 Tzfira, T., Vaidya, M., Cytovsky, V. (2002) Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis VIP1 gene. Proc Natl Acad Sci USA. 99, 10435-10440 Tzfira, T., Li, J., Lacroix, B., Citovsky, V. (2004) Agrobacterium T-DNA integration: molecules and models. Trends in Genetics. 20, 375-383 Tzfira, T., Vaidya, M., Citovsky, V. (2004b) Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature. 431, 8792 Tzfira, T., Citovsky, V. (2005) Nuclear import of Agrobacterium T-DNA in nuclear import and export in plants and animals. Springer Publishing, page 8399 Tzfira, T. (2006) On tracks and locomotives: the long route of DNA to the nucleus. Trends in Microbiology. 14, 61-63 Umeda, M., Izaddoost, S., Cushman, I., Moore, M.S., Sazer, S. (2005) The fission yeast Schizosaccharomyces pombe has two importin-
proteins, Imp1p and Cut15p, which have common and unique functions in nucleocytoplasmic transport and cell cycle progression. Genetics. 171, 7-21 Vandromme, M. Gauthier-Rouviere, C., Lamb, N., Fernandez, A. (1996) Regulation of transcription factor localization: fine-tuning of gene expression. Trends Biochem Sci. 21, 59-64 Veena, Jiang, H., Doerge, R.W., Gelvin, S.B. (2003) Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression . Plant J. 35, 219-236 Vaughan, M.A. (1991) Evaluation of phylogenetic relationships among fission yeast by nDNA/nDNA reassociation and conventional taxonomic criteria. Yeast. 7, 73-78 Veluthambi, K., Ream, W., Gelvin, S.B. (1988) Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens. J Bacteriol. 170, 1523-1532 Vergunst, A.C., Schrammeijer, B., den Dulk-Ras, A., de Vlaam, C.M.T., Regensburg-Tuink, T.J.G., Hooykaas, P.J. (2000) VirB/D4 dependent protein translocation from Agrobacterium into plant cells. Science. 290, 979-982 Vergunst, A.C., van Lier, M.C., den Dulk-Ras, A., Hooykaas, P.J. (2003) Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1. Plant Physiol. 133, 978-988 Vergunst, A.C., van Lier, M.C., den Dulk-Ras, A., Stuve, T.A., Ouwehand, A., Hooykaas, P.J. (2005) Positive charge is an important feature of the Cterminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc Natl Acad Sci USA. 102, 832-837 Vichi, P., Coin, F., Renaud, J.P., Vermeulen, W., Hoeijmakers, J.H., Moras, D., Egly, J.M. (1997) Cisplatin- and UV-damaged DNA lure the basel transcription factor TFIID/TBP. EMBO J. 16, 7444-7456 de Vit, M.J., Johnston, M. (1999) The nuclear exportin Msn5 is required for nuclear export of the Mig1 glucose repressor of Saccharomyces cerevisiae. Curr Biol. 9, 1231-1241 Vogel, A.M. and Das, A. (1992) Mutational analysis of Agrobacterium tumefaciens virD2: tyrosine 29 is essential for endonuclease activity. J Bacteriol. 174, 303-308 Vogel, A.M., Yoon, J., Das, A. (1995) Mutational analysis of a conserved motif of Agrobacterium tumefaciens VirD2. Nucl Acids Res. 23, 4087-4091 Voinnet, O.,Rivas, S., Mestre, P., Baulcombe, D. (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949-956 Voinnet, O. (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet. 6, 206-220 Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I., Martienssen, R.A. (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science. 297, 1833-1837 Wagner, V.T., Matthysse, A.G. (1992) Involvement of vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells. J Bacteriol. 174, 5999-6003 Wallar, B.J., Alberts, A.S. (2003) The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 13, 435-446 Wang, K., Stachel, S.E., Timmerman, B., Van Montagu, M., Zambryski, P. (1987) Site-specific nick occurs within the 25bp transfer promoting border sequence following induction of vir gene expression in Agrobacterium tumefaciens. Science. 235, 587-591 Waston, B. Currier, T.C. Gordon, M.P., Chilton, M.D., Nester, E.W. (1975). Plasmid required for virulence in Agrobacterium tumefaciens. J Bacteriol. 123, 255-264 Weber, S., Horn, R., Friedt, W. (1998) Isolation of a low-copy plasmid from Agrobacterium using QIAprep
technology. QIAGEN News. 5, 11-12 Welch, M.D., Iwamatsu, A., Mitchison, T.J. (1997) Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature. 385, 265-269 Welch, M.D., Mullins, R.D. (2002) Cellular control of actin nucleation. Annu Rev Cell Dev Biol. 11, 370-374 Wendland, B., McCaffery, J.M., Xiao, Q., Emr, S.D. (1996) A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J Cell Biol. 135, 1485-1500 Wertman, K.F., Wyman, A.R., Botstein, D. (1986) Host/vector interactions which affect the viability of recombinant phage lambda clones. Gene. 49, 253262 Winans, S.C. (1991) An Agrobacterium two-component regulatory system for the detection of chemicals released from plant wounds. Mol Microbiol. 