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Molecular and biochemical characterization of the role of the Cucumber necrosis virus coat protein in… Hui, Elizabeth Lai-wun 2009

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MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF THE ROLE OF THE CUCUMBER NECROSIS VIRUS COAT PROTEIN IN PARTICLE STRUCTURE AND SUBCELLULAR TARGETING by ELIZABETH LAI-WUN HUI  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Plant Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2009 © Elizabeth Lai-Wun Hui, 2009  Abstract The Cucumber necrosis virus (CNV) particle is a T = 3 icosahedron composed of 180 identical coat protein (CP) subunits. Each subunit consists of: an RNA binding domain (R); a S domain that forms the shell and a protruding domain (P) that projects from the particle surface. The R and S domains are connected by a 34 aa arm. The aim of this study was to investigate the role of the CP N-terminal region in particle structure and subcellular targeting. Results of deletion CP mutant analyses indicate that mutants lacking the β region of the arm are capable of producing particles in infected plants; however, mutants lacking the complete arm or the ε region do not produce particles. β deletion mutant particles are less thermally stable and, under conditions where wild-type CNV particles “swell”, β(-) mutant particles disassemble, reinforcing the role of the β region in particle stability. In addition, it was found that β(-) mutant particles bind fungal zoospores less efficiently and are not fungally transmissible. GFP fusion protein constructs of CP R and arm deletion mutants were used in agro-infiltration and confocal analyses to assess the role of these regions in subcellular targeting. The studies show that specific regions in the CP N-terminus are involved in mitochondrial and chloroplast targeting. Western blot and organellar subfractionation studies show that the R domain associates with the mitochondrial inner membrane and that the C-terminal 27 aa of the arm along with the first four aa of the S domain are sufficient for targeting fusion proteins to the chloroplast stroma. In addition, a 22 aa region at the N-terminus of the S domain facilitates entry of the respective fusion proteins into the chloroplast stroma and mitochondrial matrix. The arm region of R/arm/S22 GFP fusion protein associates with both mitochondria and chloroplasts; these results suggest a  ii  novel mode of dual-targeting. Together, these results demonstrate a multifunctional role for the N-terminal region of the CNV CP in virus structure and function. The possible role of subcellular targeting in virus assembly and disassembly is discussed.  iii  TABLE OF CONTENTS Abstract ............................................................................................................................... ii Table of Contents............................................................................................................... iv List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Abbreviations ...........................................................................................................x Acknowledgements.......................................................................................................... xiv Dedications ........................................................................................................................xv Co-Authorship Statement................................................................................................. xvi 1 CHAPTER ONE: Literature review ................................................................................1 1.1 Introduction...................................................................................................................1 1.2 Plant virus particles.......................................................................................................3 1.2.1 Rigid, rod-shaped and filamentous (helical) particles ...................................3 1.2.1.1 Rigid, rod-shaped particles...............................................................4 1.2.1.2 Filamentous particles........................................................................7 1.2.1.3 Icosahedral (spherical) particles .......................................................8 1.2.1.3.1 Tertiary structure of the CP subunit.................................9 1.2.1.3.2 Subunit packing .............................................................13 1.2.1.3.3 Virion assembly .............................................................15 1.2.1.3.4 Particle expansion ..........................................................18 1.3 Plant virus transmission ..............................................................................................20 1.3.1 Fungal transmission .....................................................................................22 1.3.1.1 Life cycle of Olpidium spp.............................................................23 1.3.1.2 In vitro transmission.......................................................................25 1.3.1.3 In vivo transmission........................................................................25 1.4 Subcellular targeting and protein import ....................................................................26 1.4.1 Targeting and import of chloroplast proteins...............................................28 1.4.1.1 TOC ................................................................................................31 1.4.1.2 TIC..................................................................................................33 1.4.1.3 Thylakoid protein import................................................................36 1.4.1.4 Alternative import pathways ..........................................................37 1.4.2 Targeting and import of mitochondrial proteins..........................................38 1.4.2.1 TOM ...............................................................................................41 1.4.2.2 TIM.................................................................................................45 1.4.3 Dual-targeting of chloroplast and mitochondrial proteins ..........................50 1.5 Cucumber necrosis virus.............................................................................................51 1.5.1 Genomic organization..................................................................................52  iv  1.5.2 Particle structure ..........................................................................................53 1.5.3 Targeting of the CNV CP to chloroplasts ....................................................55 1.6 Summary of thesis objectives .....................................................................................58 1.7 References...................................................................................................................60 2 CHAPTER TWO: Evaluation of specific regions of Cucumber necrosis virus coat protein arm on particle accumulation and virus transmission .........93 2.1 Introduction.................................................................................................................93 2.2 Materials and methods ................................................................................................95 2.2.1 Isolation and purification of virus.................................................................95 2.2.2 In vitro transcription.....................................................................................95 2.2.3 Leaf RNA extraction ....................................................................................96 2.2.4 Site-directed mutagenesis ............................................................................96 2.2.5 Analysis of the CP subunit ...........................................................................96 2.2.6 Virion RNA extraction .................................................................................98 2.2.7 In vitro swelling ...........................................................................................98 2.2.8 Electron microscopy ....................................................................................98 2.2.9 Fungus transmission assay...........................................................................98 2.2.10 In vitro binding assay.................................................................................99 2.2.11 Thermal stability and ribonuclease sensitivity assays..............................100 2.3 Results.......................................................................................................................100 2.3.1 Role of the β- and ε-regions of the CNV CP arm in particle accumulation .............................................................................................100 2.3.2 Particles of CNV β(-) mutants are not transmissible by O. bornovanus ...105 2.3.3 CNV 18β(-) mutants show decreased binding to zoospores in vitro .........105 2.3.4 β(-) particles disassemble at alkaline pH in the presence of EDTA ..........108 2.3.5 18β(-) particles are less thermally stable than WT CNV...........................108 2.4 Discussion .................................................................................................................111 2.5 References.................................................................................................................116 3 CHAPTER THREE: The N-terminal region of the Cucumber necrosis virus coat protein targets both chloroplasts and mitochondria ..................122 3.1 Introduction...............................................................................................................122 3.2 Materials and methods ..............................................................................................124 3.2.1 Preparation of Agrobacterium binary vector containing CNV CP constructs for agroinfiltration .....................................................................................124 3.2.2 Preparation of N. benthamiana protoplasts................................................126 3.2.3 Isolation of mitochondria...........................................................................127 3.2.4 Separation of mitochondrial fractions........................................................128 3.2.5 Isolation of chloroplasts.............................................................................129 3.2.6 Trypsin and thermolysin treatment of chloroplasts ...................................130 3.2.7 Confocal microscopy .................................................................................130 3.2.8 Western blot analyses ................................................................................131 3.3 Results and discussion ..............................................................................................131 3.3.1 A CNV R domain GFP fusion protein targets mitochondria.....................131  v  3.3.2  The first 47 amino acids of the R domain are required for efficient mitochondrial localization .........................................................................133 3.3.3 CNV R domain sequences are found in specific mitochondrial subfractions in agro-infiltrated plants ............................................................................138 3.3.4 The complete arm is not required for chloroplast targeting ......................145 3.3.5 The N–terminal 22 aa sequence in the CNV CP S domain influences the efficiency of transport of CNV CP GFP fusion proteins into mitochondria and chloroplasts.........................................................................................153 3.3.6 Mitochondrial targeting of R/arm/SSVRI and R/arm/S22 appears to precede chloroplast targeting ..................................................................................156 3.3.7 Dual targeting of R/arm/SSVRI and R/arm/S22.................................................................. 159 3.3.8 CNV CP is associated with both chloroplasts and mitochondria during CNV infection.....................................................................................................160 3.4 References.................................................................................................................163 4 CHAPTER FOUR: Concluding chapter ......................................................................170 4.1 General discussion ....................................................................................................170 4.2 References.................................................................................................................179 APPENDICES .................................................................................................................183 Appendix A Local lesion analysis of two CNV CP arm mutants...................................183 Appendix B The effect of calcium on interactions between Cucumber necrosis virus and Olpidium bornavanus zoospores ...............................................................184 B.1 Introduction........................................................................................184 B.2 Materials and methods .......................................................................185 B.2.1 Virus purification................................................................185 B.2.2 Maintenance of O. bornavanus cultures .............................185 B.2.3 Confocal microscopy assay ................................................185 B.2.4 Fungus transmission ...........................................................186 B.3 Results and discussion .......................................................................186 B.4 References..........................................................................................192  vi  List of Tables Table 1.1 Comparative features of typical rigid, rod-shaped and flexuous filamentous virus particles .....................................................................................................5 Table 1.2 Summary of chloroplast import proteins ..........................................................32 Table 1.3 Summary of mitochondrial import proteins......................................................42 Table 2.1 Primers used for PCR mutagenesis of CNV CP arm mutants..........................97 Table 3.1 Primers used for production of CNV CP constructs for agro-infiltration.......125  vii  List of Figures Figure 1.1 Comparative structures of helical TMV and PVX particles..............................6 Figure 1.2 Geometrical features of the two types of T = 3 capsids ..................................10 Figure 1.3 Structural features of a T = 3 icosahedral virus particle..................................11 Figure 1.4 Life cycle of Olpidium brassicae ....................................................................24 Figure 1.5 Schematic representation of the chloroplast protein import machineries .......29 Figure 1.6 Schematic representation of the mitochondrial protein import machineries...39 Figure 1.7 The genomic organization of CNV .................................................................54 Figure 1.8 A model for import of CNV CP and CNV particle uncoating on chloroplasts ......................................................................................................57 Figure 2.1 Description of the location of the CNV CP arm and deletion mutants .........101 Figure 2.2 Gel electrophoresis of leaf RNA, virions and CP subunit of arm mutants ...103 Figure 2.3 Electron micrographs of negatively stained native and swollen β(-) mutant particles ........................................................................................................104 Figure 2.4 Summary of fungus transmission assays of β(-) mutant particles.................106 Figure 2.5 Summary of in vitro virus-zoospore binding assays .....................................107 Figure 2.6 Agarose gel electrophoresis illustrating the thermostability and ribonuclease sensitivity of WT CNV and 18β(-) particles..................................................109 Figure 3.1 Diagrammatic representation of CNV CP GFP fusion constructs ................132 Figure 3.2 Confocal analysis of the subcellular location of the CNV R GFP fusion protein in agroinfiltrated N. benthamiana plants ...........................................134 Figure 3.3 Confocal and Western blot analyses of leaves agro-infiltrated with R domain constructs .......................................................................................................136 Figure 3.4 Summary of proposed cleavage sites and mitochondrial presequence-like features of the N-terminus of the R/arm/S22 region of the CNV CP .............139  viii  Figure 3.5 Confocal and Western blot analyses of the subcellular location of R, R47, R/arm/SSVRI, R/arm/S22, R19/arm/S22 and R/arm/SSVRI in agro-infiltrated plants ..............................................................................................................140 Figure 3.6 A model for the translocation and import of the CNV CP to chloroplasts and mitochondria ..................................................................................................146 Figure 3.7 Analysis of the location of various R and arm-containing constructs in agroinfiltrated plants .............................................................................................148 Figure 3.8 Confocal images of N. benthamiana leaves agro-infiltrated with CNV R19/arm/S22 and R19/arm/SSVRI constructs at 2dpi........................................149 Figure 3.9 Confocal microscopy of chloroplasts isolated from plants agro-infiltrated with R/arm/SSVRI, R/arm/S22, R6/arm/S22, arm27/S22, R19/arm/S22 and R/arm/SSVRI constructs was conducted to determine GFP localization ............................150 Figure 3.10 Western blot analyses of total protein extracted from leaves agro-infiltrated with the indicated three sets of fusion protein constructs containing the R/arm, R19/arm and arm regions and either SVRI or the first 22 amino acids of the Nterminus of the S domain ...............................................................................155 Figure 3.11 Western blot analysis of total protein extracted from chloroplasts and mitochondria isolated from R/arm/SSVRI and R/arm/S22 infiltrated leaves at 24 and 48 hpi.......................................................................................................158 Figure 3.12 Subcellular location of CNV CP in CNV-infected leaves...........................161 Figure A.1 Local lesion analysis of two CNV CP arm mutants.....................................183 Figure B.1 Confocal images of O. bornovanus zoospores treated with Fluo-4 only (in NaPO4 buffer), Fluo-4 labelled calcium or Fluo-4 labelled CNV particles .187 Figure B.2 Effect of calcium on transmission efficiency of CNV particles ...................189 Figure B.3 Effect of calcium channel blockers on transmission efficiency of CNV particles ........................................................................................................191  ix  List of Abbreviations A260 aa AAC ADP AMV ARM Asp ATP β BaMMV BMV BNYVV BPMV BSA o C Ca2+ CaCl2 cDNA CCMV ClpC CLSV CNV CO2 CP Cpn60 ct C-terminal DAS DI DNA dpi ds ε EDTA EGTA ELISA ER EtBr FHV fig. FNR g G GDD  absorbance at a wavelength of 260 nm amino acid ADP/ATP carrier adenosine diphosphate Alfalfa mosaic virus arginine-rich motif in the context of RNA aspartate adenosine triphosphate βeta Barley mild mosaic bymovirus Brome mosaic virus Beet necrotic yellow vein benyvirus Bean pod mottle virus bovine serum albumin degrees Celsius calcium ion calcium chloride complementary DNA Cowpea chlorotic mottle virus C-type lectin-like protein Cucumber leaf spot virus Cucumber necrosis virus carbon dioxide coat protein chaperonin60 chloroplast carboxy-terminal double-antibody sandwich defective interfering deoxyribonucleic acid days post-inoculation double-stranded epsilon ethylenediaminetetraacetic acid ethyleneglycol-bis(β-amino-ethylether)N,N’-tetraacetic acid enzyme-linked immunosorbent assay endoplasmic reticulum ethidium bromide Flock house virus figure ferredoxin NADPH reductase gram(s) in the context of mass; genome in the context of viral RNA GTP in the context of a binding domain glycine-aspartate-aspartate  x  GDP GFP GIP Gly Glu GTP hr h HCl HEPES Hsp or HSP IM IMP IMS J Kb KCl kDa KOH Lf µ m M MA MdmI MES μg mg MGD1 MGDG mi MIA min μl ml mM MOM MOPS MP MPP MSF NaCl NAD NADP NADPH nm  guanosine diphosphate green fluorescent protein general import pore glycine glutamate guanosine triphosphate hour(s) hinge in the context of the CNV CP hydrochloric acid 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid heat shock protein inner membrane intermembrane space peptidase inner membrane space J in the context of a DnaJ-like domain Kilobase potassium chloride Kilodalton(s) potassium hydroxide leaf micro milli molar in the context of concentration matrix mutants defective in mitochondrial inheritance 2-(N-morpholino)ethanesulfonic acid microgram(s) milligram(s) MGDG synthase monogalactosyldiacyl glycerol mitochondria mitochondrial intermembrane space import and assembly minute(s) microlitre(s) milliliter(s) millimolar mitochondrial outer membrane 3-(N-morpholino)propanesulfonic acid movement protein mitochondrial processing peptidase mitochondrial import stimulating factor sodium chloride nicotinamide adenine nicotinamide adenine dinucleotide phosphate nicotinamide adenine dinucleotide phosphate reduced nanometer(s)  xi  N-terminal OM ORF P PAGE PAM PCR PEG pK2/M5 pmol POTRA Pro PVDF PVX R RdRp redox RNA RT s S SAM SBMV SBWMV SDS Sec SeMV sg sH20 SPP SRP ss STNV T Tat TAE TB TBE TBSV TCV TEM TGBp1 TIC or Tic TIM or Tim TMV TNV  amino-terminal outer membrane open reading frame Protruding domain in the context of the CNV CP polyacrylamide gel electrophoresis presequence translocase-associated motor polymerase chain reaction polyethylene glycol full-length infectious clone of CNV picomoles polypeptide translocase proline polyvinylidene difluoride Potato virus X RNA-binding domain in the context of the CNV CP RNA dependent RNA polymerase reduction/oxidation ribonucleic acid readthrough seconds Shell domain in the context of the CNV CP sorting and assembly machinery Southern bean mosaic virus Soilborne wheat mosaic virus Sodium dodecyl sulphate secretory in the context of the thylakoid pathway Sesbania mosaic virus subgenomic sterile water stromal processing peptidase signal recognition particle single-stranded Satellite tobacco necrosis virus triangulation number Twin arginine translocation Tris acetate EDTA Tris borate Tris borate EDTA Tomato bushy stunt virus Turnip crinkle virus transmission electron microscopy triple gene block protein1 translocon at the inner membrane of chloroplasts translocon at the inner membrane of mitochondria Tobacco mosaic virus Tobacco necrosis virus  xii  Tob TOC or Toc TOM or Tom TP Tris TRP V WT xg  Tom β barrel translocon at the outer membrane of chloroplasts translocon at the outer membrane of mitochondria transit peptide tris(hydroxymethyl)aminomethane tetratricopeptide voltage wild type unit of rotational speed based on gravitational force  xiii  Acknowledgements I wish to thank my Ph.D. supervisor, D’Ann Rochon, for her expertise and guidance during the course of my thesis research. I would like to thank the members of the Rochon lab who have been there for me throughout my thesis: Jane Theilmann for her continual support, both professionally and personally, Ron Reade and Steve Orban for their technical support. I wish to acknowledge Kishore Kakani for his assistance and scientific input in the beginning of my thesis. I am also most appreciative to Michael Weis for his technical expertise in microscopic imagery and analysis. Much appreciation goes out to my supervisory committee, Drs. Mary Berbee, George Mackie, Andrew Riseman and Helene Sanfaçon for their advice and input. I would like to thank the Natural Sciences and Engineering Research Council of Canada, UBC for a University Graduate Fellowship and the various other private awards for financial assistance. I would like to acknowledge the Pacific Agri-Food Research Centre (PARC) for use of their facilities. I would also like to express my appreciation for the many members of the PARC staff whose friendship and support I could not have done without; a special thanks goes to Lynn Boyd, PARC’s librarian, Barry Butler and Jim Wild for their ITS assistance, Chris Gustavsen for keeping the envirocons functioning, Phil Schwab for keeping the paper towel supply in our lab going and the many PARC commissionaires whose presence has always reassured me that I am not alone in the building late at night. I wish to thank Scott Smith and Anne Hargrave and especially to our little Tahltan Bear Dog, Vinny, who has been my constant companion throughout all this. Finally, I wish to thank my family whose patience and support has been invaluable to me. “No amount of experimentation can ever prove me right; a single experiment can prove me wrong.” — Albert Einstein  xiv  Dedications  I dedicate this to all my family and friends who have been patience in letting this happen.  xv  Co-authorship Statement Ms. Hui was the main contributor in the design and execution of studies conducted in chapters 2, 3 and 5 which consists of the majority of research described in this thesis. She has also contributed to the experimental analyses and manuscript preparation for each chapter. The contributions of the co-authors are listed below:  1. Dr. Y. Xiang designed some of the CNV CP mutants and was co-author contributing to approximately 5% of the research in Chapter 3.  2. Dr. D. Rochon acted as the lead supervising scientist. She contributed significantly in the development of research goals and participated in the design and interpretation of thesis data as well as manuscript review.  xvi  1 CHAPTER ONE: LITERATURE REVIEW 1.1 Introduction Plant viruses are capable of replicating and inducing disease in a variety of food crops world-wide. The genomes of plant viruses are small ranging in size from approximately 3.5 to 20 Kb. The genome encodes a limited number of proteins and therefore, plant viruses rely heavily on host components to carry out the multiplication process. The process of infection by a positive sense RNA plant virus requires: (i) dispersal, transmission and entry into a plant host via specific vectors such as insects, nematodes and fungi; (ii) intracellular particle uncoating, possibly facilitated by host factors; (iii) expression of viral proteins from either virion RNA or following positive sense viral RNA synthesis; (iv) viral RNA replication; (v) particle assembly and genome encapsidation; (vi) cell-to-cell and systemic movement; and (vii) transmission to new hosts (106, 132). During infection, the virus particle must, at least partially, disassemble before viral replication can occur; however, the events leading up to, including the site of, disassembly are largely unknown. Viruses are known to recruit a variety of intracellular components during infection including specific membrane-bound organelles. For example, viral RNA replication is known to occur on the endoplasmic reticulum (ER) (172), peroxisomes (134, 153); nucleus (49) and mitochondria (233). Previous findings suggest that specific events brought about by virus/vector interactions may be important for particle disassembly (95) and that the coat protein (CP) contains information that targets specific sites within the host cell possibly for disassembly and/or assembly (243).  1  The viral CP serves many functions: it encapsidates genomic RNA and protects virion RNA during intracellular, intercellular (though not required for all viruses) and systemic movement; provides site(s) of recognition and attachment to host cell components; and is an important determinant of vector transmission (26, 132, 178). However, details of the specific means by which the CP carries out these functions remain largely unknown, particularly at the molecular level. A greater understanding of the host and viral components involved in virus infection would expand our fundamental knowledge of vector and virus-host interactions and thus contribute to the design of new strategies for controlling viral disease. Cucumber necrosis virus (CNV) is an icosahedral virus which is vectored by zoospores of the root-inhabiting fungus, Olpidium bornovanus (50, 51). Recently, studies on the CNV CP have provided much to our understanding of vector recognition and transmission (95, 96). Similar studies on related spherical viruses have revealed that distinct differences exist in CP-mediated functions (175, 176). This literature review is organized into four sections, each providing a background and rationale for the main areas undertaken by this thesis research. The first section describes the basic principles of virus particle structure with a special focus on virions with T = 3 icosahedral symmetry. The second section briefly describes virus transmission with a particular emphasis on fungal transmission. The third section provides a brief outline of what is currently known about subcellular targeting with a special interest in chloroplast and mitochondrial targeting and the fourth is a brief overview of molecular and biological aspects of CNV.  2  1.2 Plant virus particles A plant virus particle consists of a set of nucleic acids normally encased in a protective proteineous coat which requires suitable hosts for replication. The nucleic acid may be DNA or RNA and single- or double-stranded. If the nucleic acid is singlestranded it may be of positive or negative sense (83). Most plant viruses have positive sense single-stranded RNA genomes. Plant virus particle structure can be divided into two major types based on symmetry of the particle formed during infection: helical, which can be rigid, rod-shaped or filamentous (=flexuous) and icosahedral or spherical particles (83). Crick and Watson (47) proposed that the amount of nucleic acid in small virions were insufficient to code for more than a few protein molecules of limited size. They concluded that the only logical way to build a spherical protein shell was to use the same type of molecule many times over. They then speculated that these identical subunits must be packed symmetrically, aggregating around the genome in a regular way. Upon infection, virus particles must disassemble or uncoat within the host cell so that the genome may be expressed. Although particle disassembly is understandably considered an important step in the initiation of a viral infection, very little is known about the uncoating process. The following provides additional details on particle structure, CP structure and the virus assembly/disassembly process.  1.2.1 Rigid, rod-shaped and filamentous (helical) particles Plant RNA viruses with helical capsids can be divided into rod-shaped (tobamo-, tobra-, hordei-, and furoviruses) and filamentous (poty-, bymo-, potex-, carla-, and  3  closteroviruses) viruses (83) in which the structurally equivalent protein subunits are set in a helical array about a central axis (Table 1.1). The helical symmetry implies that all protein subunits in the virus occupy the same environment and that the same contact points between neighbouring subunits are used repetitively along the helix. In theory, there is no restriction on the number of protein subunits that can be packed into a helical virus, with the length of the encapsidated RNA determining the final particle size. The repetitive packing arrangement also assumes that the symmetry of the encapsidated RNA (along the sugar-phosphate backbone) is the same as the symmetry of the protein (i.e., helical).  1.2.1.1 Rigid, rod-shaped particles Tobacco mosaic virus (TMV), the type member of the genus Tobamovirus, is the classical example of a rigid, rod-shaped RNA virus (Fig. 1.1A) and is one of the few examples in which the processes associated with assembly and disassembly are known in detail. The TMV structure consists of a helical array of identical CP subunits (except for a few subunits at the ends) that have similar non-covalent contacts with each other, with a pattern that is repeated many times leading to the final symmetrical structure. Each CP subunit consists of four closely parallel α-helices which are connected by narrow β sheets with the N- and C-termini of each subunit being exposed at the virus surface (15, 105, 152). A single molecule of RNA is embedded within the helix and packed between helical turns of the capsid (83, 209). Features of the subunit arrangement of TMV are shown on Table 1.1. TMV assembly begins with the formation of an intermediate cylindrical ‘disk’  4  Table 1.1 Comparative features of typical rigid, rod-shaped and flexous filamentous virus particles Rigid, rod-shaped1  Flexuous filamentous2  Virus (type member)  TMV  PVX  Number of subunits per turn  16.3  8.9  Helical pitch (nm)  23.0  33.0  3  5  Radius of central hole (nm)  2.0  1.5-2.0  Maximum radius (nm)  9.0  6.5  Minimum radius (nm)  7.5  ND3  Number of nucleotides per subunit  1  Adapted from (90); TMV, Tobacco mosaic virus Adapted from (108); PVX, Potato virus X 3 ND=not determined 2  5  A  B  a  b  a  b  c  d  Figure 1.1 Comparative structures of helical TMV and PVX particles A. TMV (a) Electron micrograph of negatively stained TMV particles. (b) Schematic representation of RNA and CP subunit interactions and a photograph of the TMV model with major dimensions indicated (see Table 1.1). With permission from (35) and (106). B. PVX (a) Electron micrograph of PVX stained with 2% uranyl acetate. With permission from (7). (b) IHRSR2 reconstruction of PVX, section normal to viral axis. (c) IHRSR reconstruction of PVX, outside surface view. (d) IHRSR reconstruction of PVX, section through viral central axis. Scale bar = 25 nm. Colour coding in panels (a) to (d) is from red-orange (low density) to green-blue (high density). (b) to (d) with permission from (108).  6  consisting of two layers of protein unit which combine with a specific ‘origin of assembly’ initiation site on the viral RNA (33, 247, 248). This RNA initiation site is in the form of a hairpin loop which is inserted through the central hole of the disk, causing the disk to dislocate into a helical structure, entrapping the RNA (24). The protein helix elongates in a bidirectional fashion as more disks and/or CP subunits and RNA are added. TMV particles undergo cotranslational disassembly in which uncoating begins with the exposure of the capped 5’ end of the RNA and the first open reading frame being translated (197, 239). Disassembly is believed to involve the disruption of carboxylcarboxylate interactions between neighbouring subunits and of phosphate-carboxylate interactions between CP and RNA (151). Subsequently it was discovered that TMV particles are uncoated bidirectionally in which CP subunits are removed from the 3’ end of the RNA minutes after the uncoating has begun at the 5’ end (241, 242). A “cotranslational-disassembly” mechanism has the advantage of ensuring that the viral capsid continues to protect the nucleic acid as it uncoats during the translational process.  1.2.1.2 Filamentous particles The most detailed structural studies of filamentous viruses have been on the Potexvirus (101, 215), Potato virus X (PVX) (Fig. 1.1B). The PVX particle consists of CP subunits arranged in a helical array with 8.9 CP subunits per turn of the helix (162), with RNA packed between turns and with five nucleotides of RNA per subunit. The capsid tertiary structure consists of CP subunits with a single-domain structure whereby the N-terminus is on the outer virion surface above the C-terminus (similar to TMV) (5). Features of the CP subunit arrangement of a PVX particle are shown on Table 1.1.  7  Two mechanisms of PVX particle disassembly have been suggested: (i) cotranslational disassembly activated by CP phosphorylation (6) and (ii) disassembly associated with the binding of the viral-encoded Triple gene block protein1 (TGBp1) to the end of the virus particle associated with the 5’ end of the RNA. It was shown that such binding to TGBp1 leads to particle destabilization and a fully translatable virion (7). TGBp1-triggered translational activation involves particle “remodelling” that results in rapid and complete particle disassembly and RNA release (182). This translational activation is different from canonical “co-translational disassembly” of TMV which involves a gradual and partial removal of the CP subunits by the ribosome (239, 240).  1.2.1.3 Icosahedral (spherical) particles All known spherical viruses have icosahedral symmetry (from the Greek “icosa-” meaning “20” and “hedr-” meaning “faces” in the context of a geometrical solid) with the interior viral genome completely encapsidated. In contrast to helically symmetrical viruses, the number of subunits in an icosahedron is restricted. A regular capsid with icosahedral symmetry has 60 asymmetric units (121). However, most spherical viruses contain more than 60 subunits in their capsids, yet are still able to maintain icosahedral symmetry. Caspar and Klug (32) proposed a theory involving quasi-equivalence to describe how multiples of 60 subunits could be arranged to form an icosahedral structure. The theory of quasi-equivalence is based on the assumption that even if exact symmetry cannot be achieved, a stable complex is still possible if every protein subunit of the capsid is packed in a very similar (but not identical) way, as closely as possible utilizing the same set of packing interactions (32). All possible structures leading to a quasi-  8  equivalent arrangement of protein subunits can be described by a triangulation number, T (83, 187). This number represents the number of equilateral triangles into which each triangular face of an icosahedron can be divided. With 60 asymmetric subunits in an icosahedron, the number of quasi-equivalent protein subunits into which each triangular face can be divided will produce capsids with 60T subunits. T can only assume certain values, 1, 3, 4 and 7, etc. as only these values lead to quasi-equivalent packing. Most known spherical plant viruses have virions with T = 3 symmetry; each virion contains 180 copies of three chemically identical but conformationally distinct subunits: A, B and C. There are two morphologically different types of T = 3 icosahedra (88, 205): the truncated icosahedron, e.g., Cowpea chlorotic mottle virus (CCMV) (Family: Bromoviridae) and the rhombic triacontahedron, e.g., Tomato bushy stunt virus (TBSV) (Family: Tombusviridae) (Fig. 1.2). Virions with T = 3 symmetry have 20 hexamers and 12 pentamers. In the truncated icosahedron, B/C subunits are arranged with nearly exact six-fold symmetry around the icosahedral three-fold axes. In the rhombic triacontahedron, there are two conformationally distinct B/C dimer contacts (direct and divided) around the quasi-sixfold axis (see below); as a result the hexamers are more appropriately described as trimers of dimers.  1.2.1.3.1 Tertiary structure of the CP subunit The first structure of an icosahedral virus determined with X-ray crystallographic techniques was that of TBSV (70, 159) (Fig 1.3A). TBSV is a T = 3 virus with 180 chemically identical copies of a CP subunit encapsulating a single-stranded RNA molecule. Each CP subunit occupies three distinct positions in the icosahedral capsid.  9  a  b  Figure 1.2 Geometrical features of the two types of T = 3 capsids. (a) A truncated icosahedron, e.g. CCMV. Positions of icosahedral axes marked by yellow symbols (pentagons: five-fold axes; triangles: three-fold axes; ovals: two-fold axes). White triangles denotes quasi three-fold with A, B and C subunits. (On right) Side view of dimer interaction between B2-C and C-B5 which are co-planar whereas bent angles occur at the B-C and C-A interfaces. (b) A rhombic triacontahedron model, e.g. TBSV and labelled as in (a). The A, B and C subunits are co-planar within each asymmetric unit. Two such asymmetric units are co-planar by icosahedral two-fold symmetry. The interaction between B2-C forms a planar orientation whereas the interaction between C-B5 forms a bent surface. With permission from (215).  10  A  B R  β-annulus 18 aa  C B C  C  B B  Part of ε-region  C  Figure 1.3 Structural features of a T = 3 icosahedral virus particle. A. Schematic representation of the TBSV particle showing arrangement of CP subunits. A, B and C designate the three structural isoforms of the subunit. The opening at the top of the particle shows the interior R/arm regions. With permission from (167). B. Structural features of the virus CP subunit showing the three major structural domains: R, S and P and the connecting arm(a) and hinge(h) (see text for details). C. Diagrammatic representation of the β-annulus plus six residues of the ε-region (partial arms of the three C subunits are in black, blue and red) viewed down the particle three-fold axis. Adapted from (89).  11  The asymmetric units consist of three co-planarly arranged subunits, denoted as A, B and C. Five A subunits are in contact around the five-fold axis and six subunits (three B/C dimers) are in similar contact around the three-fold axis. The two-fold axis is comprised of C/C dimers and a quasi two-fold axis is comprised of A/B dimers that are arranged slightly differently than the C/C dimers. The CP subunit consists of three main structural domains (Fig. 1.3B). The Nterminal RNA-binding (R) domain resides within the particle interior. Most of this domain is not visible in electron density maps and is therefore disordered relative to the rest of the subunit. A flexible, interiorly extending arm region joins the R domain to the Shell (S) domain. The S domain forms the shell of the capsid. The Protruding (P) domain forms dimeric protrusions that extend outward from the shell. The overall topology of the P domain is similar to the S domain but consists of ten β strands instead of eight (see below) (159). The S domain is connected to the P domain by a small, extended hinge (h). The conformation of the polypeptide chain differs only in the arm and hinge regions which connect the domains and enables quasi-equivalent interactions among the CP subunits. The shell consists mainly of an eight stranded antiparallel β sandwich forming a β barrel or “jellyroll” (83). The sheets are connected by loops; four short loops at one end make the five- or six-fold contacts and three long loops at the other end are involved in dimeric and trimeric contacts. One end of the β barrel is narrower when compared with the other, giving it a wedge shape, allowing closer contact of subunits at the five- and three-fold axes.  12  1.2.1.3.2 Subunit packing Most of the residues from the disordered R and arm regions of the A and B subunits of TBSV are not visible in electron density maps; however, residues comprising the arms of the C subunits can be seen because they are ordered. The arm, which consists of an N-terminal beta “β” region and a C-terminal epsilon “ε” region, passes along the region where the B and C subunits contact and arms of three C subunits meet to form a structure called the β annulus (Fig. 1.3C). The β annulus is stabilized by β interactions, each involving 19 N-terminal residues from each β region of the C subunit arm. As a result there are two types of dimer contacts around the three-fold axis: a divided C/C dimer contact with the arm inserted (resulting in a bent formation) and a direct A/B contact without the arm (resulting in a flat formation). This latter interaction is similar to the A/A contact at the five-fold axis. The difference between the divided and direct contacts can be described by a fulcrum-like rotation of about 40 degrees around an axis parallel to the subunit interface. The trimer contacts between A, B and C subunits around the quasi-threefold axis are all similar. Some subunit interactions are presented in two different states although some subunit-subunit interactions near the fulcrum are retained. The distinction between these two states is controlled by a switch, namely the insertion of the arm which is ordered only in the C subunits. The CCMV CP has a similar tertiary structure to that of TBSV in that it has three chemically identical subunits, denoted as A, B and C, in each asymmetric unit (205). However, the subunits in the CCMV asymmetric unit are not co-planarly arranged (Fig. 1.2C). The arm region in each subunit is visible; the six arms of the B and C subunit which converge at the quasi six-fold axes intertwine to form a hexameric tubular  13  structure called the β hexamer which is functionally analogous to the β annulus of TBSV (238).  Protein-nucleic acid interactions. More direct structural information is beginning to emerge about nucleic acid packing in icosahedral viruses. Bipartite Bean pod mottle virus (BPMV) was the first virus for which a portion of the packaged RNA genome was visualized by X-ray crystallography (40). The genome of BPMV consists of two separately packaged RNA molecules. The structure of the particle encapsidating BPMV RNA2 showed density for six well-ordered ribonucleotides near the three-fold axes of the virion. The icosahedral structures of TBSV and CNV particles suggest that most of the viral nucleic acid lies along the inner face of the shell within the capsid (39, 100, 214). The volume occupied by nucleic acid in the virions of studied viruses is much smaller than its volume as a free molecule, indicating that the encapsidated nucleic acid is highly condensed. Details of how the nucleic acid molecule achieves this densely packed state are not known. In CNV and TBSV, the R domain and the arm of the A and B subunits are disordered in relation to the shell and neutron scattering studies using contrast variation indicate that most of the R domains of the C subunits form a layer inside the RNA shell (39, 100). In T = 3 Southern bean mosaic virus (SBMV), the R domain residues do not form a layer but are distributed throughout the interior of the shell (113). The R domain, arm and inward facing portions of the shells of both TBSV and SBMV contain many positively charged residues that neutralize most of the phosphate groups of the RNA (73, 81). In T = 1 Satelite tobacco necrosis virus (STNV), the N-terminal region is partly  14  ordered, positively charged and forms a helix that interacts with the RNA (93, 115). Particles which have a T = 1 icosahedral symmetry consist of 60 identical CP subunits with each subunit occupying similar packing environments and no requirement for quasiequivalent CP interactions (93, 122). Neutralization of negatively charged RNA contributes to the dense packing of the nucleic acid.  1.2.1.3.3 Virion assembly The assembly of plant virus T = 3 icosahedral particles can occur in vitro by a spontaneous aggregation of the CP components, without the aid of enzymes or other supporting materials (83). Therefore, the viral CP has an inherent capacity to assemble into the correct, icosahedral structure which is in a state of lowest energy. In contrast to the assembly of TMV, the details of the assembly process for small icosahedral viruses are less understood. The following describes three models of assembly that have been proposed for different T = 3 plant viruses. The first mechanism for assembly had been suggested by Sorger et al. (204) and is based on in vitro studies using Turnip crinkle virus (TCV). In this case, it is proposed that C/C dimers associate with viral RNA and the β annulus is formed by the cooperative ordering of the arms of both C subunits. Additional CP dimers are then added adapting to the necessary A/B or C/C conformations. The correct packing of CP subunits, assisted by RNA binding, will follow until the shell is complete. This mechanism of assembly is supported by the observation that a trimer of dimers-RNA complex can be isolated and is relatively stable (204). Electron micrographs show that the growing CP-RNA capsid has  15  the same curvature as the final virion, indicating that the added dimers are capable of achieving the correct conformation when they associate (204). CCMV was the first icosahedral virus to be assembled in vitro, both as empty capsids (9) and with RNA (8). Based on studies with CCMV, the second assembly mechanism supports the initial formation of a subunit dimer; six dimers then interact to form a hexamer of dimers (12-mer) (205). The binding of metal ions and RNA will induce curvature at each subunit dimer interface within each hexamer; subsequently more dimers will be added to the 12-mer, leading to a closed shell. Particle assembly relies on the addition of dimers because two hexamers cannot directly assemble with each other. The study conducted by Speir et al. (205) also showed ordered RNA within the capsid that displays quasi icosahedral symmetry. In the models represented by TCV and CCMV, hexamers are considered to be essential for initiation of the assembly process. The mechanism of CCMV assembly has been challenged by Zlotnick et al. (249). They proposed that capsid assembly is nucleated by a pentamer (rather than a hexamer) of dimers and were able to demonstrate that a CCMV 10-mer can exist in solution. More recently, this model has been refined by Johnson et al. (89). They found that during the early stages of CCMV infection low levels of CP dimers bind with excess RNA. Increased concentrations of CP dimers cause cooperative compact RNA folding and subsequent packaging, a process which is dependent on CP capsid structure and independent of RNA sequence. This pathway takes advantage of the limited concentrations of CP to bind excess viral RNA present early in infection, minimizing the chance of encapsidating cellular RNA.  16  The third model for capsid formation that initiates with A/B dimers of pentamers (a 10-mer) at the five-fold axes was originally proposed for the assembly of SBMV (183). Additional CP dimers are then progressively added to the 10-mer. This model has been supported by studies conducted by Satheshkumar et al. (186) on the related T = 3 Sesbania mosaic virus (SeMV). The assembly of SeMV capsids has been proposed to initiate with dimers interacting to form a 10-mer intermediate. The growing capsid is stabilized with the binding of RNA and the inclusion of subsequent dimers. Lokesh et al. (125) were able to generate stable pseudo T = 2 (120 subunits) particles with icosahedral symmetry by using a SeMV CP arm mutant. They argue that such particles could have only originated from dimers of pentamers and not from dimers of hexamers since that would have required additional complex conformational changes. Despite our limited understanding of spherical virus particle assembly, the mechanisms involved must ensure specific interactions to attain icosahedral symmetry. For example, specific regions of the CP R domain of CNV have been shown to be required for T = 3 particle formation (94). In TBSV, different subunit contacts such as the direct and divided C/B interactions at the six-fold axes, in which the divided contact is stabilized by the insertion of an ordered arm, requires that the switching between the different packing interactions of the assembling subunits be correct (69).  