5, 23452350 Winans, S.C. (1992) Two-way chemical signalling in Agrobacterium-plant interactions. Microbiol Rev. 56, 12-31 Winkler, R.G., Frank, M.R., Galbraith, D.W., Feyereisen, R., Feldmann, K.A. (1998) Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Plant Physiol. 118, 743-750 Winsor, B., Schiebel, E. (1997) Review: An overview of the Saccharomyces cerevisiae microtubule and microfilament cytoskeleton. Yeast. 13, 399-434 Winter, D.C., Choe, E.Y., Li, R. (1999) Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits. Proc Natl Acad Sci USA. 96, 7288-7293 Wood, V., et al. (2002) The genome sequence of Schizosaccharomyces pombe. Nature. 415. 871-880 Xiang, C., Han, P., Lutziger, I., Wang, K., Oliver, D.J. (1999) A mini binary vector series for plant transformation. Plant Mol Biol. 40, 711-717 Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis, A.D., Zilberman, D., Jacobsen, S.E., Carrington, J.C. (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, E104 Yanisch-Perron, C., Vieira, J., Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 33, 103-119 Yano, R., Oaks, M., Yamagishi, M., Vu, L., Nomura, M. (1992) Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae. Mol Cell Biol. 12, 56405651 Yano, R., Oaks, M., Tabb, M.M., Nomura, M. (1994) Yeast Srp1p has homology to armadillo/plakoglobin/
-catenin and participates in apparently multiple nuclear functions including the maintenance of nucleolar structure. Proc Natl Acad Sci USA. 91, 6880-6884 Yanofsky, M.F., Porter, S.G., Young, C., Albright, L.M., Gordon, M.P., Nester, E.W. (1986) The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell, 47, 471-477 Yi, H., Mysore, K.S., Gelvin, S.B. (2002) Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation. Plant J. 32, 285-298 Yoshida, K., Blobel, G. (2001) The karyopherin Kap142pMsn5p mediates nuclear import and nuclear export of different cargo proteins. J Cell Biol. 152, 729-740 Yoshida, M., Sazer, S. (2004) Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe. Methods. 33, 226-238 Yusibov, V.M., Steck, T.R., Gupta, V. Gelvin, S.B. (1994) Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc Natl Acad Sci USA. 91, 2994-2998 Zaenen, I., Van Larebeke, N., Teuchy, H., Van Montagu, M., Schell, J. (1974) Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. J Mol Biol. 86, 109-127 Zambryski, P. (1992) Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol. 43, 465-490 Zamore, P.D., Tuschi, T., Sharp, P.A., Bartel, D.P. (2000) RNAi: Doublestranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 101, 25-33 Zamore, P.D. (2002) Ancient pathways programmed by small RNAs. Science. 296, 1265-1269 Zeilinger, S. (2004) Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation. Curr Genet. 45, 54-60 Zhang, A., Tkacz, J.S., An, Z. (2003) Efficient disruption of a polyketide synthase gene (psk1) required for melanin synthesis through Agrobacteriummediated transformation of Glarea lozoyensis. Mol Genet Genomics. 268, 645655 Zhao, Z., Sagulenko, E., Ding, Z., Christie, P.J. (2001) Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via Agrobacterium type IV secretion pathway. J Bacteriol. 183, 38553865 Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, Y. et al. (2003) Identification of Arabidopsis rat mutants. Plant Physiol. 132, 494-505 Zhu, Y., Nam, J., Carpita, N.C., Matthysse, A.G., Gelvin, S.B. (2003b) Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene. Plant Physiol. 133, 1000-1010 Ziemienowicz, A., Gorlich, D., Lanka, E., Hohn, B., Rossi, L. (1999) Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium. Proc Natl Aacd Sci USA 96, 3729-3733 Ziemienowicz, A., Tinland, B., Bryant, J., Gloeckler, V., Hohn, B. (2000) Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro. Mol. Cell. Biol. 20, 63176322 Ziemienowicz, A., Merkle, T., Schoumacher, F., Hohn, B., Rossi, L. (2001) Import of the Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins. Plant Cell. 13, 369-384 Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller, T., Felix, G. (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell. 125, 749-760 Zupan, J., Citovsky, V., Zambryski, P. (1996) Agrobacterium VirE2 protein mediates nuclear uptake of ssDNA in plant cells. Proc Natl Acad Sci USA. 93, 2392-2397 Zupan, J., Zambryski, P. (1997) The Agrobacterium DNA transfer complex. Crit Rev Plant Sci. 16, 279-295 Zupan, J.R., Ward, D., Zambryski, P. (1998) Assembly of the VirB transport complex for DNA transfer from Agrobacteria tumefaciens to plant cells. Curr Opin Microbiol. 1, 649-655 Zupan, J., Muth, T.R., Draper, O., Zambryski, P. (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J. 23, 11-28 Zwiers, L.H., De Waard, M.A. (2001) Efficient Agrobacterium tumefaciensmediated gene disruption in the phytopathogen Mycosphaerella graminicola. Curr Genet. 39, 388-393 [...]