Role of RNA in icosahedral virus capsid formation. In addition to the role of RNA in carrying genetic information, it may play an important structural role in capsid assembly and possibly influence the ability of the CP to form a particle with icosahedral symmetry (191). In vitro studies on T = 3 particles of Brome mosaic virus (BMV) have  17  shown that specificity of RNA encapsidation is associated with an arginine-rich motif (ARM) located within the CP N-terminus (169). Mutagenesis studies on the N-terminus of CP subunits of CNV and the insect virus, Flock house virus (FHV), have shown that RNAs of various lengths were packaged into a heterogeneous collection of particles of different shapes and sizes (53, 94). This demonstrated that changes to the RNA binding portion of the CP N-terminus are critical in controlling the geometry of the particle. Similar RNA-controlled particle polymorphism has been found in BMV (112) and TMV (43). In the absence of RNA, SBMV (56, 57) and TCV (116) CPs, in which the Nterminus was proteolytically cleaved, assembled into empty T = 1 icosahedral particles. Competition experiments with FHV CP and either native or cellular RNA revealed preference for encapsidation of the correct RNA (192). Together, these findings suggest that there are specific interactions between the CP and viral RNA in the assembly process, and that specific interactions appear essential for T = 3 particle formation and RNA encapsidation.  1.2.1.3.4 Particle expansion Divalent cations in many plant viruses play a role in maintaining capsid stability. Removal of divalent cations results in pH-dependent virion swelling and at higher pH levels many particles become sensitive to proteolysis (219). In TBSV, there are two calcium ion binding sites per subunit on the S domain at the quasi-three fold axis (159). They link neighbouring subunits at the A/B, B/C and C/A interfaces whereby the Ca2+ ions join carboxylate groups from the side chains of Glu and Asp residues and thus stabilize subunit-subunit contacts. When Ca2+ ions are removed by EDTA chelation in an  18  alkaline pH, the negative charges on the Glu and Asp groups repel and result in particle swelling (69). It has been suggested that the sequestering of Ca2+ ions may promote particle disassembly in plant cells (83, 95). The X-ray structure of swollen TBSV virions treated with EDTA at pH 7.5 was determined by Robinson and Harrison (175). With the removal of calcium ions, the radius of a swollen particle was found to increase by approximately 10%. In addition, dimer contacts between S domains are geometrically altered and new ordered dimeric contacts between the A and B arms are formed. Openings (approximately 2 nm in diameter) were created at the quasi three-fold axes and the angle of the P and S domains shifted relative to that found in compact particles. However, the five-fold and six-fold contacts of the S domains and the two-fold interaction of the P domains remained intact, indicating that the virus particle retains icosahedral symmetry in the expanded state. Particle expansion was found to be reversible indicating that the internal scaffold contacts are conserved in the swollen state. The potential biological significance of swelling has been provided by studies conducted by Kakani et al. (95) who showed that CNV particles bound to zoospores of its fungal vector are conformationally different from the native particle and that such an altered conformational state is essential for virus transmission. The biological significance of particle swelling has also been inferred by studies conducted by Brisco et al. (21) who showed that the RNA of swollen SBMV [and CCMV, BMV and Alfalfa mosaic virus (AMV)] virions becomes translatable in vitro, suggesting that swelling may enable the initial stages of infection to occur. Swollen SBMV virions have also been found to bind to ribosomes in a cell-free translational system (22). Thus it is possible that  19  like TMV, swollen particles of spherical plant viruses disassemble co-translationally. Currently, there is no information regarding the subcellular site(s) and mechanism(s) associated with spherical particle disassembly.  1.3 Plant virus transmission Plant viruses are dependent on their hosts for replication yet lack the means to move between hosts and the ability to penetrate the plant cuticle or cell wall. Consequently, they must rely on vectors for transmission, host entry and in some cases, long-term protection in the absence of suitable hosts (83). Plant viruses are vectored predominantly by arthropods (156) and also by nematodes, fungi, and plasmodiophorids; transmissibility also occurs by seeds, pollen and vegetative propagation (for reviews see 14, 63-65, 83, 147, 156, 176, 178, 184). A successful virus transmission consists of: virus acquisition from an infected source, stable retention of acquired virions at specific sites through binding of virions to ligands and virion release and delivery from retention sites to susceptible host cells (4, 63, 220). Mechanisms of transmission are categorized based on virus retention time, site(s) of virus retention in the vector and the ability of the virus to replicate in the vector (156). The terms “non-persistent” and “persistent” were proposed by Watson and Roberts (232). Currently, “non-persistent” transmission applies to viruses which have very short acquisition times (seconds to minutes) and very short retention times (minutes to hours). “Persistent” transmission applies to viruses that are retained over longer periods of time (hours) and where retention can last from hours to indefinitely. “Semipersistent” transmission applies to viruses with intermediate features of the two  20  processes. Viruses are also described as “noncirculative” and “circulative”. Noncirculative transmission applies to viruses that are retained in the mouth parts and gut of the insect vector but that do not enter the haemocoel. In circulative transmission the virus can enter the hemocoel and cross cell membranes of its insect vector prior to being transmitted to a new host via the insect mouthparts (63). The circulative mode can be further divided into two subcategories depending on whether the virus can replicate in its vector (propagative) or not (non-propagative) (63, 66). These terms are applied more appropriately to viruses transmitted by arthropods and to a lesser extent by nematodes and does not reflect transmission processes of fungi or plasmodiophorid vectors. The virus-vector relationship is specific in that a plant virus can be transmitted by one or a few vector species but not by others (for reviews see 4, 14, 156, 176, 178, 184). The specificity of vector-mediated transmission of plant viruses is associated with various characteristics, such as ligand-receptor interactions, sites of virus retention and use of virus-encoded determinants (14, 96, 156, 176, 220). Studies using transmissible and nontransmissible isolates of the same virus species, chimeric clone analyses of suspected regions and virus-vector overlay assays have helped identify viral and vector components that are involved in vector transmission. Virus-encoded determinants that facilitate transmission include the coat protein (CP) and less often other transmission factors referred to as “helper components” (176). The CP has been demonstrated as the sole contributor to vector transmission in several insect-transmitted viruses including Cucumoviruses, Alfamoviruses, Carlaviruses and Criniviruses (65, 128, 156, 157, 176, 234) and in fungally-transmitted members of the Family Tombusviridae (176, 178). Empirical evidence for an association between the  21  viral CP and vector receptors has been obtained in fungal transmission studies of CNV (97, 174). In these studies specific amino acids located at the particle quasi three-fold axes were found to be important for fungal transmission. Subsequently, Kakani et al. (96) found that binding of CNV CP to fungal zoospores is mediated by specific mannose and/or fucose-containing oligosaccharides and that transmission requires conformational change of the virus particle (95). In the following sections, I will focus primarily on the fungal transmission of plant viruses within which the only known true fungal vectors are two species of Olpidium in the phylum Chytridiomycota (87, 178).  1.3.1 Fungal transmission Currently, there are two species of Olpidium known to vector plant viruses: O. bornavanus (=O. radicale) and O. brassicae, both of which are obligate intracellular parasites of plant roots (2, 27, 178). Two virus-fungal relationships are recognized: in vitro and in vivo (see Sections 1.3.1.2. and 1.3.1.3.) (27). They are distinguished by the method of virus acquisition by the fungus and the location of the virus relative to the fungal resting spore. Viruses transmitted in vitro by both Olpidium sp. are spherical and are specific members of the Tombusviridae family. Viruses transmitted in vivo solely by O. brassicae are either rod-shaped or filamentous and belong to the Ophiovirus and Varicosavirus genera. Studies using host specific strains of O. bornavanus have demonstrated high virus-vector specificity of transmission (27, 31, 178); similarly, specificity of transmission was found using different strains of O. brassicae as vectors of TNV (212). The role of the CP in the specificity of O. bornavanus transmission has also  22  been demonstrated by using reciprocal exchanges of the CP ORF between Olpidiumtransmissible CNV and the nontransmissible TBSV (136). In addition Kakani et al. (96) showed that the binding of CNV and that of other plant viruses occur more efficiently to zoospores of its natural vector than to a non-vector.  1.3.1.1 Life cycle of Olpidium spp. Olipidium bornavanus and O. brassicae produce motile zoospores which serve as a means of dispersal and resting spores that allow long-term survival in the soil (2, 27, 178) (Fig. 1.4). Thick-walled resting spores germinate and release motile zoospores which attach and encyst upon contact with cells of root hairs or other epidermal cells. It is believed that prior to zoospore encystment, the flagellum, which includes the axoneme and the axonemal sheath, is withdrawn into the zoospore body (2, 27, 176, 211). Electron micrographs of infected root cells show membranous structures that may be remnants of the axonemal sheath inside the encysted zoospore (207). Thereafter, contents of the encysted zoospore which include a newly formed plasmalemma are released into the host cell cytoplasm via an opening in the cyst (211). Once inside the cell, the thallus ( = cyst cytoplasm surrounded by the plasmalemma) enlarges and undergoes several nuclear divisions, developing into a multinucleate structure. A cell wall is formed around the thallus outside of the plasmalemma. The thallus forms cleavage vesicles which fuse to become zoospores contained within a zoosporangium. Maturation of the zoosporangium involves the formation of an exit tube from which zoospores are released (211). The thallus will also develop into a thick-walled resting spore but does not become multinucleated structure (27, 178, 211).  23  root cell penetration  mature thalli (zoosporangia)  encystment and root attachment  zoospore discharge  zoospores  resting spores Figure 1.4 Life cycle of Olpidium brassicae (see text for details). Adapted from (221)  24  1.3.1.2 In vitro transmission Both O. bornavanus and O. brassicae transmit viruses in the in vitro manner (178) which involves independent release of zoospores and virus into the soil followed by adsorption of virus particle onto the surface of the zoospore (27). The acquisition time is relatively short (~5-15 min) and the virus is stably adsorbed onto both the plasmalemma of the zoospore body and the axonemal sheath of the flagellum (207, 211). It is believed that the virus present on the axonemal sheath enters the zoospore protoplasm during the retraction of the flagellum (207, 211). In studies of CNV transmission by O. bornavanus, Stobbs et al. (207) showed that virus particles were found in “whorl of membranes” within encysted zoospores following flagellar retraction. The means by which the virus within the zoospore protoplasm enters the root cell cytoplasm is unknown; however, it is assumed that this event occurs prior to thallus cell wall formation (27, 178). Once the virus is released into the host cytoplasm, it replicates independently from its fungal vector. As the fungal thallus matures, the virions do not associate with resting spores or zoospores during coinfection. Zoospores and free virions are released into the soil for further transmission to new host plants. Alternatively, resting spores may germinate after which the resulting zoospores will bind virus particles present in the soil.  1.3.1.3 In vivo transmission Olipidium brassicae is the only fungal vector known to transmit plant viruses utilizing the in vivo mechanism. Viruses which use this method of transmission are taken up by zoospores within co-infected root cells. The virus is believed to be located within zoospore protoplasm and later in resting spores. The virus is transmitted when the  25  zoospores infect roots of another plant. Virus acquisition is believed to occur as the fungus develops within a virus-infected host cell. Similar to in vitro transmission, the method of virus acquisition within co-infected root cells or of cyst protoplast release into the root cytoplasm is unknown. Obtaining direct evidence for the presence of virus in resting spores has been difficult and is based primarily on the observation that harsh chemical treatment or long-term storage of resting spores does not result in the loss of virus infectivity (28-30, 79). It is worthy to note that direct evidence for the presence of plant virus in resting spores has been found in plasmodiophorid vectors (i.e., Polymyxa graminis, P. betae and Spongospora subterranean) which transmit virus in the in vivo manner. For example, the movement protein and RNA of Soilborne wheat mosaic furovirus (SBWMV) were discovered in both zoospores and resting spores of its plasmodiophorid vector, P. graminis (54). However, no virions were found in the resting spores. Verchot-Lubicz et al. (224) found all Beet necrotic yellow vein benyvirus (BNYVV) encoded proteins in resting spores of its plasmodiophorid vector, P. betae, suggesting that the virus may replicate in its vector.  1.4 Subcellular targeting and protein import Many nuclear-encoded proteins destined for subcellular organelles are synthesized in the cytosol, most as precursor proteins 1 with signals or targeting peptide(s) that direct the proteins to the correct subcellular site. The precursors must possess the means to be translocated across membranes (sometimes more than one) and be sorted into the various subcompartments. The translocation and import mechanisms for 1  The term precursor protein(s) is used in the same manner as the term preprotein(s) 26  precursor proteins require an ordered and regulated system which may include additional components such as chaperones and energy from the hydrolysis of ATP and/or GTP. Viruses exploit a variety of cellular components and processing mechanisms to facilitate infection. Such opportunistic invaders have acquired the necessary signals and the ability to present their own proteins in a manner that is recognized by cellular machinery. For example, viruses have usurped cellular ribosomes for translation of structural and non-structural proteins (107), and peroxisomes (134, 153, 161, 185), chloroplasts (166), mitochondria (233), and the endoplasmic reticulum (218) for replication. Further examples of a variety of host proteins implicated in plant virus infections have been described (149, 235). Chloroplasts and mitochondria are cellular organelles which consist of hundreds to thousands of nuclear-encoded proteins. Both are believed to have originated from independent endosymbiotic events, in which an ancestral bacterium was taken up by a heterotrophic host cell (130); this event was followed by a large unidirectional transfer of genetic information to the nucleus. Mitochondria originated much earlier than plastids; therefore, plastids evolved in cells that already contained an efficient system for targeting cytosolically synthesized proteins to mitochondria (130). Analogous functions required by both chloroplasts and mitochondria resulted in the sharing of proteins and the emergence of mechanisms to facilitate dual-targeting of nuclear-encoded products to both compartments (129, 198). The targeting and import pathways for cytoplasmically synthesized chloroplast and mitochondrial proteins will be described in more detail below.  27  1.4.1 Targeting and import of chloroplast proteins Most chloroplast proteins are synthesized as preproteins in the cytosol and are imported posttranslationally. Chloroplasts consist of three distinct membrane systems: the outer (OM) and inner membranes (IM) which surround the organelle and the internally located thylakoid membrane system which contains important components for photosynthetic activity. There are also three soluble compartments: the space between the two membranes (IMS), the stroma which is bound by the IM and the thylakoid lumen. Most proteins are translocated into chloroplasts by the general import pathway, mediated by the TOC (translocon at the outer membrane of chloroplasts) and TIC (translocon at the inner membrane of chloroplasts) complexes (see Fig. 1.5 for schematics for TOC/TIC proteins; for reviews see 67, 84, 160, 203, 206 and references therein). TOC and TIC complexes are physically associated with one another at the membrane contact sites of the chloroplast envelope providing a direct pathway to the stroma (84). Most proteins that are destined for the IM, stroma and thylakoids are synthesized as preproteins in which each contains an N-terminal extension called a transit peptide (TP) that is proteolytically cleaved upon stromal import. The TP is both necessary and sufficient for organelle recognition and translocation initiation. General features of the TP include: (i) sequences that can vary in length from 20-150 amino acids; (ii) an overall positive charge and (iii) a sequence enriched in serine and threonine amino acids (203). Various cytoplasmic factors have been implicated to assist in the targeting of the preprotein to the TOC receptors. For example, 14-3-3 and/or Hsp70 proteins in the cytosol can form guidance complexes with phosphorylated chloroplast preproteins (133). The preprotein-bound guidance complexes are directed to chloroplasts by the N-terminal  28  29  64  +  SPP  ATP  22  TPP  A B  C  Tat  A  ?pH/Tat-dependent  -  + ?ψ  Stromal factors  20 21  TOC complex  SRP-dependent  -  GTP  ?ψ Alb3  + + +  40 32 Hsp93  110  ATP  12 Hsp70  75  P  NADH  159  GTP  55 FeS  Spontaneous  TPP  62 FNR  NADH  TIC complex  34  P GTP  cytosolic Hsp70/14-3-3  TPP  E  Sec-dependent  Y  ATP  Sec  Figure 1.5 Schematic representation of the chloroplast protein import machineries. The components of the TOC and TIC complexes are in blue and green, respectively. Tic110 is the designated IM translocation channel. Components of the thylakoid pathways are in shades of yellow. For each pathway the respective energy forces are indicated. Further details are described in the text. Adapted from (74); (215).  Thylakoid lumen  Thylakoid membrane  Stroma  Inner membrane  Intermembrane space  Outer membrane  Cytosol  preprotein  TP which is recognized in a GTP-dependent manner by surface receptors of the TOC complex (245). Interactions with molecular chaperones allow preproteins to be maintained with minimal structure to allow their translocation through the membrane channels in a relatively unstructured, extended conformation (229). Some preproteins, especially those destined for the chloroplast OM and IM (see below), are synthesized without a cleavable TP and the targeting signal is located within the mature part of the protein (206). ATP is required to bring the preprotein into contact with the TIC complex (85). Hydrolysis of ATP is possibly due to an Hsp70-like chaperone in the IMS. IM preprotein penetration and its interaction with TIC components stimulate the recruitment of IM-bound Hsp93 in an ATP-dependent manner. This complex interacts with the preprotein as it exits the TIC translocon and enters the stroma (44). Entry into the stroma is accompanied by cleavage of the TP by the stromal processing peptidase (SPP). Thus, GTP hydrolysis initiates OM translocation whereas ATP hydrolysis is most likely the driving force for OM and IM channel translocation. Proteins imported via the TOC/TIC machinery are regulated according to the metabolic requirements of the chloroplast. Regulation of the TOC complex is mediated by GTP/GDP binding and by phosphorylation of Toc receptors (103). Moreover, some Toc receptors have a higher affinity for phosphorylated than non-phosphorylated preproteins (see below). Therefore, phosphorylation of preproteins can positively influence the rate of translocation (230).  30  1.4.1.1 TOC Properties of five known TOC proteins of the TOC complex are summarized in Table 1.2. Toc159 2 is a GTP-binding protein which can be divided into three regions: an N-terminal acidic (A) domain, a central GTP-binding (G) domain and a C-terminal membrane (M) domain (78, 102). Toc159 has been proposed to serve two functions: (i) a receptor which can recognize presequences in a GTP-dependent manner and (ii) a role in translocation by initial contact with the incoming preprotein (189). Toc34 is anchored to the OM by its C-terminus; however, most of the protein which includes a G domain is exposed to the cytosol (196). Toc34 is also a receptor for which the GTP-bound form has a high affinity for preproteins (190). Although controversy exists regarding the relative roles of the two receptors in binding and OM penetration, it is clear that they are both responsible for TP recognition at the TOC complex and that TPs interact directly with their G domains. Transfer of the preprotein from the TOC GTPase receptors to the TOC channel requires very low levels of ATP. Toc75 is the most abundant OM protein and forms a β barrel, cation-selective, high conductance ion channel in the OM (76) and has a cytosolic, low affinity preprotein-binding site (77). The channel is estimated to be approximately 2.5 nm wide at the entrance and narrows to approximately 1.5-1.7 nm wide inside the channel (77). Toc34, Toc75 and Toc159 form a stable core complex with a stoichiometric ratio of 4:4:1, respectively (189). The Toc64 protein has a C-terminus that contains three tetratricopeptide (TRP) motifs that are exposed to the cytosol (202) and, although its function is unclear, the TRP motif is usually associated with proteinprotein interactions including mediating interactions with Hsp70/90. Evidence for the  2  TocXX/TicXX proteins are designated based on molecular weight “XX”; Toc/Tic proteins will be underlined when first described 31  Table 1.2 Summary of chloroplast import proteins (see text for further details) TOC ( = translocon at the outer membrane of chloroplasts) proteins Toc protein  Function(s)  Structural Features  Toc159  receptor and translocator  GTP-binding site  Toc75  translocation channel  β barrel protein, cation-selective and voltage dependent ; pore size ~2-3 nm  Toc64  chaperone docking site, dual-targeting recognition*, receptor*  C-terminal 3 tetratricopeptide repeat (TPR) motifs exposed to cytosol  Toc34  (initial*) receptor; regulator  GTP-binding site  Toc12  IMS translocation  J domain, associated with Toc64, exposed to IMS  TIC ( = translocon at the inner membrane of chloroplasts) proteins Tic protein  Function(s)  Structural Features  Tic110  translocation channel*  hydrophobic N-terminus and hydrophilic C-terminus; pore size ~1.5-2 nm  Tic62  redox-dependent regulation of protein import  NADPH-binding site; C-terminus interacts with FNR and exposed to stroma  Tic55  redox-dependent regulation of protein import  iron-sulfur centre and a mononuclear iron-binding site  Tic40  IM protein translocation  Hip and Hop homology and C-terminal TPR domains exposed to stroma  Tic32  redox-dependent regulation of protein import  N-terminal NADPH-binding site and C-terminal calmodulin-binding domain  Tic22  IMS protein translocation  loosely bound to the IM; exposed into the IMS  Tic20  *  translocation channel  four putative transmembrane α-helices  Tic21  translocation channel*  four putative transmembrane α-helices  *  Function(s) unclear  32  proposed role of Toc64 as a docking site for cytosolic chaperone Hsp90 interacting with precursor proteins was provided by Qbadou et al. (167). Toc64 is proposed to mediate the transfer of the preprotein from Hsp90 to the Toc34 receptor. Interestingly, an Arabidopsis homolog, Toc64-V, has been found in association with mitochondria which has led to the hypothesis that Toc64 may be involved in the recognition of dual-targeted proteins (206). In addition, Toc64 has also been proposed as an alternative receptor for proteins lacking a cleavable TP (206). The smallest protein subunit of the TOC complex is Toc12 which extends into the IMS. It consists of a J domain (DnaJ-like domain) that interacts with an imsHsp70 homologue to prevent preproteins from misfolding or mislocalizing to the IMS (13). The close association between Toc12, Toc64 and Tic22 (see below), suggests that these proteins may form a complex to assist in the transfer of precursor proteins across the IMS, a step which is mediated by ATP.  1.4.1.2 TIC The translocation of preproteins across the chloroplast IM requires energy, likely due to the requirement of ATP-dependent chaperones in the stroma. The eight known TIC proteins of the TIC complex are summarized in Table 1.2. Tic110 consists of two domains: an N-terminal domain with two hydrophobic transmembrane α-helices, which are important to anchor the protein to the IM, and a hydrophilic stromal C-terminal domain. Tic110 is the most abundant subunit of the TIC complex and is believed to form the translocation channel with a predicted pore size of approximately 1.5-2.0 nm (71). Tic110 is able to recruit stromal molecular chaperones, Cpn60 and ClpC, to the import site. Both chaperones may function in the folding of the imported protein and/or serve a  33  role as an import motor, possibly by stabilizing the imported protein and preventing retrograde movement. Tic20 and Tic21 are structurally similar hydrophobic proteins, consisting of four putative transmembrane α-helices that are deeply embedded in the IM (108, 213). The possibility that they also form translocation channels is supported mainly by their distant sequence similarity to mitochondrial Tim channel proteins (see Section 1.4.2.2). There is some uncertainty as to which protein serves as the TIC channel. It has not been ruled out that perhaps all three subunits, Tic110, Tic20 and Tic21, participate in the formation of the TIC channel. Tic62 and Tic55 have the capacity to catalyze electron-transfer reactions. Tic62 has a conserved NADPH-binding site at the N-terminus. The C-terminus is exposed to the stroma and can interact with ferredoxin NADPH reductase (=FNR) (114). FNR is involved in CO2 fixation and is thus an important component of photosynthesis; the interaction between Tic62 and FNR therefore links chloroplast metabolism to the import capacity of the TIC complex. Tic55 contains a Rieske iron-sulfur centre and a mononuclear iron-binding site (25). Tic55 is predicted to contain a transmembrane helical region, most of which is exposed to the stroma. Rieske-type proteins are known to be involved in electron transfer. Tic110, Tic62 and Tic55 form the TIC core complex. Tic32 was shown to have NADPH-dependent dehydrogenase activity at the N-terminus with a central β sheet motif and a calmodulin-binding domain at the C-terminus (42). For example, NADP/NADPH binding to Tic32 is important for TIC translocation (42). Tic32 interaction with Tic110 significantly decreases with higher NADPH levels; therefore, the interaction between Tic32 and Tic110 can change based on the NADP+/NADPH ratio in chloroplasts. It has also been identified as an IM calmodulin-binding protein and is thus  34  possibly involved in calcium regulation of protein import. Thus, it appears that Tic62, Tic55 and Tic32 may play a role in redox-dependent and calcium regulation of protein import and may not be directly involved in the translocation of preproteins across the import channels. Tic40 is an integral protein and is closely associated with both Tic110 and Hsp93. The C-terminus is exposed to the stroma and consists of two domains: one is homologous to Hsp70-interacting proteins (Hip) and Hsp70/Hsp90-organizing proteins (Hop) and the other has a TRP domain (45). It has been proposed that Tic40 functions to recruit molecular chaperones to the TIC complex. As the preprotein emerges from the TIC channel, the TP first binds to Tic110. The resulting conformational change in Tic110 leads to binding with Tic40. The association of Tic40 and Tic110 results in the dissociation of the precursor protein from Tic110 and exposure of the Hip/Hop domain of Tic40 which leads to the ATP hydrolysis of Hsp93 and translocation of the preprotein across the IM. Lastly, Tic22 is mainly exposed to the IMS and is only loosely bound to the IM. It may function to coordinate TOC/TIC activities and/or to guide preproteins across the IMS. Upon preprotein translocation to the stroma through the TOC/TIC complexes, the N-terminal extension of the TP is removed in an ATP-dependent manner by the SPP (223). Proteins destined for the stroma are usually folded into their active conformation by stromal chaperones. Other proteins which contain an appropriate second targeting signal will be translocated to the thylakoids.  35  1.4.1.3 Thylakoid protein import The thylakoid membrane contains photosynthetic protein complexes; therefore, most of the proteins that target thylakoids are found exclusively in organisms that undergo photosynthesis. Four pathways have been identified that are associated with translocation across the thylakoid membrane: the signal recognition particle (SRP)dependent and “spontaneous” for insertion of proteins into the thylakoid membrane and the Sec-dependent and ∆pH/Tat-dependent which function to transport proteins into the thylakoid lumen (67, 195). Known thylakoid membrane proteins using the SRP-dependent pathway contain a TP for stromal targeting which is removed by the SPP (158). Protein targeting and insertion into the thylakoid membrane is mediated by internal uncleaved signals in the mature part of the protein and requires GTP hydrolysis and additional stromal and thylakoid factors. Direct or “spontaneous” protein insertion into the thylakoid membrane (188) requires a preprotein with a bipartite TP for stromal (with no SPP processing) and thylakoid import and the presence of two hydrophobic domains flanking the hydrophilic N-terminal segment which will span the membrane upon insertion (143). Upon insertion, the preprotein is processed by the thylakoidal processing peptidase (TPP), generating the mature protein (195). Nuclear-encoded proteins that are destined for the thylakoid lumen are synthesized in the cytosol, contain a bipartite TP carrying two signals in tandem; a cleavable N-terminal TP signal for stromal import followed by a second transport signal that mediates transport across the thylakoid membrane and is subsequently removed by a lumen TPP. The Sec-dependent pathway is similar to the secretory pathway in bacteria,  36  i.e., requiring stromal homologs of bacterial SecA (246), ATP and involving integral thylakoid membrane homologs of SecY and SecE which may form the translocation pore (194). Transport of proteins using the ∆pH/Tat-dependent pathway is mediated by targeting signals which have a twin pair of arginine residues (Tat = Twin arginine translocation) upstream of a hydrophobic core domain (37). The pathway is energized solely by the proton gradient generated across the thylakoid membrane (3) and is able to transport fully folded proteins across the membrane (46).  1.4.1.4 Alternative import pathways The general import pathway (i.e., TOC/TIC) is estimated to mediate about 90% of the trafficking to the chloroplasts; the remaining proteins use alternative pathways (84). Most of these proteins localize to the OM and IM. OM proteins are generally synthesized without an N-terminal cleavable TP and include most of the proteins for the TOC complex. These proteins are thought to be inserted directly (without energy requirements) and independently into the membrane from the cytosolic side and the signals for localization are within the mature parts of these proteins (118). The most interesting exception is Toc75 which contains a bipartite targeting signal (86). The cleavable N-terminal TP is responsible for chloroplast targeting and translocation initiation. Translocation is halted at the TIC complex but the TP is the still processed by the SPP; thereafter, the protein is redirected to the OM. Different pathways have been found for the import of different IMS proteins. For example, both MGD1, which is a MGDG (monogalactosyldiacyl glycerol) synthase  37  (226), and Tic22 (109) are translocated across the OM; however, only MGD1 is processed by the SPP before being released in to the IMS. Two pathways have been suggested for protein translocation to the IM, both of which require the TOC/TIC complex. One involves hydrophobic stop-transfer signals within the mature parts of the proteins that cause lateral movement from the TIC channel to the IM (20). The other involves complete translocation to the stroma followed by cleavage of the TP and a redirection to the IM (e.g., Tic40) (119). It should be noted that not all precursor IM proteins have cleavable TPs. It is noteworthy to mention that a novel chloroplast-targeting pathway involving the Golgi apparatus has recently been proposed. In this pathway, the protein first enters the endoplasmic reticulum (ER) probably via an ER signal peptide; the protein is secreted into Golgi vesicles which are then transported to the chloroplast as vesicle cargo (168).  1.4.2 Targeting and import of mitochondrial proteins Similar to chloroplasts, most mitochondrial proteins are synthesized as preproteins in the cytosol and are imported posttranslationally. Mitochondria consist of two distinct membrane systems: the outer (OM) and inner membranes (IM) which surround the organelle and two soluble compartments, the intermembrane space (IMS) and the matrix (MA). Most proteins translocated to mitochondria utilize import mechanisms mediated by the TOM (translocon at the outer membrane of mitochondria) and TIM (translocon at the inner membrane of mitochondria) complexes (see Fig. 1.6 for schematics for TOM/TIM proteins; for reviews see 16, 62, 124, 154, 155, 165, 170, 231 and references therein). Sequence analyses of plant mitochondrial TOM/TIM proteins  38  39  22  12  9/10  Mdm 10  Carrier pathway  TIM22  35 50  54 18  37  SAM  β-barrel pathway  -  ?ψ  +  MPP +++  Mge1  mtHsp70  ++  ATP  21  20  +  44  23/17  22  18 16 17  50  40  tiny Toms  MIA  TIM23  Mia 40 Erv1  Presequence pathway  PAM  TIM 9/10 chaperone complex  70  TOM  cytosolic chaperone  MIA pathway  Figure 1.6 Schematic representation of the mitochondrial protein import machineries. The components of the TOM and TIM complexes are in blue and green, respectively. The PAM complex-associated Tim23/17 is shown in orange. For each pathway the respective energy forces are indicated. Further details are described in the text. Adapted from (18).  Matrix  Inner membrane  Intermembrane space  Outer membrane  Cytosol  preprotein  show clear homologues of TIM proteins in animal and fungal mitochondria whereas possible homologues for TOM proteins have been identified (126). This may reflect the differences in the mitochondrial import systems in plants (which must contend with similar targeting signals associated with chloroplast import systems) and those of animal/fungi. Some of these differences in mitochondrial TOM proteins will be discussed. More than 99% of all known mitochondrial proteins are synthesized as precursors in the cytosol (16). Similar to chloroplasts, most mitochondrial proteins are synthesized with an N-terminal extension called a presequence that is proteolytically cleaved upon import. The presequence is both necessary and sufficient for organelle recognition and translocation initiation. General features of plant mitochondrial presequences include: (i) sequence lengths of approximately 10-85 amino acids (which are longer than those of animal and fungi); (ii) a cleavable presequence that is highly basic resulting in an overall positive charge and; (iii) a potential to form amphipathic α-helices (117, 200, 227, 228). Most proteins destined for the mitochondria must pass through the TOM complex; thereafter, protein import branches into at least four discrete protein sorting pathways (16). β-Barrel proteins of the OM are passed on to the SAM (sorting and assembly machinery) complex which mediates their membrane integration and assemblage into larger complexes (236). Many IMS proteins that contain characteristic cysteine motifs utilize the MIA (mitochondrial intermembrane space import and assembly) pathway (35). There are two distinct IM translocases which mediate IM protein import. Some preproteins destined for the IM, most of which are metabolite carrier proteins [e.g., ADP/ATP carrier (AAC)] which mediate the transport of  40  metabolites into and out of the MA, are translocated from the TOM complex, with the assistance of TIM chaperones, to the TIM22 complex (see below) via the carrier translocase pathway (155). Similar to that in chloroplasts, the role of molecular chaperones is to maintain the preprotein in an import-competent form. Most preproteins with a cleavable N-terminal presequence that are translocated from the TOM complex to the TIM23 complex (see below) are imported via the presequence pathway (sometimes referred to as the general import pathway) (36). These preproteins are sorted in the IMS and are either halted in the IM in response to a hydrophobic stop-transfer sequence downstream of the N-terminal import signal or translocated into the matrix. Translocation into the matrix requires the ATP-driven PAM (presequence translocaseassociated motor) complex in association with Hsp70 which operates in cooperation with several regulatory proteins.  1.4.2.1 TOM There are seven known TOM proteins in the TOM complex based mainly on studies using Saccharomyces, Arabidopsis and rats (Table 1.3). The animal and fungal Tom70 3 functions as a preprotein receptor with a preference for proteins with internal targeting sequences (23). In mammals, preprotein binding to Tom70 may be assisted by cytosolic chaperones such as MSF (mitochondrial import stimulating factor), Hsp70 and Hsp90 (244). The animal and fungal Tom70 is an N-terminal transmembrane anchored protein with a soluble cytosolic region comprised of 11 TPR motifs (38). Structural studies on yeast Tom70 revealed that the 11 TPRs form two domains: an N-terminal  3  TomXX/TicXX proteins are designated based on molecular weight “XX”; Tom/Tic proteins will be underlined when first described 41  Table 1.3 Summary of mitochondrial import proteins (see text for further details) TOM (= translocon at the outer membrane of mitochondria) proteins Tom protein  Function(s)  Structural Features  Tom70/mtOM64  receptor  N-terminal transmembrane, TPR cytosolic domain, Hsp70 binding  Tom40  translocation channel  cation selective, pore ~2.2-2.5 nm  Tom22  receptor, TOM organizer  1  negatively charged N- and C-termini, exposed to IMS and cytosol, respectively  *  Tom9  (hydrophobic) receptor  negatively charged N- and C-termini, exposed to cytosol and IMS, respectively  Tom20  receptor  transmembrane, helical binding grooves  Tom7  protein destabilizer  transmembrane, N-terminally anchored, small cytosolic C-terminus  Tom6  protein stabilizer  transmembrane, N-terminally anchored, small cytosolic C-terminus  Tom5  intermediate receptor?  transmembrane, N-terminally anchored, negatively charged cytosolic domain  TIM (= translocon at the inner membrane of mitochondria) proteins Tim protein  Function(s)  Structural Features *  Tim54  receptor and/or docking site  transmembrane, large IMS C-terminus  Tim50  receptor, maintains electrochemical gradient  transmembrane, N-terminally anchored, IMS C-terminus  Tim44  adaptor  mostly exposed to MA  Tim23  translocation channel, presequence IM receptor  four transmembrane segments with IMS N- and C-termini, cation selective, pore ~1.3-2.4 nm  Tim22  translocation channel  cation selective, high conductance, pore ~1.1-1.8 nm  Tim21  promotes preprotein release and transfer  transmembrane, large IMS C-terminus with positively charged surface  Tim18  unknown  three transmembrane segments, IMS C-terminus and MA N-terminus  Tim17  IM sorting, Tim23 gating-modulator  four transmembrane segments with IMS N- and C-termini  Tim12  adaptor  mostly exposed to IMS; β barrel protein with four conserved Cys residues  Tim9 and 10  chaperone  free in IMS; β barrel proteins with four conserved Cys residues  *  Function(s) unclear Tom9 is plant equivalent of Tom22  1  42  domain composed of TPRs 1-3 which may be involved in Hsp70 binding (244) and a Cterminal domain composed of TPRs 4-11 which may be involved in preprotein binding (242). However, no clear homologues of Tom70 have been found in plants. Chew et al. (41) discovered a protein called mtOM64 in the mitochondrial OM of Arabidopsis which showed 67% sequence identity with Toc64 (see Section 1.4.1.1). mtOM64 consists of an N-terminal transmembrane domain with three TPR motifs at the C-terminus, but both Western blot analysis with antibodies to Toc64 and C-terminal GFP tagging showed that mtOM64 resides in the mitochondria. A homologue Tom70 function for mtOM64 has yet to be shown. The β barrel protein Tom40 forms a cation selective translocation channel in the OM with an estimated pore diameter of ~2.2-2.5 nm (75); two Tom40 pores are believed to exist per native TOM complex (12). During translocation through the OM, the preprotein is in direct contact with the Tom40 channels, continuously to the IMS site of Tom40 suggesting that Tom40 is involved in forming the IMS presequence binding site along with the IMS domain of Tom22 (98). Tom22 (=MOM22) which functions as an N-terminal presequence receptor, is an important structural organizer of the TOM complex (104, 222) and associates with the TIM23 complex during preprotein import (see below). Tom22 has an N-terminal cytosolic domain, a transmembrane region and a C-terminal IMS domain. Antibodies to Tom22 inhibited preprotein import but did not inhibit OM surface binding (104). This indicates that Tom22 may function downstream of other initial receptors. Acidic residues in the primary sequence of both the N- and Ctermini suggest that electrostatic interactions between Tom22 and positively charged sequences of the presequence may be important for translocation through the channel  43  (18). The Tom22 homologue in plants is a 9 kDa protein (Tom9) which lacks the Nterminal cytosolic domain found in animal Tom22 (127). With fewer acidic residues, Tom9 may have a less important role as an electrostatic receptor than its animal counterpart. It is also possible that hydrophobic interactions may be more important to precursor binding on the cytosolic side of Tom9 whereas electrostatic interactions are important to binding on the IMS side of Tom9 (165). The animal and fungal Tom20 receptor consists of an N-terminal transmembrane domain with a soluble cytosolic C-terminal domain and binds preferentially to preproteins with N-terminal targeting sequences (23, 193). Animal Tom20 consists of five helices: the first two helices form a single TPR motif and together with the third helix form a hydrophobic groove which is the binding site for N-terminal precursor proteins (1). In the plant Tom20 receptor, the domain arrangement is reversed with the C-terminus anchored to the membrane and an acidic cytosolic N-terminal domain homologous to the TPR proteins of those of animal Tom20 and Tom70 (72). The Arabidopsis Tom20 consists of seven helices with the N-terminal helix and the two Cterminal helices flanking two TPR motifs (formed from α2 to α5) to form a groove that binds presequence peptides (164). Three small Tom proteins, “tiny toms”, are transmembrane proteins which, with Tom40 and Tom22, form the general import pore (GIP) (141). They are each Nterminally anchored with a small cytosolic domain. The mechanism that drives preproteins across the OM is unclear but is has been suggested that Tom5, with its negatively charged cytosolic domain, can receive positively charged preproteins from other receptors and cause the insertion of proteins into the GIP (52). Tom6 supports the  44  stable interaction of Tom22 and Tom40 and is thought to maintain the integrity of the TOM complex (48). Tom7 was found to destabilize the interactions between the Tom22 and Tom20 receptors. It is possible that Tom7 may be involved in TOM complex regulation, e.g., a role in preprotein sorting and accumulation at the OM (80).  