... that exhibit a decreased susceptibility to such transformation (Zhu et al 2003) 1.3 The function of the translocated virulence protein in the host If Agrobacterium is to successfully and stably transfect of plant tissue post TDNA translocation it is important that, the T- DNA be protected from nuclease activity, the T- DNA be transported to the nucleus where processing to dsDNA and integration into the... products are responsible for the generation and transport of the transfer DNA (T- DNA) into the host cell The T- DNA itself is located between two flanking 25bp imperfect direct repeats termed the T- borders” Virulence proteins that associate with the T- DNA are said to be constituents of the T- complex Another subset of 12 virulence proteins form a membrane associated molecular needle” to transport the T- complex... Interestingly these mutant VirE2 proteins do not interfere with T- DNA or wild-type VirE2 export again supporting the hypothesis that VirE2 and the T- strand are exported independently (Simone et al 2001) This corroborative evidence supports the theory that VirE2 and the T- strand can be translocated into the host independently of one another and that VirE2 association with the T- strand can occur post translocation... inoculated on wild type tobacco plants (Citovsky et al 1992) This complementation study demonstrates the ability of VirE2 to function within the plant cell, independently of co-production and translocation with the T- strand In addition, a VirE2 harbouring a C-terminal mutation prevents recruitment and secretion through the VirB/D4 channel yet it retains the ability to bind single stranded DNA Interestingly... the T- DNA to the host nucleus is a product of the protein constituents of the T- complex As previously discussed the current evidence leans towards the theory that VirE2 associates with the T- strand once inside the host cell as oppose to pre-translocation form Regardless, it is assumed that the binding of VirE2 to the single strand T- DNA occurs prior to nuclear import This assumption is supported by the... indicating VirD4 role in substrate recruitment from the cytoplasm (Pantoja et al 2002; Kumar and Das 2002) 1.2.7 Mechanism of T- DNA Export Recent advances have not only shed light on how substrates are targeted but also their passage through the transport channel For efficient transformation of the host to occur it is essential that the Agrobacterium select the T- strand for export along with the additional... Additional complementation studies have shown that mutant virE1 Agrobacterium strains can still transfer T- DNA into the host but not the VirE2 protein (Sundberg et al 1996) Furthermore, virE2 mutants retain the ability to transform tobacco protoplasts (Yusibo et al 1994) However, virE mutant Agrobacterium can still incite tumor production if inoculated on VirE2 expressing transgenic tobacco but not if... 1995) It is the T- strand (T- DNA with VirD2 covalently 5’bound) in conjunction with VirE2 that is collectively known as the T- complex This T- complex is prediceted to conform to a coiled “telephone cord” like structure under certain conditions (Citovsky et al 1997) One point of contention is whether or not the T- complex is formed prior to, or after T- strand translocation into the host cell Originally it was... separate entity from VirE2 Numerous independent studies have highlighted the fact that VirE2 and the Tstrand are translocated into the host cell separately prior to T- complex formation Initial suspicions that VirE2 associates with the T- DNA within the host cell were raised over 2 decades ago (Otten et al 1984) A plant wound site was inoculated with two individually avirulent Agrobacterium strains One strain... have significantly clarified our understanding by which the T- strand is transported The research supported VirD4 as the first point of contact for the T- strand in addition to proposing the sequence of subsequent VirB -T- strand interactions The data is consistent with the preperceived position of Vir proteins within the channel and indicates the T- strand interacts with VirD4 followed by VirB11, VirB6, VirB8 . 25bp imperfect direct repeats termed the T- borders”. Virulence proteins that associate with the T- DNA are said to be constituents of the T- complex. Another subset of 12 virulence proteins form. events occur that leads from the expression of virulence genes and processing of the single stranded T- DNA from the Ti-plasmid to the transport of the T- DNA and associated proteins via a type. independent studies have highlighted the fact that VirE2 and the T- strand are translocated into the host cell separately prior to T- complex formation. Initial suspicions that VirE2 associates with