1.4.2.2 TIM Based mainly on studies using Saccharomyces, Arabidopsis and rats, there are eleven known TIM proteins (Tim9, Tim10, Tim12, Tim17, Tim18, Tim21, Tim22, Tim23, Tim44, Tim50 and Tim54) involved in the transport of mitochondrial proteins (Table 1.3). As mentioned above, most mitochondrial proteins after emerging from the TOM complex will enter one of four discrete protein sorting pathways. β barrel pathway. Import of integral OM proteins requires a β barrel transmembrane topology and the preproteins must first be translocated through the TOM complex into the IMS. Upon entering the IMS, β barrel proteins bind to the TIM chaperone complex which consists of a hexameric assembly of “tiny tims”, Tim9 and Tim10 (3 Tim9s and 3 Tim10s). The “tiny tims’ both guide the proteins to the SAM complex and prevent aggregation of these hydrophobic β barrel proteins in the soluble IMS (237). Including Tim12 (see below), the “tiny tims” contain four conserved cysteine residues that are believed important for protein binding (144). The SAM complex consists of four subunits: Sam50, Sam35, Sam37 and Mdm10. The core component is Sam50 (=Tob55) (111); the hydrophilic N-terminus faces the IMS and contains a polypeptide translocase (POTRA) domain which has been suggested to function as a β barrel preprotein receptor domain whereby substrates are handed over from the TIM  45  chaperone complex (68). Although the specific functions of Sam35 and Sam37 are not clear, temperature-sensitive Sam35 (=Tob38) mutants and deletion Sam37 (=Mas37) mutants show defective import of β barrel preproteins (145, 236). The fourth subunit is Mdm10 which is involved in the maintenance of mitochondrial morphology and in the assembly of Tom40 into TOM complexes but is not important for the import of other β barrel proteins (140). It is not known how mature proteins become partitioned into the lipid bilayers of the OM, what is the molecular nature of the targeting signals with β barrel proteins and if external energy is required for membrane insertion and assembly in this pathway. MIA pathway. Soluble IMS proteins contain characteristic cysteine motifs: a twin Cx9C motif, a twin Cx3C motif (e.g., Tim8, Tim9, Tim10, Tim13) or twin Cx2C motif (e.g., Erv1). Import of these proteins requires Mia40 and the sulfhydryl oxidase Erv1 (60, 142). Upon entering the IMS through the TOM complex, these proteins bind to Mia40 via a transient disulfide bridge (35). The means by which the preproteins are transferred from the TOM complex to Mia40 is unknown. During maturation and assembly the disulfide bridge is transferred to the proteins in a process called disulfide relay (142) that requires the cooperation of Erv1 (173, 208). Recently, studies on precursors for IMS Tom9 and Tom10 proteins have revealed that these preproteins contain a nine aa MISS (mitochondrial intermembrane space sorting) signal that is essential for IMS precursor protein binding to Mia40 and to their correct IMS location (146). Carrier pathway. Many carrier preproteins destined for the IM contain internal targeting signals and are very hydrophobic. Upon entering the IMS through the TOM  46  complex, they are recognized by and bound to the hexameric Tim9/Tim10 chaperone complex (see above). Tim9/Tim10 are required for the release of carrier proteins from the TOM complex and the delivery to the TIM22 complex. Tim22, Tim18 and Tim54 make up the core of the TIM22 complex to which Tim9/Tim10 bind (with the carrier protein) via the adaptor protein Tim12 (110, 123). Tim22 is the central component and forms the IM channel (pore size ~1.8 nm) that specifically responds to internal targeting signals. The Tim22 complex consists of two such pores that cooperate during protein import (171). Protein insertion via the TIM22 complex depends on the electrical potential (∆ψ) across the IM as the sole energy source. It is assumed that the ∆ψ exerts an electrophoretic effect on the positively charged parts of the preprotein, regardless of whether the charges are localized at the N-terminus or in the mature parts of the preprotein. In the absence of a preprotein, the two Tim22 pores are closed. The functions of Tim18 and Tim54 are unclear. Tim54 exposes a large domain into the IMS which has lead to the suggestion that it may serve as a carrier protein receptor or a docking site for the Tim9/Tim10 chaperone complex (231). Subsequent steps of the import process are voltage-dependent which include stable insertion of the preprotein into Tim22 in loop-conformation, completion of membrane integration and assembly of the carrier protein into its mature form (171). Presequence pathway. Many mitochondrial proteins with positively charged, Nterminal presequences are destined to enter the IMS, IM and MA via the TIM23 complex. The TIM23 complex consists of four membrane-embedded proteins, Tim23, Tim17, Tim50 and Tim21 (155). Tim23 and Tim17 proteins share similar topology and sequence similarity with each having four transmembrane segments with the N- and C-termini  47  facing the IMS. Despite these similarities, Tim23 and Tim17 cannot functionally substitute for each other. These two proteins form a stable complex at the site of IM preprotein entry; however, it appears that only the C-terminal domain of Tim23 serves as the protein translocation channel. Tim23 forms a cation-selective, voltage-gated channel with a diameter of approximately 1.3-2.4 nm and specifically responds to the presence of presequence peptides (216). It has been suggested that the N-terminal IMS domain of Tim23 is involved in preprotein recognition and voltage-gating (11). Although the role of Tim17 is not well understood, it has been proposed that Tim17 may be involved in IM sorting (34) and/or Tim23 channel-gating (138). As the preprotein exits the TOM complex, the presequence is bound to the IMS domain of Tom22 (148). While most of the preprotein is still associated with the Tom40 channel, the presequence comes into contact with Tim50, the IM preprotein receptor, and directs the preprotein to the Tim23 channel (61). Tim50 also functions to maintain the electrochemical gradient across the IM by keeping the Tim23 channel in a closed state in the absence of preproteins (139). Tim21 is the only known IM protein that is in direct contact with the TOM complex; Tim21 interacts with the IMS domain of Tom22 (34). Tim21 competes with presequences for binding to Tom22 and stimulates the release of preproteins from the TOM complex. Thus, Tim21 together with Tim50 promotes the transfer of preproteins to the Tim23 channel. Most proteins with an N-terminal targeting presequence are translocated through the Tim23 channel, using ∆ψ as the sole energy source. The force exerted by the ∆ψ acts in an electrophoretic manner on the positively charged presequence. Completion of preprotein import into the MA requires the PAM complex. The TIM complex in  48  association with the PAM complex can be referred to as TIM23MOTOR. The central PAM component is the ATP-dependent mitochondrial Hsp70 (mtHsp70) which provides the driving force for movement of the preprotein into the MA (225). mtHsp70 is associated with the Tim23 complex via the adaptor protein Tim44 (155). mtHsp70 is also associated with the soluble MA protein Mge1 which functions as the nucleotide exchange factor. Mge1 is a mitochondrial homologue of the prokaryotic GrpE protein (17). The activity of mtHsp70 is regulated by at least three additional, closely associated proteins, Pam18, Pam16 and Pam17. Pam18 is a mtHsp70 co-chaperone and has a matrix located J-protein domain that is involved in stimulating ATPase activity of mtHsp70 (217). Pam16 which is an IM protein bound at the MA side appears to recruit Pam18 to import sites (59) and possibly regulates import by counteracting the Hsp70 ATPase-stimulating activity of Pam18 (120). Pam17 appears to be important for Pam18/Pam16 stability and for the TIM23 complex interaction with Pam18/Pam16 (221). Upon entering the MA, the presequence of the precursor is cleaved off by the mitochondrial processing peptidase (MPP) (62), resulting in the production of a mature protein which is then folded to achieve the correct conformation. The MA targeting signal and the signal for cleavage recognized by the MPP are probably separate; however, sites may overlap, especially for preproteins with short presequences (200). The plant MPP known to specifically process N-terminal targeting signals upon MA import is a membrane-localized metalloendopeptidase that is integrated into the cytochrome bc1 complex of the respiratory chain (19). This MPP is a general peptidase in that it is able to process several hundred mitochondrial precursor proteins whose presequences show low sequence similarity. However, the MPP is specific as it appears to cleave at distinct sites  49  in the precursor protein (62). Szigyarto et al. (210) have identified a less well characterized plant matrix-located MPP that also specifically processes precursor proteins. Some preproteins carry a bipartite signal sequence, in which the N-terminal matrix-targeting signal is followed by a hydrophobic stop-transfer sequence (“sorting signal”) that halts translocation and induces a lateral sorting of the preprotein into the IM. For some sorted proteins, the stop transfer sequence remains as part of the mature IM protein serving as a membrane anchor (e.g., D-lactate dehydrogenase). In other proteins, the stop-transfer sequence of the protein is processed by an intermembrane space peptidase (IMP) on the IMS side of the IM, after which the mature protein is released into the IMS (e.g., cytochrome b2) (74). Preprotein sorting in the IM does not require the PAM complex indicating that the IM sorting and MA import are mechanistically different processes. Chacinska et al. (34) demonstrated that two different forms of the TIM23 complex exist: one which contains the TIM23/PAM supercomplex and catalyzes MA import (i.e., TIM23MOTOR) and the other a PAM-free, Tim21-bound TIM23 complex that is responsible for preprotein sorting into the IM and has been called TIM23SORT. The ability of the TIM23 complex to switch between the two different forms is controlled by the targeting information within the incoming preproteins (34). Without the PAM complex, the sole energy source for TIM23SORT is the proton motive force across the IM.  1.4.3 Dual-targeting of chloroplast and mitochondrial proteins Although targeting to chloroplasts and mitochondria is highly specific there are many examples where the same protein is imported into both organelles (198). Although  50  both chloroplast and mitochondrial targeting sequences have N-terminal presequences that are enriched with basic amino acids and deficient in acidic ones (228), they do not share other characteristic features (55, 201). Protein import systems in plants must have evolved the ability to discriminate between chloroplast and mitochondrial targeting sequences. In recent years, the number of proteins found to target chloroplasts and mitochondria has increased and analyses of targeting peptides that affect specificity shows that they are highly variable and often modulated by cellular or developmental requirements (99). There are two types of dual-targeting of chloroplast and mitochondrial proteins: twin and ambiguous presequences (163). “Twin” targeting presequences contain a mitochondrial and a chloroplast targeting sequence in tandem at the N-terminus of the preprotein. “Twin” targeting presequences can be achieved by having alternative transcription or alternative translation starts, alternative exon splicing or a combination of both; the result would be two proteins made from the same gene. “Ambiguous” presequences occur when different regions within the targeting sequence are recognized by both mitochondria and chloroplasts.  1.5 Cucumber necrosis virus CNV was first discovered in 1952 on cucumber plants grown in a greenhouse in southern Ontario, Canada (135). Infected cucumbers exhibited symptoms of marked malformed and chlorotic leaves with stunted growth and a reduction in fruit size. Eventual necrosis from systemic infection resulted in death, usually in 6 to 8 weeks. McKeen (135) also noted that in all experiments using CNV-infected potted plants, the  51  virus was found consistently in roots and in greater concentrations compared with that found in stems and leaves. It had also been established that the first infections originated from root invasion. The natural host range of CNV is apparently restricted to cucumbers; however, it can experimentally infect a wide range of hosts. CNV was originally considered as a possible member of the Necrovirus group, mainly due to the development of comparable symptoms to that of the type member Tobacco necrosis virus (TNV) and the fact that it was found to be fungally transmissible as is TNV (131, 207). However, sequence homology to TBSV and some cross-protection against TBSV-infected plants established CNV as a Tombusvirus (Family: Tombusviridae) (179). CNV is serologically distinct from other tombusviruses, possibly due to the considerable variability of the tombusvirus CP protruding domain which may be the most antigenic region of the particle. Of 13 recognized species, CNV is the only tombusvirus known to have a specific fungal vector (178). CNV is transmitted by zoospores of the soil-habiting, root-infecting, chytrid fungus, Olpidium bornavanus (=radicale) (10, 51, 207). CNV is known to produce defective interfering (DI) RNAs which are natural deletion mutants of the viral genome generated by errors made during replication. DI RNAs are unable to replicate without the parent virus, CNV (58). A fulllength infectious cDNA clone of CNV has been produced by Rochon and Johnston (180), enabling extensive mutational analysis of the genome.  1.5.1 Genomic organization CNV contains a single-stranded (ss) monopartite 4.7 kb RNA genome of which the complete nucleotide sequence has been determined (181). The genome consists of  52  five open reading frames (ORFs) which express proteins of 33, 92, 41, 21 and 20 kDa (Fig. 1.7) (177). p33 (ORF1) is an auxiliary protein for viral replication (161). The p33 protein has been shown to localize to peroxisomal membranes which are believed to be the sites of viral replication (134, 161; D. Rochon, unpublished observations). The 92 kDa protein (ORF2) is produced from a readthrough of the UAG amber stop codon at the 3’ terminus of ORF1. Comparative amino acid sequence analysis and presence of a known glycine-aspartate-aspartate (GDD) replicase motif within the 92 kDa protein indicates that it is the viral RNA dependent RNA polymerase (150, 181). Two 3’ coterminal subgenomic RNAs (sgRNA1 and sgRNA2) are generated from CNV genomic RNA during infection (92). p41 (ORF3) is expressed from sgRNA1; comparative amino acid sequence analysis of p41 indicates that it encodes the coat protein (CP) (181). The CNV CP is a multifunctional protein with roles in systemic movement (137, 199), fungal transmission (see Section 1.3.1) as well as encapsidation of viral RNA (94). sgRNA2 is a bifunctional mRNA that encodes p21 and p20 from two distinct, overlapping ORFs (4 and 5, respectively) (180). Expression of p20 occurs via a leaky scanning mechanism (90-92). p20 is involved in symptom development (180) and is a suppressor of gene silencing (Rochon, D., unpublished observations). p21 (ORF4) is required for cell-to-cell movement (91, 177).  1.5.2 Particle structure The structure of CNV virions is similar to that of the type member of the Tombusvirus group, TBSV (see Section 1.2.1.2.1.) and has recently been determined using cryo-TEM to a resolution of ~12Å (100). The particle is arranged as a T = 3  53  ORF5 5’  20 kDa  UAG 33 kDa  92 kDa  41 kDa  21 kDa  ORF1  ORF2  ORF3  ORF4  3’  sgRNA1 sgRNA2  Figure 1.7 The genomic organization of CNV. The five open reading frames (ORFs) with their respective protein sizes are indicated (see text for details). The 33 and 92 kDa read-through (RT) proteins are translated from the genomic length RNA, the CP is translated from subgenomic (sg) RNA1 and the 21/20 kDa proteins are translated from sgRNA2. Subgenomic RNA2 is bicistronic with the 20 kDa protein being translated by leaky scanning (97).  54  icosahedron and consists of 180 copies of a single 41K CP subunit of which there are three structural conformations, A, B and C. Each subunit contains three major domains (Fig. 1.3B): a 58 amino acid (aa) RNA-binding (R), a 167 aa Shell (S) that encapsidates the RNA and a 116 aa Protruding (P) that extends beyond the S. The R and S domains are connected by a 34 aa flexible arm (a) and the P domain is connected to the S by a small 5 aa hinge (h). The CP arm of CNV consists of two distinct regions: an Nterminal 18 aa “β” and a 16 aa C-terminal “ε” region (see Fig. 1.3C). An internal βannular structure formed by the interdigitating arms from three ordered C subunits at the particle three-fold axis is believed to contribute to virus stability (this thesis; 82, 175). Most of the virion RNA lies along the inner face of the outermost protein shell; moreover, an inner RNA core with distinctive icosahedral features was found which has been attributed to the internal scaffolding effect of the R domain (100).  1.5.3. Targeting of the CNV CP to chloroplasts It has recently been shown that the CNV CP targets to chloroplasts during virus infection (243). Agroinfiltration studies using the N-terminal R and arm regions of the CP showed that a 39 aa region encompassing the arm region is sufficient for chloroplast targeting. Analysis of this region showed that it contained features typical of chloroplast transit peptides, suggesting that CNV arm has a transit peptide-like function. The CNV CP was found to undergo specific cleavage prior to and following chloroplast import: cleavage near the R/arm junction (which was proposed to occur in the cytoplasm) and cleavage at the arm/shell junction which was found to occur upon CP uptake into the  55  chloroplast stroma. Figure 1.8A shows a model for CNV CP import into chloroplasts previously proposed by Xiang et al. (243). The biological significance of CP uptake into chloroplasts is not known; however, it is believed to be associated with either the assembly or disassembly process of virus particles. A model describing a possible role for chloroplast targeting of the CNV particle in the uncoating process is shown in Fig. 1.8B. The model incorporates both the findings of Xiang et al. (243) and other known features of CNV particles. As described in Section 1.2.1.3.4 and by Kakani et al (95), CNV particles are believed to “breath” in vivo, that is, to undergo transient swelling within the plant cell during infection. Such breathing leads to externalization of the R and arm domain (95). The model proposes that the exposed R/arm region of the swollen particle is cleaved in the cytoplasm near the R/arm junction resulting in exposure of the chloroplast TP-like region of the CP arm at the N-terminal region. The arm, due to its TP-like function, then directs the particle to chloroplasts, likely in conjunction with host factors known to be involved in chloroplast precursor protein transport. Binding of the CP arm to chloroplasts and the ensuing stromal uptake via the Tic/Toc translocon (see Section 1.4.1) result in destabilization and disassembly of the virus particle. Chloroplast uptake of CP sequences and subsequent stromal cleavage would lead to particle disassembly. Recent studies have shown that the CP R and arm regions of two other members of the Tombusviridae family also act as chloroplast TP-like sequences (D. Rochon, unpublished observations), demonstrating that chloroplast targeting is not restricted to the CNV CP. For example, high levels of CP or virus particles have been found in chloroplasts purified from leaves infiltrated or inoculated with Cucumber leaf spot virus  56  A  B  Native CNV particle  Extrusion of “Breathing” R/arm Cleavage in arm at R/arm boundary  Swollen particle  Cleavage in arm at R/arm boundary Host factors (HSP70, 14-3-3…)  Binding of host factors Transport to site of translation, replication?  Transport to chloroplast  Host factors (HSP70, 14-3-3…)  Binding of host factors  Toc Tic  Cleavage in R domain arm/S domain cleavage  Disassembly in cytoplasm  Cytoplasm  (or)  Transport to chloroplast Disassembly on chloroplasts  Chloroplast stroma  arm/S domain cleavage  To cT ic  To cT R arm  S  ic  P Cytoplasm  Chloroplast stroma  Figure 1.8 A model for import of CNV CP and CNV particle uncoating on chloroplasts. A) Import of the CNV CP subunit. A diagram of the folded CNV coat protein subunit is shown with the R, arm, S and P domains differentially coloured (R=red; arm=yellow; S and P=gray) (see inset at bottom). The R domain is shown as containing two regions (designated with a line and closed circle) that correspond to the two fragments that are released following proteolytic cleavage. Cleavage in the arm at the R/arm boundary followed by binding of the arm region (the CNV cpTP) by host factors involved in cellular chloroplast preprotein targeting. Transport to and uptake by the chloroplast Tic/Toc translocon occurs and cleavage by a stromal protease ensues. B) Uncoating of the CNV particle. The internal location of the R and arm and the encased virion RNA (blue line) are shown (see part A for color scheme used for the CP subunit domains). Extrusion of the arm and part of the R domain occurs (due to “breathing” of particles). This is followed by proteolytic cleavage in the arm near the R/arm junction, and binding by host factors involved in cellular chloroplast protein import as described in part A for the CP subunit. Two potential routes for disassembly of the host-protein bound particle are shown. Disassembly in the cytoplasm would be driven by binding of host factors such as HSP70 and 14-3-3. Alternatively, the particle complex could be transported to the chloroplast whereupon uptake of the arm/S/P region of the particle facilitates the disassembly process on the cytoplasmic side. Virion RNA is released as an R domain/RNA complex wherein the R domain is proteolytically cleaved and transported to the site of RNA translation and/or replication. The imported arm/S/P region of the particle subunit is cleaved as in part A above. From Xiang et al. (257) 57  (CLSV). More recent studies with the CNV CP, show that full-length CNV CP fused to GFP also targets chloroplasts, undergoing specific cleavage events as predicted by Xiang et al. (243; D. Rochon, unpublished observations). In addition, CNV CP which is not fused to GFP also targets chloroplasts indicating that CP fusion to GFP is not a consequence of an artifactual exposure of a cryptic TP-like sequence (D.Rochon, unpublished observations).  1.6 Summary of thesis objectives The overall goal of this thesis is to further the understanding of the role of the CNV CP in the infection process. In particular, the structural importance of the CNV CP arm during viral infection, in relation to fungus transmission and particle stability, is examined. In addition, the subcellular location of the CP is examined as part of a larger goal to investigate the subcellular site(s) of particle assembly/disassembly. Details of the thesis objectives are described below.  1. To identify specific regions of the CNV CP arm that are important for particle structure, stability and fungus transmission. A. Site-directed mutational analysis. Site-specific in vitro mutagenesis of the CP arm was conducted to determine the importance of the two regions of the arm for particle accumulation and stability in infected leaves. The CNV CP arm consists of two distinct regions: the ε region which extends from the N-terminal portion of the CP interiorly and consists of residues postulated to interact closely with viral RNA in the particle interior  58  and the β region in which similar regions from three ordered C subunits interact to form an internal β annular structure proposed to function in virion stability. B. Fungal transmission and in vitro zoospore/virus binding studies. Mutant particles from the site-directed mutagenesis studies were tested to assess the role of the arm regions in fungal transmission and the ability to bind zoospores in vitro. The results of these studies are described in Chapter 2.  2. To characterize the role of specific sequences of the N-terminal region of the CNV CP in subcellular targeting during infection. A. Analysis of the subcellular location of the CNV CP using green fluorescent protein (GFP)-tagged CP constructs. Previous confocal and biochemical studies in our laboratory showed that specific regions of the CP N-terminus target chloroplasts and other unidentified subcellular sites (243). Deletion analysis of specific regions of the CNV CP N-terminus was conducted to further delineate the role of the CP R, arm and S regions in subcellular targeting. Studies were conducted by tagging select CNV CP regions with GFP and by examining agro-infiltrated plants using confocal microscopy. B. Analysis of isolated and subfractionated organelles to assess the location of GFP-tagged CP fragments. 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Virology 277:4506.  92  2 CHAPTER TWO: Evaluation of specific regions of Cucumber necrosis virus coat protein arm on particle accumulation and virus transmission* 2.1 Introduction The capsids of many plant and animal viruses are multifunctional (2) playing roles in genome protection, cell-to-cell and long distance movement within plants (4), vector transmission (9, 21, 28) replication (1) and suppression of gene silencing (22, 34). Structural studies of several plant viruses have revealed that the overall architecture of the T = 3 capsid can be highly conserved between otherwise divergent virus groups, including those of several animal viruses (14). The structures of many plant virus particles have been obtained and several in vitro studies have been conducted to assess the role of the different structural domains in particle integrity and assembly but few in vivo studies have been conducted. Moreover, fewer studies that relate the various structural domains to other possible functions of viral capsids have been reported. Cucumber necrosis virus (CNV), a member of the Tombusviridae, is a 33 nm spherical virus that encapsidates a monopartite, positive-sense RNA genome (27). In nature, transmission of CNV occurs via zoospores of the fungus, Olpidium bornovanus, in which zoospore-bound particles are transmitted to cucumber following zoospore entry into root cells (3, 28). Based on structural homology to Tomato bushy stunt virus (TBSV) (12, 15), CNV is a T = 3 icosahedron consisting of 180 identical 41 kDa CP subunits. Each subunit consists of three major structural domains: the R domain, which extends interiorly in the capsid; the S domain, which forms the shell of the capsid; and the P *  A version of this chapter has been published. Hui, E. and Rochon, D. (2006) Evaluation of specific regions of the Cucumber necrosis virus coat protein arm on particle accumulation and fungus transmission. J. Virol. 80:5968-5975. 93  domain, which projects outward from the capsid. The P and S domains are joined by a 5 amino acid (aa) hinge and the R and S domains are connected by a 34 aa arm (Fig.2.1A). The CP subunit exists in three different conformations designated A, B, and C. In the C subunit, the arm is ordered and the R domain is spatially disordered; whereas in the A and B subunits, both the arm and R domain are disordered. The CP arm can be divided into two structurally distinct regions: “β” and “ε” (Fig. 2.1B). The β-regions from three C subunits interdigitate at the particle three-fold axis to form a β-annular structure (Fig. 1C) that contributes to particle stability and possibly to assembly as well. Similar structural regions within the CP of various other T = 3 icosahedral viruses are believed to function as scaffolds important for particle stability and virus assembly (5, 32, 33). The ε-region of the CNV arm extends from the β-region and continues along the inner face of the S domain. It has been suggested that the ε-region may serve to bind RNA within the capsid, contribute to the stability of the swollen form of the particle at the quasi two-fold axis and, due to its ordered/disordered state in C and A/B subunits, respectively, contribute to quasi-equivalence (12, 24, 35). The CP of CNV, as well as that of several other tombusviruses is not required for cell-to-cell movement (29, 31); however, systemic movement of unencapsidated viral RNA is delayed compared with that of encapsidated RNA (31). Previous work has shown that specific regions of the S and P domains of the CNV capsid are involved in fungal transmission (15, 23) and that the defects are due, at least in part, to inefficient attachment of virions to the zoospore surface (15, 23). Kakani et al. (17) have shown that zoospore-bound CNV is conformationally different from native CNV particles and that a CP mutant with a Pro to Gly change in the arm near the β and ε junction that is incapable  94  of swelling was not being fungally transmitted, suggesting that swelling is required for transmission. These findings have been related to the possible role of particle swelling during virus disassembly as has been suggested by others (14, 17). In this study, we have generated CNV CP arm mutants to examine the role of “β” and “ε”-regions of the arm in virus infection, focusing on particle accumulation, stability and vector transmission.  2.2 Materials and methods 2.2.1 Isolation and purification of virus CNV (pK2/M5) was maintained by mechanical passage in Nicotiana benthamiana or N. clevelandii as previously described (23). A “miniprep” procedure was employed to partially purify wild type (WT) CNV and CNV mutants from single infected leaves for use in the initial screenings and for subsequent assays (23). The concentration of virus was determined by electrophoresis of several dilutions through a 1% agarose gel buffered in 80 mM Tris/80 mM borate, pH 8.3, followed by staining with ethidium bromide in electrophoresis buffer containing 1 mM EDTA. Purification of virus from larger amounts of leaf tissue (100-250 g) was carried out by a differential centrifugation method previously described (15). Virus pellets were resuspended in 10 mM sodium acetate buffer (pH 5.0) and virus concentration was determined spectrophotometrically (A260 of a 1 mg/ml suspension of CNV is 4.5.)  2.2.2 In vitro transcription T7 polymerase run-off transcripts from full-length cDNA clones of CNV were generated by a method previously described (25). Transcripts were used immediately to  95  inoculate leaves of 3- to 4-week old N. benthamiana plants. Aliquots of transcript reactions were routinely examined by agarose gel electrophoresis to assess the quality and quantity of transcripts produced.  2.2.3 Leaf RNA extraction Infected leaves were ground to a powder in liquid nitrogen and RNA was extracted with phenol/chloroform in buffer containing 100 mM Tris, 100 mM NaCl and 10 mM EDTA, pH 7.5, along with 0.5% SDS and 5% 2-mercaptoethanol.  2.2.4 Site-directed mutagenesis In vitro mutagenesis was used to produce the following CNV CP arm mutants: arm(-), 15β(-), 18β(-), 16ε(-) and 19ε(-). An EcoRI/ NcoI fragment, encompassing the CNV CP and flanking regions in a full-length infectious cDNA clone of CNV (pK2/M5; 25), was excised and subcloned into EcoRI/NcoI-digested pT7 Blue (Novagen) and used as a template for oligonucleotide-directed in vitro mutagenesis (8). Primers used to create the deletion mutants are described in Table 2.1. Subclones bearing mutations were digested with EcoRI and NcoI and cloned into pK2/M5 which was then used as a template to form full-length infectious clones. The sequences between the EcoRI and NcoI sites were confirmed by sequence analysis.  2.2.5 Analysis of the CP subunit Electrophoresis of protein from purified mutant particles was performed using sodium dodecyl sulfate (SDS)-containing12% polyacrylamide gels according to Laemmli  96  Table 2.1 Primers used for PCR mutagenesis of CNV CP arm mutants Construct name  Primer sequence  Amino acids deleted1  arm(-)  Negative sense 5’ AGCACCGTTTCCATTCTTACC 3’ Positive sense 5’ TCTGTGCGAATAACCCATAGAG 3’  59-92 deleted  15β(-)  Negative sense 5’ AGCACCGTTTCCATTCTTACC 3’ Positive sense 5’ ATCTCTTATGCCTATGCGG 3’  59-73 deleted  18β(-)  Negative sense 5’ AGCACCGTTTCCATTCTTACC 3’ Positive sense 5’ GCCTATGCGGTTAAAGGAAGG 3’  59-76 deleted  16ε(-)  Negative sense 5’ ATAAGAGATTGGCGCGGC 3’ Positive sense 5’ TCTGTGCGAATAACCCATAGAG 3’  77-92 deleted  19ε(-)  Negative sense 5’ TGGAGCAGCAATAGCCCCAGG 3’ Positive sense 5’ TCTGTGCGAATAACCCATAGAG 3’  74-92 deleted  1  refers to CP amino acids beginning at the amino-terminus  97  (18) to verify correct protein size.  2.2.6 Virion RNA extraction RNA was extracted from purified β(-) mutant particles using 10 mM EDTA and phenol/chloroform/1%-SDS in 50 mM Tris buffer (pH 8.8) as previously described (26).  2.2.7 In vitro swelling WT CNV, 15β(-) and 18β(-) virions (500 ng virus/20 ul) were swollen in 50 mM sodium phosphate buffer (pH 7.6), containing 25 mM EDTA at room temperature for 30 min.  2.2.8 Electron microscopy Purified virus was negatively stained with 2% uranyl acetate. Each sample was placed onto a collodion-covered copper grid upon which uranyl acetate was applied and left for 1 min. Samples were viewed at 27,000X to 80,000X magnification in a JEOL 100CX transmission electron microscope (TEM) operated at 80 kV.  2.2.9 Fungus transmission assay O. bornovanus isolate SS196 was maintained on cucumber roots (Cucumis sativis cv. Poinsette 76) as previously described (23). Purified 15β(-) and 18β(-) virions were tested for transmission by O. bornovanus zoospores as previously described (15). Virus (1 μg) was incubated with 10 ml of zoospores (105/ml in 50 mM glycine, pH 7.6). After a 15-min acquisition period, the mixture was poured into pots containing 14-16 day-old  98  cucumber seedlings. Five days later, roots of cucumber seedlings were tested for the presence of CNV by double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) using polyclonal antisera raised to CNV particles. Each transmission experiment included a wild-type (WT) CNV control, to determine any background level of CNV transmission in the absence of zoospores, and an aviruliferous zoospore control to confirm the absence of contaminating virus in zoospore preparations. Each assay was replicated three times.  2.2.10 In vitro binding assay An in vitro binding assay was performed to determine the ability of mutant virus to bind to zoospores of O. bornovanus (15). One hundred micrograms of purified virus were incubated with 5 X 105 zoospores in 1 ml of 50 mM sodium phosphate binding buffer (pH 7.6) for 1 h at room temperature. Following incubation, zoospores were pelleted by centrifugation at ~2,700 × g for 7 min. The pellet was washed with 1.5 ml of binding buffer and then resuspended in sterile, deionized water. The zoospore pellet was assayed for the presence of virus by Western blot analysis using CNV monoclonal antibody 57.2 (specific to the CNV R domain) (23) and an enhanced chemiluminescent detection system (Amersham Biosciences). The quantity of virus in the pellet was assessed by densitometric analysis of exposed film using the ImageQuant TL program (Amersham Biosciences).  99  2.2.11 Thermal stability and ribonuclease sensitivity assays Purified WT CNV and 18β(-) virions were assayed for thermostability by incubating 1 μg of virus (in 10 mM sodium acetate, pH 7.2) for 30 min at 4oC, 26 oC, 37 o  C, 42 oC, 55 oC, 60 oC, 70 oC and 100oC. Particles were immediately examined by  electrophoresis using a 0.7% agarose gel at 90 V for 1.5 h. Virion RNA was visualized by staining with ethidium bromide and virion protein by subsequent staining with Coomassie brilliant blue. Ribonuclease assays were conducted by incubating 1 μg of 18β(-) virions at 70°C or at 26oC for 30 min, followed by incubation at 26oC for 30 min with 10 ng of pancreatic RNase A. A negative control was performed in which particles were incubated without RNase A at 70oC for 30 min, followed by incubation at 26oC for 30 min. Virions were electrophoresed and stained as above.  2.3 Results 2.3.1 Role of the β- and ε-regions of the CNV CP arm in particle accumulation Five CP arm deletion mutants were produced to assess the role of the β- and εregions of the arm in particle accumulation (Fig. 2.1A). Transcripts of each arm mutant were used to inoculate leaves of N. benthamiana plants and plants were monitored for symptom induction. Both the 15β(-) and 18β(-) mutants produced symptoms typical of WT CNV, resulting in necrotic lesions on inoculated leaves and subsequent systemic necrosis and death of the plants within 5 to 6 days post-inoculation (dpi) (data not shown). The arm(-), 16ε(-) and 19ε(-) mutants produced necrosis on the inoculated  100  WT CNV coat protein  (A)  h(5)  arm(34) R(58)  P(116)  S(167)  arm Mutants arm(-) 15β(-) 18β(-) 19ε(-) 16ε(-)  ε(16)  β(18)  (B)  P  S  ε  β  arm  β-annulus 18 aa deletion  (C) B C  C  B B  Part of ε-region  C  Figure 2.1 Description of the location of the CNV CP arm and deletion mutants. (A) Linear order of CP domains and the β- and ε-regions of the arm as well as the locations of deleted regions of each arm mutant. The number of amino acids comprising each CP domain and the β- and ε-regions are shown in parentheses. R = RNA binding; S = shell; h = hinge; and P = protruding domain. The dotted regions indicate deleted regions in the arm. The number of amino acids comprising each CP domain and the βand ε-regions are shown in parentheses. (B) Ball and stick representation of the C subunit of CNV CP showing location of ε- (in yellow) and β-regions (in green) of the arm. The R domain is not shown. (C) Schematic representation of the β-annulus plus six residues of the ε-region (partial arms of the three C subunits in black, blue and red) viewed down the particle three-fold axis. The region within the circle (------) indicates the deleted residues of the 18β(-) mutant. The balls represent α-carbon positions. (Diagram adapted from Ref. 12). 101  leaves, but systemic necrosis was delayed in comparison to WT CNV, appearing 10 to 14 dpi (data not shown). Mutant-infected plants were then tested for the presence of viral genomic RNA and production of virions. Fig. 2.2A shows an electropherogram of total leaf RNA extracts of each of the mutants and indicates that viral genomic RNA is readily observed in each mutant-infected plant. In addition, truncated genomic RNA (which lacks the CP coding regions, data not shown) was occasionally observed in ε(-) mutants (Fig. 2.2A, lane 8). Agarose gel electrophoresis of partially purified virus particles showed that particles accumulated in leaves infected with 15β(-) and 18β(-) mutants (Fig. 2.2B, lanes 4 and 5), whereas particles were not observed in leaves infected with arm(-), 16ε(-) and 19ε(-) mutants (Fig. 2.2B, lanes 3, 6 and 7). The yield of 15β(-) and 18β(-) virions from infected plants at 5 dpi was determined and yields of both were found to be approximately 18% that of WT CNV. SDS-PAGE analysis was conducted to confirmed the presence of the deletions in the CP subunit of purified 15β(-) and18β(-) particles. As seen in Fig. 2.2C, the CP of both mutants show the expected increase in mobility relative to that of WT CNV CP. Virions of β(-) deletion mutants purified from infected plants were analyzed for the presence of viral genomic RNA. Agarose gel electrophoresis demonstrated that genomic RNA was present in both both 15β(-) and18β(-) virions (data not shown). β(-) mutant particles were also visualized using transmission electron microscopy (TEM). Negatively stained particles were found to be grossly similar to those of WT CNV particles; however, β(-) mutant particles appeared more hexagonal in shape than CNV particles, and also exhibited more densely-stained centers (Fig. 2.3).  102  oc W k T ar m 15 (-) β 18 (-) β 19 (-) ε( 16 -) ε 19 (-) ε( -)  m  (A)  del ds gRNA del ss gRNA  ds gRNA ss gRNA  m  (B)  4 5 6 7 8  oc W k T ar m 15 (-) β( 18 ) β( 19 -) ε( 16 -) ε()  1 2 3  41.0 kDa 39.4 kDa 39.0 kDa  4  5  6  7  T 15 β( -) 18 β( -) PS S  (C)  3  W  1 2  48.8 kDa 37.1 kDa 25.9 kDa  Figure 2.2 Gel electrophoresis of leaf RNA, virions and CP subunit of arm mutants. (A) Agarose gel electrophoresis of total RNA extracted from inoculated leaves at 4 dpi and visualized with ethidium bromide (EtBr). Lane 8 corresponds to an independent extract of 19ε(-) in which genomic RNA was found to contain a deletion in the CP ORF. Positions of the double-stranded (ds) and single-stranded (ss) genomic RNAs (gRNA) are indicated on the left and the deleted (del) ds and ss gRNAs are indicated on the right. (B) Agarose gel electrophoresis of partially purified virions Virions in 1% gel buffered in Tris-borate buffer (pH 8.3) were visualized by EtBr staining in the presence of 1 mM EDTA. (C) SDS-PAGE gel of 5 μg of purified WT CNV, 15β(-) and 18β(-) virions. Gels were stained with Coomassie brilliant blue. The numbers on the right indicate the molecular mass (in kDa) of the protein size standard (PSS). The numbers on the left indicate the predicted sizes of WT, 15β(-) and 18β(-) CP subunits.  103  WT CNV  15β(-)  18β(-)  Native  Swollen  Figure 2.3 Electron micrographs of negatively stained native and swollen β(-) mutant particles. Virions were swollen in 50 mM Tris (pH 8.0), 25 mM EDTA for 30 min at room temperature and immediately examined by TEM. Native particles are in the left column and swollen particles are on the right column. Virus particles used are shown on the left of each pair of treatments.  104  2.3.2 Particles of CNV β(-) mutants are not transmissible by O. bornovanus Particles purified from plants infected with 15β(-) and18β(-) mutants were analyzed for their ability to be transmitted by O. bornovanus to roots of cucumber seedlings. Transmission efficiency was scored by assessing the number of pots infected with virus (as determined by ELISA of cucumber root extracts) versus the number of pots inoculated. Results of the transmission assays indicated that 30 of 30 pots (100% efficiency) inoculated with CNV/zoospore mixtures became infected with WT CNV, whereas 0 of 30 pots (0% efficiency) became infected when either 15β(-) or 18β(-) zoospore mixtures were used (Fig. 2.4). These results suggest that the β-annulus may have an important role in virus transmission, an effect that may be confounded by the relatively low level of particle accumulation as observed above.  2.3.3 CNV 18β(-) mutants show decreased binding to zoospores in vitro The 18β(-) mutants were assessed to determined if the lack of transmissibility may be at least partly due to an inefficient ability of mutant particles to bind zoospores. To do this, a previously developed in vitro virus/zoospore binding assay was utilized (15, 23). One hundred micrograms of either WT CNV or 18β(-) particles were incubated with 5 X 105 zoospores for 1 h, followed by low-speed centrifugation to pellet zoospores and washing to remove unbound or non-specifically bound virus (15). The amount of bound virus in the pellet was then determined by Western blot analysis, followed by densitometry. Figure 2.5 shows that a lower level of 18β(-) particles (approx. 41% of that of WT CNV) were found in the zoospore pellet, suggesting that the lack of transmission  105  (%) Transmission efficiency  120 100  30/30  80 60 40 20 0 WT WT  0/30 15β15β(-)  0/30 18β18β(-)  Virus  Figure 2.4 Summary of fungus transmission assays of β(-) mutant particles. 1 μg of either WT CNV, 15β(-) or 18β(-) particles was incubated with 1 X 106 zoospores. After 15 min, the mixture was poured into pots containing cucumber seedlings. Five days later, roots of the seedlings were tested for the presence of CNV by DAS-ELISA using polyclonal antisera raised to CNV particles. The percentage of pots showing transmission for each virus is indicated on the y-axis. The numbers on the columns indicate the number of pots showing transmission versus the number of pots inoculated with virus/zoospore mixtures. The data represent a compilation of three separate experiments with two replicates per experiment.  106  % WTCNV CNV binding % WT binding  120 100 80 60 40 20 0 WT CNV CNV WT  Virus Virus  18β(-) 18β(-)  Figure 2.5 Summary of in vitro virus-zoospore binding assays. 100 μg of either WT CNV or 18β(-) particles were added to 4 X 105 O. bornovanus zoospores and incubated for 1 h. Following incubation, zoospores were pelleted and washed. The amount of zoospore-bound virus was determined by Western blot analysis using a previously described CNV monoclonal antibody (57.2) corresponding to the R domain (23; unpublished data). The quantity of virus in the pellet was assessed by densitometric analysis of the exposed film. Percent 18β(-) binding values were normalized against WT CNV. The results are averages of samples from three separate experiments, using triplicate samples of each virus per experiment.  107  of CNV 18β(-) particles may be at least partly due to a reduced ability to stably attach to zoospores during the transmission process.  2.3.4 β(-) particles disassemble at alkaline pH in the presence of EDTA Many icosahedral viruses undergo structural expansion in vitro when incubated at alkaline pH in the presence of EDTA (24). To assess the role of the β-annulus in particle stability under swelling conditions, β(-) mutant particles were incubated with EDTA at pH 8.0 and analyzed by TEM. WT CNV particles were slightly enlarged with electron dense centers as expected. However, few intact particles were observed in similarlytreated β(-) particles; instead discrete areas of stained material with no specific structural features were observed (Fig. 2.3). These results suggest that particles lacking the CP βannulus disassemble under swelling conditions.  2.3.5 18β(-) particles are less thermally stable than WT CNV The stability of 18β(-) mutant particles was further investigated by analyzing virions subjected to heat treatment. Mutant particles were incubated at increasing temperatures, followed by assessment of particle integrity by agarose gel electrophoresis. Virion RNA was visualized by ethidium bromide (EtBr) staining and capsids by subsequent staining with Coomassie brilliant blue (Fig. 2.6). Stained gels of WT CNV revealed a single band at all temperatures examined (except at 100oC in which no band was observed). However, stained gels of 18β(-) mutant particles incubated at 55oC, 60oC and 70oC revealed two bands, one that comigrated with WT CNV particles and a second that migrated slightly faster (Fig. 2.6A, panels a and b, lanes 13-15). The faster moving  108  (A)  WT CNV  18β(-)  Virion Virion Temperatures (oC) 26 37 42 55 60 70 RNA 4 100 26 37 42 55 60 70 RNA 4 100  (a) EtBr stained  (b) Coomassie blue stained  1  (B)  2  3  4  WT CNV Treatments  5  6  7  8  9  10 11 12 13 14 15 16  17 18  18β(-)  no RNase RNase no RNase added RNase added  70  26 70 70 26 70 (oC)  1  2  (a) EtBr stained  (b) Coomassie blue stained 3  4  5  6  Figure 2.6 Agarose gel electrophoresis illustrating the thermostability and ribonuclease sensitivity of WT CNV and 18β(-) particles. (A) 1 μg of WT CNV and 18β(-) virions were incubated at a range of temperatures for 30 min in 10 mM sodium acetate buffer, pH 7.2. Immediately thereafter, virions were electrophoresed through a 0.7% agarose gel buffered in Tris-borate, pH 8.3, and visualized with ethidium bromide (EtBr) (panel a). The gel was then stained with Coomassie brilliant blue (panel b). (B) Ribonuclease sensitivity assays were conducted in which 1 μg of either WT CNV or 18β(-) particles were incubated with 10 ng of pancreatic RNase A, after incubation at 70°C for 30 min or after incubation at 26oC for 30 min, followed by an additional incubation at 26oC for 30 min. Virions were electrophoresed and stained with EtBr (panel a) followed by Coomassie blue (panel b) as in (A). (-) and (+) denotes absence and presence of RNase, respectively. Arrows point to the two bands that are discernible in the EtBr and Coomassie blue stained gels of 18β(-) mutants at 55oC, 60oC and 70oC.  109  band stained with both EtBr and Coomassie blue indicating that it is a ribonucleoprotein (Fig. 2.6A, panels a and b, lanes 13-15). In addition, the second band did not comigrate with virion RNA of 18β(-) mutant particles (Fig. 2.6A, panel a, compare lanes 13-15 with lane 16), indicating that it is not simply viral RNA that was released from the heated particles. To further evaluate the nature of the second band, 18β(-) mutant particles were subjected to RNase treatment after incubation at 70oC. Agarose gel electrophoresis showed that the addition of RNase to 18β(-) mutant particles immediately after incubation at 70oC resulted in the disappearance of the faster moving ribonucleoprotein species in both the EtBr- and Coomassie blue-stained gels (Fig. 2.6B, panels a and b, compare lane 4 with lane 6); however, particles incubated at 26oC were not ribonuclease sensitive (Fig. 2.6B, panel a, lane 5). Also, WT CNV particles were not ribonuclease sensitive at either temperature (Fig. 2.6B, lanes 2 and 3). Together, these results suggest that the β-region of the CP arm contributes to thermal stability of CNV virions and that virion RNA of 18β(-) virions becomes exposed in the thermally destabilized state. In addition, the complete loss of 18β(-) particle integrity following ribonuclease treatment indicates that virion RNA contributes, at least in part, to structural stability. Heating WT CNV particles to 70oC resulted in slightly slower mobility on agarose gels (Fig. 2.6A, panels a and b, lane 6 and Fig. 2.6B, panels a and b, lanes 1 and 3). The slower mobility might be due to expansion similar to that observed when WT particles are subjected to alkaline pH and EDTA (Fig 2.3). TEM analysis was performed to directly visualize the effects of 55oC and 70oC treatment on 18β(-) mutant particles (data not shown). Alterations to the particles were not apparent. However, due to our observation that the majority of the particles remain  110  unchanged as determined by agarose gel electrophoresis (Fig. 2.6A and B, lanes 13-15 and lane 4, respectively), and the possibility that the heat treatment could make the particles morphologically unidentifiable, it would be difficult to assess if any change in structure could actually be observed.  2.4 Discussion There are few in vivo studies that delineate the role of the individual CP domains of icosahedral plant viruses in particle assembly and transmission. Based on structural studies, the tombusvirus particle has been postulated to be stabilized, in part, by the βannulus located at the three-fold axis as well as by calcium-mediated subunit-subunit interactions and RNA (10, 12). The remaining residues of the arm (the ε-region) are likely important for virion RNA binding (35) and for the conformational switching required for T = 3 particle formation (10). In this study we examined the significance of the β- and ε-regions of the CNV arm in particle accumulation in plants as well as in the ability of the particle to be fungally transmitted. Our studies indicate that the β-region is not essential for CNV particle accumulation in plants, whereas the ε-region is. However, the presence of the β-region does contribute to both particle stability and fungal transmissibility. CNV β(-) particles accumulate to 18% of that of WT virus in N. benthamiana, suggesting that the β-region of the CP arm plays an important role in particle assembly and/or stability, but is not absolutely required. It has been suggested that Turnip crinkle virus assembly initiates with the formation of a complex consisting of a trimer of C/C dimers that is stabilized by the β-annulus and possibly virion RNA attachment as well  111  (13, 32, 36). The formation of CNV T = 3 particles that lack the CP β-region in plants demonstrates that the β-annulus is not absolutely required for CNV particle stability or assembly initiation and therefore additional factors, such as interactions with viral RNA, may also play an important role. The CP β-region is highly hydrophobic and therefore is unlikely to interact directly with the viral RNA backbone. However, the upstream R domain sequences and the downstream ε-region that immediately flank the β-region likely do bind RNA as they contain clusters of basic amino acids (32, 35); thus, RNAprotein interactions that are expected to occur in each of the three C-C subunits at the 3fold axis could substantially increase the stability of this complex during assembly initiation and/or within assembled particles. However, it is still possible that CNV assembly may be initiated by a pentamer of A/B subunits, possibly as an alternative mode of assembly. Comparable to our findings, mutational analyses of Cowpea chlorotic mosaic virus (CCMV) mutants lacking the β-hexamer (which is functionally similar to the CNV β-annulus) revealed that the β-hexamer is not required for virion formation suggesting that assembly initiation can occur via pentamers of A/B dimers (33, 37). Similar results obtained by in vitro studies of assembly of a sobemovirus capsid (Sesbania mosaic virus) have recently been reported where it was shown that the βannulus is also not required for formation of T = 3 capsids suggesting that assembly could initiate via pentamers as suggested for CCMV (30). Our observation that β(-) mutant particles accumulate at a relatively low level could be due to reduced particle stability following assembly. This is supported by our findings that β(-) mutant particles disassemble under in vitro swelling conditions (Fig. 2.3) and that they are more heat labile than WT CNV particles (Fig. 2.6A) (see below).  112  The lack of particle accumulation in plants infected with either arm(-) or ε(-) mutants is consistent with the role of the ε-region of the arm in virion RNA binding and quasiequivalence (10, 11). This result is also consistent with our previous finding (17) that a proline to glycine mutation at CNV CP aa 85 which lies in the ε-region close to the arm/shell junction and which may control the flexibility of the arm required for quasi equivalent interactions also results in little or no observable particle accumulation. The 18 amino acid sequence of the ε-region is highly basic, containing five Lys or Arg residues, and is likely to bind RNA along the inner lining of the shell (35). Thus an additional crucial role of this region may be in assembly initiation and/or stabilization of subunit interactions during or following assembly as has been suggested in the case of TCV (32, 37). Our in vivo experiments would not be able to distinguish between an alternate or dual role of the ε-region. In vitro transmission assays showed that β(-) mutant particles are not transmitted by zoospores of O. bornovanus (Fig. 2.4). In addition, in vitro binding assays indicate that fewer β(-) particles bound zoospores (41% of that found using WT CNV virions) (Fig. 2.5). Loss of transmissibility could be partly a result of reduced accumulation of virus following transmission, although the 85% reduction in accumulation observed in N. benthamiana plants may not be sufficient on its own to completely account for the loss of transmission. Figure 2.3 shows that β(-) mutant particles partially disassemble under swelling conditions. This observation, in conjunction with the observation that WT CNV particles undergo swelling upon zoospore binding (17), suggests that at least part of the basis for reduced binding to zoospores is due to instability of β(-) mutant particles following zoospore binding. We have recently postulated that the β-region of CNV A or  113  B subunits may contribute to the stability of virus/zoospore binding similar to that suggested for the analogous region of the poliovirus CP upon virion binding to host cells (17). Thus the approximate two-fold reduction of zoospore binding may be a result of the absence of the postulated stabilizing effect of the β-region. Our observation that 18β(-) particles heated to high temperatures have altered electrophoretic mobility and are ribonuclease-sensitive suggests that the β-region contributes to particle stability and that virion RNA is exposed in the heat destabilized form. It is likely that the absence of the β-annulus is largely responsible for the decreased stability similar to that observed under swelling conditions. The absence of the β-region in the A and B subunits might also contribute to some loss of stability by influencing other aspects of virus particle structure. Attempts were made to observe the morphology of the heat-destabilized form using TEM. Particles with a discernibly different structure were not observed; however, as Fig. 2.6 shows, these may simply correspond to the unaltered particles present in 50oC and 70oC treated preparations. It is noted that in vivo thermal particle stability was assessed at temperatures not typical for normal virus replication and, therefore, the significance of these results for virion assembly or stability under natural conditions is not known. Interestingly, WT CNV virions heated to 70oC result in a slower migrating form of the particles that is not ribonuclease sensitive (Fig. 2.6A and Fig. 2.6B, lanes 6 and 3, respectively). WT CNV particles that have been swollen in vitro also migrate more slowly on agarose gels (data not shown), raising the possibility that the heat labile form of WT CNV is similar in some respects to the expanded form.  114  It has been found that T = 1 particles can form from the modified CPs of several T = 3 viruses by removal of the portion of the capsid that enables the quasi-equivalent interactions between subunits required for T = 3 particle formation (6, 19, 20, 32). Flexibility of the ε-region of the CNV CP arm is required for quasi-equivalent subunit interactions in T = 3 particle formation whereas such flexibility is not required for T = 1 formation. The presence of T = 1 particles in arm(-) and ε(-) mutant infections was evaluated using a variety of methods including TEM and sucrose gradient fractionation; however, unequivocal evidence for the presence of T = 1 particles was not obtained. T = 1 particle formation also was not observed following deletion of a Flock house virus (FHV) CP region believed responsible for ordered subunit contacts (5). It has been estimated that a T = 1 capsid can accommodate a maximum of approximately 1 kb of RNA (5). Therefore, it is possible that the inability to form T = 1 particles in arm(-) and ε(-) infections is due to the lack of small viral RNA species in infected leaves. To test this possibility plants were co-inoculated with the arm(-) or ε(-) mutants and either of two CNV DI RNAs (425 and 622 nt) (7). Although both DI RNAs were found to accumulate to high levels in infections with either the arm(-) or ε(-) mutant, T = 1 particles were not observed (unpublished observations). Since the ε-region is highly basic and is likely to play a role in binding RNA (35), it is unlikely that T = 1 particles can be formed during infection. It should be noted that many of the studies that have demonstrated the production of T = 1 particles by various virus capsids were conducted in vitro and thus the conditions leading to assembly of these particles may not be as stringent as those encountered in vivo.  115  Our study is unique in that it provides important in vivo information and refines our understanding of the role of the β-and ε-regions of the CP arm in virus particle accumulation and stability. Our data are also consistent with the notion that viral RNA plays an important role in particle assembly and/or stability. In addition, our findings further suggest that the β-annulus and/or the β-region itself may be important for the stability of the expanded form of the CNV particle during fungal transmission.  2.5 References 1. Bol, J. F. 1999. 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The role of arginine-rich motif and β-annulus in the assembly and stability of Sesbania mosaic virus capsids. J. Mol. Biol. 353:447-458. 31. Sit, T. L., J. C. Johnston, M. G. ter Borg, E. Frison, M. A. McLean, and D. Rochon. 1995. Mutational analysis of the cucumber necrosis virus coat protein gene. Virology 206:38-48. 32. Sorger, P. K., P. G. Stockley and, S. C. Harrison. 1986. Structure and assembly of turnip crinkle virus. II. Mechanism of reassembly in vitro. J. Mol. Biol. 191:639-658. 33. Speir, J. A., S. Munshi, G. Wang, T. S. Baker, and J. E. Johnson. 1995. Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy. Structure 3:63-78. 34. Thomas, C. L., V. Leh, C. Lederer, and A. J. Maule. 2003. Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306:33-41. 35. Timmins, P. A., D. Wild, and J. Witz. 1994. The three-dimensional distribution of RNA and protein in the interior of tomato bushy stunt virus: a neutron low-resolution single-crystal diffraction study. Structure 2:1191-1201.  120  36. Wei, N., L.A. Heaton, and T.J. Morris. 1990. Structure and assembly of Turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA. J. Mol. Biol. 214:85-95. 37. Willits, D., X. Zhao, N. Olson, T. S. Baker, A. Zlotnick, J. E. Johnson, T. Douglas, and M. J. Young. 2003. Effects of the cowpea chlorotic mottle bromovirus βhexamer structure on virion assembly. Virology 306:280-288.  121  3 CHAPTER THREE: The N-terminal region of the Cucumber necrosis virus coat protein targets both chloroplasts and mitochondria * 3.1 Introduction It is well known that viruses recruit a variety of cellular components to facilitate the infection process. Investigation into the various interactions that have evolved is fundamental towards an understanding of virus multiplication. A number of membranebound cellular organelles have been identified as crucial for viral infection. For example, proposed subcellular sites of viral replication include peroxisomes (21, 25, 28, 38), chloroplasts (30), mitochondria (49) and the endoplasmic reticulum (43). Microtubules have been proposed as a means for viral RNA transport (4), plasmodesmata have been found to interact with viral movement proteins (20) and the cytoskeleton has been found in association with viral coat protein (CP) for intercellular movement (1, 31). Despite the considerable knowledge we have on the role of the viral CP during infection (for reviews on CP functions see 2, 8), very little is known about viral CP-host interactions. The Turnip yellow mosaic virus particle has been found in association with chloroplasts (12) which has led to the suggestion that chloroplasts may be the site of virion assembly. It has recently been found that the Cucumber necrosis virus (CNV) CP targets chloroplasts during infection and it was hypothesized that chloroplasts may, therefore, be involved in particle disassembly and/or assembly (50). Additionally, the association of the Beet necrotic yellow vein virus CP with the outer mitochondrial membrane has suggested that mitochondria may be involved in particle assembly (44).  *  A version of this chapter will be submitted for publication. Hui, E., Xiang, Y., and Rochon, D. The Nterminal region of the Cucumber necrosis virus coat protein targets both chloroplasts and mitochondria. 122  CNV, a member of the Tombusvirus genus in the family Tombusviridae, is a T = 3 icosahedral virus consisting of 180 identical CP subunits (36). Each subunit consists of three major domains: an N-terminal 58 aa R domain which is joined to a 167 aa S domain by a 34 aa arm and a 116 aa C-terminal P domain which is connected to the S domain by a 5 aa flexible hinge (Fig. 3.1A). The R domain which resides in the particle interior contains positively charged residues that serve to neutralize most of the phosphate groups of the virion RNA (18). The arm which is also found in the particle interior is involved in particle stabilization at the 3-fold axis and in the formation of CP dimer interactions. During particle expansion, the R domain and the arm are externalized, a process that is believed to be part of the particle uncoating process (16, 35, 50). Using Nicotiana benthamiana plants agro-infiltrated with CNV CP/GFP fusion protein constructs, Xiang et al. (50) demonstrated that the CNV CP arm region targets the chloroplast stroma during infection. The arm region and the first four amino acids of the S domain were found to be sufficient for targeting and analyses of this region showed that it contains features typical of chloroplast transit peptides. It was further found that cleaved CP was present in chloroplasts isolated from CNV-infected plants, indicating that CP targets chloroplasts during the infection process. A hypothetical model was proposed in which chloroplast targeting of the virus particle serves as a means for virus particle disassembly (50). Confocal microscopy of leaves infiltrated with an R/arm/SSVRI/GFP fusion protein construct showed localization to both chloroplasts and to discrete sites in the cytoplasm. A fusion protein construct consisting of only the R domain showed localization to only  123  those specific sites in the cytoplasm. This suggested that, in addition to chloroplasts, other subcellular organelles are targeted by the CP during infection. We wished to further investigate interactions between the CNV CP and subcellular components to gain further insight into the virus disassembly/assembly process. In this study, we show that the CNV R domain specifically targets mitochondria. We also demonstrate that the CNV CP arm region of an R/arm GFP fusion protein localizes to both chloroplasts and mitochondria in infiltrated plants and that dual import into the stroma and matrix of these organelles is facilitated by a region at the N-terminus of the S domain.  3.2 Materials and methods 3.2.1 Preparation of an Agrobacterium binary vector containing CNV CP constructs for agroinfiltration CNV CP mutants R19/arm/S22, R6/arm/S22, arm30/S22, arm27/S22, and arm12/S22 were prepared by PCR using a full-length clone of CNV (pK2/M5) as template. Each PCR reaction utilized a forward and reverse primer which included an NcoI site and a SstII site, respectively (Table 3.1). The PCR products were digested with NcoI and SstII and cloned into an intermediate vector containing the GFP coding sequence such that the 3’terminus of the CNV CP sequence was in-frame with the coding sequence of GFP. The GFP-tagged CP sequences were excised using NcoI and BamHI sites and inserted into the corresponding sites of a second intermediate vector, pBBI525. These clones were then digested with HindIII and EcoI and cloned into the corresponding sites of the binary vector pBIN(+). In vitro mutagenesis was used to produce the CP mutants R47,  124  Table 3.1 Primers used for production of CNV CP constructs for agro-infiltration Construct name  Forward primer  Reverse Primer  R23  5’ AAACCATGGCACTCGTAAGCAGGAACAACAATATGCGA ACACTTGCAAAGTTAGCCGCCCCATTGGCTACGGCAGTGA GCAAGGGCGAGGAG 3’*  5’ GTGGGATCCTTACTTGTACAGCTCGTCCAT 3’  R39  5’ CCGCGGGTGAGCAAGG 3’  5’ TACTCCATTCCAGATAGCTTC 3’  R47  5’ CCGCGGGTGAGCAAGG 3’  5’ CGGTAACTTTCCCCAGATCC 3’  R47(W41A, W43A)  5’ CCGCGGGTGAGCAAGG 3’  5’ CGGTAACTTTCCCGCGATCGCTTTTACTCC 3’  R/arm/S22  5’ CACCATGGCACTCGTAAGCAGGAAC 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  arm/S22  5’ GAACCATGGCTCTCATAGCCCACCCACA 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  R19/arm/S22  5’ CTAACCATGGCAAAATGGATCTGGGGAAAGTTACCGA 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  R19/arm/SSVRI  5’ CCGCGGGTGAGCAAGG 3’  5’ TATTCGCACAGATCCTTTTGC 3’  R6/arm/S22  5’ CTAACCATGGCAAAGAATGGAAACGGTGCTCTC 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  arm30/S22  5’ CTAACCATGGCACCACAGGCTTTTCCTGGGGCT 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  arm27/S22  5’ CTAACCATGGCATTTCCTGGGGCTATC 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  arm12/S22  5’ ATCCATGGCAAAAGGAAGGAAACCTAGG 3’  5’ AGTCCCCCGCGGGCGTAGGAACTCCCCATTAG 3’  *The underlined areas indicate the NcoI (CCATGG) and SstII (CCGCGG) restriction enzyme sites.  125  R47(W41A, W43A), R39 and R19/arm/SSVRI using a forward mutagenic primer that that included an NcoI site and a reverse primer containing a BamHI site (Table 3.1) and a pBBI525 vector that contained the complete R domain fusion protein coding sequence was used as a template except in the case of mutant R19/arm/SSVRI which used the above described pBBI525(R19/arm/S22/GFP) as a template. Mutagenized PCR fragments were digested with NcoI and BamHI and inserted into the corresponding sites of pBBI525. These clones were subsequently digested with HindIII and EcoRI and cloned into pBIN(+) as described above. CP mutants R/arm/S22 and arm/S22 were prepared by PCR using clone pBBI525(CP/GFP) as a template and primers containing NcoI and SstII sites at the 5’ and 3’ termini (Table 3.1). PCR fragments were digested with NcoI and SstII as described above and a directional 3-fragment ligation was performed using a GFP sequence flanked by SstII and BamHI at the 5’ and 3’ termini, respectively and a pBBI525 vector that had been digested with NcoI and BamHI. Subsequently, they were cloned into pBIN(+) as described above. Constructs of CP mutants R, R23, R/arm/SSVRI, and arm/SSVRI were previously described (50). All constructs were confirmed by sequencing for proper construction and nucleotide sequence. The various pBIN(+) constructs were transformed into Agrobacterium strain GV3101/C58C1 (pMP90) and infiltrated into leaves of 4- to 5-week old Nicotiana benthamiana plants as previously described (50).  3.2.2 Preparation of N. benthamiana protoplasts Two to three agro-infiltrated leaves were cut lengthwise and placed, epidermal side down into 8 mL of enzyme solution (0.1% macerozyme, 1% cellulase, 0.25 mM  126  polyvinylpyrrolidone, 3.85 mM CaCl2, 0.5 M sucrose and 5 mM KCl) and left overnight. On the following day, the leaves were gently broken up in the enzyme solution using a clean glass rod and filtered through two layers of cheesecloth. Residual filtrate was rinsed through the cheesecloth with a sucrose solution (0.6 M sucrose, 0.1% MES and 1.5 mM CaCl2). The leaf-sucrose filtrate was overlaid with a one-tenth volume of W5 media (154 mM NaCl, 5 mM KCl, 125 mM CaCl2 and 5 mM glucose) and centrifuged for 10 min at 250 x g at 4oC with the brake off. The interface layer containing the protoplasts was removed and gently mixed with 6.5 to 8.0 ml of W5 media. The mixture was pelleted for 5 min at 250 x g at 4oC with the brake off and resuspended in a solution of 0.5 ml of W5 media and 0.4 M mannitol.  3.2.3 Isolation of mitochondria Mitochondria were isolated from agro-infiltrated N. benthamiana leaves using a method modified from Millar et al. (22). Four to ten grams of leaves agro-infiltrated with R, R47, R19/arm/S22, R19/arm/SSVRI, R/arm/S22 or R/arm/SSVRI -GFP fusion protein constructs were homogenized in a cooled Waring blender using 3 5s bursts at low speed. A homogenization medium (0.4 M mannitol, 1.0 mM EDTA, 25 mM MOPS-KOH, pH 7.8, 10 mM Tricine, 8 mM cysteine, 0.1% BSA and 1.0% PVP40) with COMPLETE® protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA), at a concentration of one COMPLETE® tablet per 50 ml volume, was added in a ratio of 4 ml/g fresh weight of leaf tissue. The homogenate was filtered through four layers of Miracloth (Calbiochem Corp., La Jolla, CA) and the filtrate was centrifuged at 1,000 x g for 5 min at 4oC. The supernatant was centrifuged at 12,000 x g for 15 min at 4oC. The resulting pellet was  127  gently resuspended in 6.4 to 8 ml of wash medium (0.4 M mannitol, 1.0 mM EGTA, pH 7.2, 10 mM MOPS-KOH, pH 7.2 and 0.1% BSA) and centrifuged at 1,000 x g for 5 min at 4oC. The supernatant was centrifuged at 12, 000 x g for 15 min at 4oC and the pellet was resuspended in 500µl wash medium. The suspension was layered onto a selfgenerating Percoll gradient which was prepared from a solution of 28% Percoll/0.4 M mannitol and centrifuged at 40,000 x g for 45 min at 4oC. The mitochondrial layer was removed and washed twice: once with four volumes of wash medium and again with two volumes. The pellet was resuspended into a final volume of 100 µl with resuspension buffer (0.4 M mannitol, 10 mM Tricine and 1.0 mM EGTA, pH 7.2).  3.2.4 Separation of mitochondrial fractions Intact mitochondria were purified as above and subfractionated into the outer membrane (OM), inner membrane space (IMS), inner membrane (IM) and matrix (MA) as described in Millar et al. (22). The mitochondrial suspension was diluted 50 times with 86 mM sucrose in MOPS-KOH, pH 7.2 and rotated gently at 4oC for 15 min. Using a stock solution of 2 M sucrose, the sucrose concentration of the suspension was brought to a final concentration of 0.3 M and rotation was continued for 15 min at 4oC. The resulting mitoplasts were pelleted by centrifugation of the suspension at 15,000 x g for 15 min at 4oC. The mitoplast pellet was washed once in 500 µl of 100 mM TRIS-HCl, pH 7.4 and then re-centrifuged at 15,000 x g for 10 min at 4oC. The supernatant was centrifuged at 100,000 x g for 90 min at 4oC. The resulting supernatant which corresponds to the IMS was precipitated with acetone in three times the volume and resuspended in 1X Laemmli buffer (19). The 100,000 x g-pellet which contained the OM  128  was resuspended in 100 µl of TRIS-HCl, pH 7.4 and layered onto a 0.6 M/0.9 M sucrose step gradient and centrifuged in a swinging bucket rotor at 50,000 x g for 60 min at 4oC. The OM was collected from the 0.6 M/0.9 M interface, subjected to acetone precipitation and resuspended in 1X Laemmli buffer. The 15,000 x g-mitoplast pellet from above was resuspended in 500 µl of 100 mM TRIS-HCl, pH 7.4 and sonicated at full power using 3 5s bursts with 20s rest periods on ice. The sonicated mixture was made up to a final concentration of 1 ml using 100 mM TRIS-HCl, pH 7.4 and was centrifuged at 80,000 x g for 60 min at 4oC. The pellet which corresponded to the IM was resuspended in 1X Laemmli buffer whereas the supernatant containing the MA was subjected to precipitation with acetone (see above) and resuspended in 1X Laemmli buffer.  3.2.5 Isolation of chloroplasts Chloroplasts were isolated from leaves agro-infiltrated with CP constructs using a method originally described by Bruce et al. (7) and modified by Xiang et al. (50). Briefly, 4 – 8 g of fresh leaf tissue was homogenized in a Waring Blender in 35 ml icecold grinding buffer. Crude chloroplasts were obtained by centrifugation of the filtered homogenate at 2,000 x g for 5 min at 4oC. Chloroplasts were further purified by centrifugation through a preformed 50% Percoll gradient. Isolated chloroplasts were washed with 3 volumes with import buffer (50 mM HEPES-KOH, pH 8.0 and 330 mM sorbitol) after which the final chloroplast concentration was determined by measuring the concentration of chlorophyll (7).  129  3.2.6 Trypsin and thermolysin treatment of chloroplasts Intact chloroplasts were purified as described above and treated with trypsin and thermolysin as described by Bruce et al. (7). Chloroplast suspensions were adjusted to final concentration of 0.5 mg chlorophyll/ml in import buffer as described by Xiang et al. (50) and 100 µl were either mock incubated or incubated with 5 µg of trypsin (Sigma) or 20 µg of thermolysin (Sigma) on ice for 30 min in the dark. Ten micrograms of trypsin inhibitor was added to trypsin-treated chloroplasts and thermolysin-treated chloroplasts were adjusted to a final concentration of 10 mM EDTA. These inhibitors were included in all subsequent treatments. Chloroplasts were reisolated as described in Xiang et al. (50) and vortexed at 4oC for 10 min in hypotonic lysis buffer (25 mM HEPES-KOH, pH 8.0). Lysed chloroplasts were separated into the membrane (pellet) and soluble (supernatant) fractions by centrifugation at 165,000 x g at 4oC for 10 min. Proteins in the soluble fraction were precipitated with acetone as described in Xiang et al. (50) but were pelleted at 16,000 x g at 4oC for 20 min. Pellets were resuspended in 100 µl 1X Laemmli buffer and analyzed by Western blotting as described below.  3.2.7 Confocal microscopy Confocal microscopy of agro-infiltrated N. benthamiana leaves, protoplasts, isolated mitochondria or isolated chloroplasts was conducted using a Leica SP2-AOBS confocal microscope. Leaves and isolated mitochondria were infused with 1.0 µM MitoTracker® Red CMXRos (Molecular Probes) diluted in a solution of 10 mM MgCl2, 10 mM MES and 0.2 mM acetosyringone and observed after a minimum of 30 min. Mitochondria in protoplasts were observed by staining with 0.5 µM MitoTracker® Red  130  CMXRos. An excitation wavelength of 488 nm (using an argon laser) was used for GFP fluorescence and chloroplast auto-fluorescence simultaneously whereas an excitation wavelength of 579 nm (using a helium-neon laser) was used to view MitoTracker® Red CMXRos fluorescence (red). All samples were observed under a 63X water immersion objective.  3.2.8 Western blot analyses Total leaf protein samples were prepared as previously described (50). All samples were boiled in 1X Laemmli buffer (19) for 5 min and residual materials were removed by centrifugation at 14,000 x g for 1 min. Samples were electrophoresed through 15% SDS-polyacrylamide gel, blotted onto PVDF membranes (BioRad, Mississauga, ON, Canada) and probed with either a monoclonal antibody specific to GFP (BD Biosciences) or with a polyclonal antibody specific to the CNV S and P domains (50). Thereafter, blots were stained with Ponceau S (Sigma-Aldrich, Oakville, ON). Antigen-antibody complexes were detected as previously described (50).  3.3 Results and discussion 3.3.1 A CNV R domain GFP fusion protein targets mitochondria A variety of CNV R and arm domain GFP fusion protein constructs was prepared and used for agro-infiltration studies (Fig. 3.1). Previous work showed that CNV R/arm/SSVRI targeted both chloroplasts and specific unidentified sites in the cytoplasm (50). The R domain fusion protein construct was also found to target specific sites in the cytoplasm suggesting that sequences within the R domain are responsible for the  131  A  1  59  hinge 260 265  93 arm  R  CNV CP (380 aa)  P  S  MALVSRNNNMRTLAKLAAPLATAGTRTIVDNKEAIWNGVKWIWGKLPKGKKGKNGNGALIAHPQAFPGAIAAPISYAYAVKGRKPRFQTAKGSVRITHREYVSVLSGTNGEFLR  1  59  B  NcoI  HindIII  //  35S  R23 (29.3K)  //  R39 (31.1K)  // // // 1  R (33.1K)  // 1  arm12/S22 (30.9K) arm27/S22 (32.5K)  66  GFP 114  // 63  arm/SSVRI (31.1K)  //  arm/S22 (33.1K)  //  GFP 93  59  93  GFP 114  53 59  93  GFP 114  40  59  93  40  59  93  1  59  93  1  59  93  //  R19/arm/SSVRI (33.1K)  //  R19/arm/S22 (35.1K)  //  GFP  // // //  -  -  +  -  +  -  +  -  +  //  -  -  //  +  -  GFP  //  //  weakly  -  +  -  //  +  -  //  +  -  //  59  R6/arm/S22 (33.7K)  GFP (27.0K)  93  93  //  arm30/S22 (32.7K)  R/arm/S22 (39.1K)  93  GFP 114 GFP 114  // 81  R/arm/SSVRI (37.1K)  -  //  W41A  1  R47 (32.1K)  Chloroplasts Mitochondria  GFP // 39 W43A GFP // 47 GFP // 47 GFP // 59  1  R47(W41A,W43A) (32.1K)  Targets:  // pBin(+)  NOS GFP 23  1  114  EcoRI  BamH1  AMV  93  //  GFP 114 GFP //  //  GFP 114  //  weakly  +  weakly  +  +  +  +  +  -  -  Figure 3.1 Diagrammatic representation of CNV CP GFP fusion constructs. (A) Linear structure of the CNV CP subunit. The locations of the three major domains (R, S and P), along with the connecting arm and hinge (h) regions, are shown. The amino acid (aa) sequence of the R and arm regions and the sequence of the N-terminal 22 aa of the S domain are shown below the linear structure. The numbers indicate the beginning of each domain and region. (B) Structure of the pBIN(+)/GFP fusion vector constructs used for expressing R, arm and S domain GFP fusion proteins. The various CP sequences are inserted in-frame with GFP. The R, arm or S regions retained in each construct are indicated by numbers and correspond to the aa numbering system shown in A. The small black box indicates the SVRI sequence of the S domain whereas the grey rectangular box indicates the first 22 amino acids of the S domain. The ovals represent the fused GFP protein. Construct names are indicated on the left. The predicted sizes in kilodaltons of the fusion proteins are indicated in parentheses next to the construct name. A summary of the subcellular locations of the various fusion proteins in N. benthamiana cells is shown on the right. 132  cytoplasmic localization. Based on confocal analysis, the pattern of localization of the R domain construct was consistent with the possibility that R domain sequences target either mitochondria or peroxisomes. To assess these possibilities, protoplasts prepared from R domain-infiltrated plants were stained with a mitochondria-specific dye, MitoTracker® Red. Figure 3.2 shows that GFP fluorescence of the R domain fusion protein is confined to distinct sites in the cytosol and co-localizes with MitoTracker® Red fluorescence. These findings therefore demonstrate that R domain sequences target mitochondria. Experiments to assess the possible co-localization with peroxisomes were also conducted. Plants were co-infiltrated with the R domain fused to GFP and a YFP construct (Clonetech Laboratories Inc., Mountain View, CA) that specifically targets peroxisomes due to the presence of a C-terminal SKL sequence. The infiltrated leaves were observed directly under confocal microscopy within which the GFP and YFP clearly targeted distinct regions in the infected cells (data not shown) indicating that the R domain does not associate with peroxisomes.  3.3.2 The first 47 amino acids of the R domain are required for efficient mitochondrial localization To determine the specific CNV R domain sequences involved in mitochondrial localization, four R domain GFP fusion protein constructs, R47, R47(W41A, W43A), R39, and R23 (Fig. 3.1; includes full-length construct sizes) were used in agro-infiltration studies. The R47(W41A, W43A) construct was used to determine whether the targeting results of constructs R47 and R19/arm/S22 (as will be described below) was due to the two Trp residues within the overlapping region of these two constructs. At 24 and 48 hours  133  R  GFP  GFP  MitoTracker  Merged  Figure 3.2 Confocal analysis of the subcellular location of the CNV R GFP fusion protein in agroinfiltrated N. benthamiana plants. Protoplasts from plants agroinfiltrated with the R GFP fusion protein were stained with MitoTracker® Red and visualized using confocal microscopy. GFP fluorescence (green) is shown in the first column, MitoTracker fluorescence in the second column and the third column is a merged image of the GFP and MitoTracker fluorescence.  134  post-infiltration (hpi), N. benthamiana leaves were stained with MitoTracker® Red and analyzed by confocal microscopy. As described above, the R domain fusion protein localized to mitochondria (with some cytosolic labelling) and localization was observed as early as 24 hpi (Fig. 3.3A, panel a). The two R47 fusion proteins also localized to mitochondria but localization was not apparent until 48 hpi (Fig. 3.3A, compare panels b, c with h, i, respectively) although the GFP colocalization with mitochondria by the R47(W41A, W43A) fusion protein was less clear at 48 hpi compared to that of R47. These data suggest that the two tryptophan residues may not be necessary for efficient chloroplast targeting. Localization of the R39 construct was not apparent at 48 hpi (Fig. 3.3A, panel j); however, some targeting was observed at approximately 72 hpi (data not shown). Similar to the GFP control, the R23 fusion protein was consistently found only in the cytosol (Fig. 3.3A, panels e, k and f, l, respectively). Total protein was extracted from leaves at 24 and 48 hpi and analyzed by SDSPAGE, followed by Western blotting using a GFP monoclonal antibody. Two protein species of approximately 33.1K and 30.5K [a revised estimate for the corresponding 31.5K species described by Xiang et al. (50)] were detected in leaf extracts of the R domain infiltrated leaves at 24 and 48 hpi (Fig. 3.3B, lanes 1, 2). As described previously (50), and also shown here, the ratio of the full-length 33.1K to 30.5K protein species decreases over the 48 hour period analyzed, suggesting that the 30.5K protein is a specific cleavage product of the 33.1K fusion protein. Based on the 30.5K size, we estimate that cleavage occurs approximately 2.6 kDa [~24-28 amino acids (aa)] from the N-terminus of the R-domain. Total leaf extracts from either the R47 construct or the R47(W41A, W43A) construct show the expected full-length 32.1K protein at 24 hpi and  135  A  24 hpi  a  48 hpi  g  R  b  h  c  i  d  j  e  k  f  l  R47  R47(W41A,  W43A)  R39  R23  GFP  GFP  B  MitoTracker  R 24  R47 48  24  Merged  GFP  R47(W41A,W43A) R39 48  24  48  24  24  48  48  48 R  33.1 32.1 31.1  R47  •  • 1  2  3  4  R47(W41A,  + +  + 5  6  W43A)  R39 R23  7  8  9  Merged  GFP mock  R23 48  MitoTracker  10  11  •* (30.5K) + (29.5K) * + + (29.5K)  (33.1K) (32.1K)  (32.1K) * (28.6K) (31.1K) * (29.3K)  12  Figure 3.3 Confocal and Western blot analyses of leaves agro-infiltrated with R domain constructs. (A) Confocal images of leaves agro-infiltrated with R, R47, R47(W41A, W43A), R39, R23 and a GFP protein fusion construct at 24 and 48 hpi as indicated. The first column of each set of three columns shows GFP fluorescence (green), the second shows MitoTracker® Red fluorescence (red), and the third shows a merged image. Constructs used for agro-infiltration are indicated on the left. (B) Total protein from leaves infiltrated with R, R47, R47(W41A, W43A), R39 and R23 were electrophoresed through an SDS-polyacrylamide gel, followed by Western blot analysis using a GFP monoclonal antibody. Constructs used for agro-infiltration are indicated above the blot. The numbers on the left correspond to the predicted masses in kilodaltons (K) of the full-length proteins of the indicated constructs. The numbers in italics in the diagram on the right correspond to the estimated sizes of cleavage products obtained from the various constructs. The symbols next to the italicized numbers correspond to the symbols on the blot, indicating the cleavage product band. The 28.6 kDa cleavage product was not observed, likely due to inefficient targeting of R39 up to 48 hpi. Sizes were estimated by coelectrophoresis with MagicMark™XP (Invitrogen, Burlington, ON) and BenchMark™ Prestained (Invitrogen, Burlington, ON) as molecular mass standards (not shown). 136  an additional species of approximately 29.5K at 48 hpi (Fig. 3.3B, compare lanes 3, 5 with 4, 6, respectively). Based on the size of this cleavage product, cleavage occurs at a site similar to that predicted for the R domain fusion protein construct (see above). We note that the second species produced following agro-infiltration with both R47 and, less abundantly, R47(W41A, W43A) is not apparent until 48 hpi. The appearance of this species in the two R47 constructs at 48 hpi but not 24 hpi is in agreement with the results of confocal analyses described above (Fig. 3.3A, panels h and i). These results are also consistent with the notion that cleavage in the R domain occurs upon mitochondrial import. Total leaf extracts showed one prominent full-length 31.1K protein species in leaves agro-infiltrated with the R39 construct (Fig. 3.3B, lanes 7, 8); however, a small amount of cleavage product was visible at 3-4 dpi concurrent with detectable GFP fluorescence in mitochondria (data not shown). A single prominent 29.3K species was observed in the R23 agro-infiltrated plants at both 24 and 48 hpi (and at later time points; data not shown) (Fig. 3.3B, lanes 9, 10). The absence of detectable cleavage products is consistent with slow or absent mitochondrial uptake. It is known that N-terminal presequences direct mitochondrial localization of cytoplasmically synthesized mitochondrial proteins and that these sequences are proteolytically removed by specific mitochondrial processing peptidases (MPPs) upon import of the precursor protein into mitochondria (11, 14, 40, 42). Our data suggest that the N-terminus of the CNV R domain may resemble plant mitochondrial targeting sequences with efficient uptake requiring at least the N-terminal 39 aa. Although the primary structure of known mitochondrial matrix targeting presequences is highly variable, common features exist. These include a presequence ranging in size from approximately 20-60 aa in length that  137  has an overall positive charge and the presence of amphipathic α-helices (9, 10, 23, 32, 46, 47). Analysis of the R domain sequence by TargetP (41) suggests that there is a high probability (p = 0.674) that a mitochondrial presequence is located within this region. As shown in Figs. 3.1 and 3.4, the N-terminal region of the CNV R domain has three Arg and one Lys residue and no acidic residues within the first 28 aa, giving the sequence a net positive charge. Analysis of this region by PredictProtein (http://cubic.bioc.columbia.edu; 37) indicates the possible presence of a α-helix upstream of the proposed MPP cleavage site which is approximately 24-28 aa from the N-terminus. Features of the R domain that resemble cellular mitochondrial presequences are summarized in Fig. 3.4 (B, C).  3.3.3 CNV R domain sequences are found in specific mitochondrial subfractions in agro-infiltrated plants To further evaluate the role of the R domain in mitochondrial localization, confocal analyses were conducted using four additional GFP fusion protein constructs (Fig. 3.1): R/arm/SSVRI and R/arm/S22 which contain the complete R domain plus sequences from the arm and either the first four or the first 22 aa of the S domain, and R19/arm/SSVRI and R19/arm/S22 which consist of the 19 C-terminal R domain residues, the complete arm region and again either the first four or the first 22 aa of the S domain. Confocal microscopy of MitoTracker® Red-stained protoplasts shows that all four constructs target mitochondria at 2 dpi (Fig. 3.5A, panels c to f). Co-localization of the GFP fluorescence of R/arm/SSVRI and R/arm/S22 fusion protein constructs with  138  A Mitochondria  Cytoplasm (Mitochondria?)  ~24-28  1  Mitochondria Chloroplasts (aa 87/88)  59  R  93 arm  S  B Positively charged  * * * * MALVSRNNNMRTLAKLAAPLATAGTRTIVDNKEAIWNGV location of predicted helix predicted cleavage site at aa26(Arg)  C  R  A  *8 A  A  1  T  *  12  K  *4  5  * 9  *11 2  7  A  *  3 L*  *P  *T  6 10  *L  *L  Figure 3.4 Summary of proposed cleavage sites and mitochondrial presequence-like features of the N-terminus of the R/arm/S22 region of the CNV CP. (A) The approximate positions of the proposed cleavage sites are shown by the brackets above the indicated R, arm and S regions. The proposed subcellular targeted sites are indicated above the CP cleavage sites. Note that the cleavage site at the R/arm junction is indicated as being immediately downstream from this junction. This site is deduced from data showing that R GFP fusion proteins do not undergo cleavage at the R domain C-terminus. The indicated CP cleavage sites that were proposed as the result of cytoplasmic and chloroplast localization were deduced from data described by Xiang et al. (50), and Fig. 3.5 of this study. Target P predicts stromal cleavage between residues Phe/Gln at aa position 87/88 (data not shown). (B) Features of the CNV R domain showing similarity to mitochondrial presequence. The minimal 39 N-terminal aa residues of the R domain that are predicted to act as a mitochondrial presequence are shown. Features that are commonly found in mitochondrial presequences are described. Basic residues are indicated by asterisks. The approximate location of the predicted MPP cleavage site is indicated. The underlined area corresponds to a region predicted to form a helix as determined by PredictProtein (37; http://cubic.bioc.columbia.edu). (C) Helical wheel diagram showing the amphipathic nature of the predicted helical region shown in (B). Hydrophobic aa are in blue, polar aa are in black and basic aa are shown in red (Helical wheel produced from http://cti.itc.virginia.edu/~cmg/Demo/wheel/wheelApp.html and http://www-nmr.cabm.rutgers.edu/bioinformatics/Proteomic tools/Helical wheel/. 139  A  B a  Lf Mi OM IMS IM MAGFPmock  R  R  b  R47  R47  33.1 30.5  *  32.1 29.5  *  *  a b  37.1  R/arm/SSVRI  c R/arm/SSVRI  *  *  30.0 27.5 39.1 36.5 R/arm/S22 32.0 29.5  * 1  2  3  4  d  * 5  6  7  c  8  d  C  R/arm/S22  R19/arm/SSVRI  e  Lf  Mi GFP mock  33.1  a  27.5  R19/arm/SSVRI R19/arm/S22  f  35.1 32.0 29.5  b  R19/arm/S22 1  GFP  MitoTracker  2  3  4  Merged  Figure 3.5 Confocal and Western blot analyses of the subcellular location of R, R47, R/arm/SSVRI, R/arm/S22, R19/arm/S22 and R/arm/SSVRI in agro-infiltrated plants. (A) Confocal images of protoplasts prepared from agro-infiltrated leaves at 48 hpi. Protoplasts were stained with MitoTracker Red®. The first column shows GFP fluorescence (green), the second MitoTracker Red fluorescence (red) and the third is a merged image of the red and green fluorescence. The insets show images of individual mitochondria demonstrating the apparent mitochondrial location of GFP from plants infiltrated with the R and R/arm/SSVRI fusion protein constructs. (B) Western blot analysis of total leaf protein (Lf), purified mitochondria (Mi) and various mitochondrial subfractions obtained from plants agro-infiltrated with R, R47, R/arm/SSVRI, R/arm/S22 (see text). Mitochondrial subfractions included: the outer membrane (OM), intermembrane space (IMS), inner membrane (IM) and matrix (MA). Lanes 7 and 8 correspond to total protein extracted from GFP- and mock-infiltrated N. benthamiana leaves, respectively. The blot was probed with a GFP monoclonal antibody. The numbers on the left correspond to the predicted sizes (in kilodaltons) of the full-length and the estimated sizes of the cleaved products (in italics), respectively. Non-specific bands in lanes of purified mitochondria isolated from mock and fusion protein construct infiltrated plants are indicated with an asterisk. (C) Western blot analysis of total leaf and mitochondrial protein (symbols used as in B) obtained from plants agro-infiltrated with R19/arm/SSVRI and R19/arm/S22. Lanes 3 and 4 correspond to total protein extracted from GFP- and mock-infiltrated leaves, respectively. Sizes of cleavage products shown in B and C were estimated by co-electrophoresis using MagicMark™XP (Invitrogen, Burlington, ON) and BenchMark™ Prestained (Invitrogen, Burlington, ON) as protein molecular size standards (not shown). 140  MitoTracker® Red fluorescence is consistent with the previous finding that the R domain contains sequences associated with mitochondrial targeting. The R19/arm/S22 construct localizes to mitochondria even though this construct lacks the N-terminal aa residues of the R domain that were deduced (see above) to be important for mitochondrial localization. Similarly, the R19/arm/SSVRI construct localized to mitochondria but was not observed until 3 dpi (data not shown). However, these sequences are highly basic and therefore may be recognized as a presequence by the mitochondrial preprotein uptake machinery. Mitochondria were purified and total protein was analyzed by Western blotting to further assess mitochondrial localization of the six R and arm domain constructs shown in Fig. 3.5A. The previously observed 33.1K and 30.5K R and the 32.1K and 29.5K R47 protein species that are present in total leaf extracts (Fig. 3.3B, lanes 2 and 4, respectively) are shown in Fig. 3.5B (lane 1, panels a, b, respectively) and are found in total mitochondrial protein (Fig. 3.5B, lane 2, panels a, b). It can be seen that isolated mitochondria are enriched for the smaller cleaved species in both constructs (Fig. 3.5B, lane 2, panels a, b) reinforcing, as described above, that cleavage indeed occurs upon mitochondrial import of the precursor protein. Total protein extracted from leaves infiltrated with R/arm/SSVRI contains detectable but low levels of the full-length 37.1K species and two abundant cleavage products of approximately 30K and 27.5K (Fig. 3.5B, lane 1, panel c). Total protein from isolated mitochondria contains predominantly the 30K cleavage product as well as the full-length 37.1K precursor (Fig. 3.5B, lane 2, panel c) and possibly low levels of the 27.5K species. Based on size, the 30K cleavage product appears to arise from cleavage near the R/arm junction (and possibly slightly downstream  141  considering that the R GFP fusion proteins do not show cleavage at the R/GFP junction) and the 27.5K protein is predicted to arise from cleavage near the arm/S junction (Fig. 3.4A). A species of approximately 34.6K which would correspond to cleavage in the R domain (as described above) may also be present (Fig. 3.4A, B). Total leaf extracts from R/arm/S22 infiltrated plants contain, in addition to the 39.1K precursor protein, a major 29.5K cleavage product whereas total mitochondrial protein contains predominantly the 29.5K cleavage product as well as low levels of the full-length precursor (Fig. 3.5B, lanes 1, 2, panel d). Based on size estimates, the 29.5K cleavage product was predicted to result from proteolysis near the arm/S junction and therefore cleavage is predicted to occur at the same site that resulted in the R/arm/SSVRI minor 27.5K protein product (Fig. 3.4A). In contrast to that of R/arm/SSVRI, cleavage near the arm/S junction of R/arm/S22 appears to occur readily in mitochondria. A protein corresponding to cleavage near the R/arm junction (~ 32K protein, Fig. 3.5B, lane 2, panel d) that was observed in R/arm/SSVRI infiltrated leaves is detected but occurs at a relatively low level. It is possible that the proposed cleavage site at the arm/S junction in this construct is more efficient and therefore precludes efficient accumulation of the 32K species. We note that a minor species of 36.5K, possibly corresponding to cleavage in the R domain near aa 24-28, may also be present (Fig. 3.5B, lane 2, panel d). Total leaf extracts from R19/arm/SSVRI infiltrated plants show primarily the 33.1K precursor protein which is also the primary species observed in total mitochondria (Fig. 3.5C, lanes 1, 2, panel a). Total leaf extracts from R19/arm/S22 infiltrated plants contain the full-length 35.1K protein and a prominent cleavage product of approximately 29.5K (Fig. 3.5C, lane 1, panel b) which would correspond to cleavage near the arm/S junction  142  as observed in the above described R/arm/S constructs. Purified mitochondria contained both the 35.1K and 29.5K species (Fig. 3.5C, lane 2, panel b). The presence of the 29.5K cleavage product indicates that unlike the corresponding R19/arm/SSVRI construct, cleavage in R19/arm/S22 construct near the arm/S boundary is very efficient. To further examine and refine the location of the R domain and its cleavage products in mitochondria, mitochondria were purified from agroinfiltrated leaves and separated into four protein fractions: outer membrane (OM), intermembrane space (IMS), inner membrane (IM) and matrix (MA). Isolated mitochondria were osmotically shocked, followed by centrifugation to separate the mitoplast pellet (IM and MA) from the supernatant (OM and IMS) fractions. Subsequently, the sonicated pellet was resuspended and centrifuged to separate the IM and MA subfractions and the supernatant was subjected to high speed centrifugation to separate the OM from the IMS subfractions. Proteins from these subfractions were then analyzed by SDS-PAGE and Western blotting using a GFP monoclonal antibody. Figure 3.5B shows that for each of the constructs analyzed (i.e., R, R47, R/arm/SSVRI and R/arm/S22), the majority of the protein species found in total mitochondrial protein are present in the IM fraction (Fig. 3.5B, lane 5). However, in the case of R47, although the 29.5K cleavage product is detected in the IM, both the full-length 32.1K and cleaved 29.5K species are found in the IMS (Fig. 3.5B, lane 4, panel b). The basis for the different localization patterns of the R and R47 fusion proteins in mitochondrial subfractions is not known. However, it is known that substrates destined for the mitochondrial inner membrane translocon (TIM23) are preferentially positively charged (3, 26, 48). Thus it is possible that a decrease in the overall positive  143  charge of R47 due to the absence of the lysine rich region between aa 47 to 58 (see Fig. 3.1) precludes efficient entry into TIM23. In the case of R/arm/S22, the fully cleaved 29.5K species is observed in both the IM and the MA fractions (Fig. 3.5B, lanes 5, 6, panel d) whereas the 30K species of the R/arm/SSVRI is only found in the IM fraction (Fig. 3.5B, lane 5, panel c). Our data suggest that the S22 sequence of R/arm/S22 promotes more efficient transport of the presequence across the IM concomitant with cleavage by the MPP (5, 11). The mitochondrial matrix HSP70 is known to assist in transport of proteins into the matrix (11, 45, 48). It is possible that the R/arm/S22 preprotein is brought to the IM facilitated by the net positively charged region of the R domain; the N-terminus of the R domain then extends into the matrix whereby it is cleaved by the MPP. The S22 sequence is possibly recognized by HSP70 and/or other matrix-associated proteins and is brought into the matrix where it is cleaved at the arm/S junction. This potential role for the S22 sequence is reinforced by our findings that R domain constructs (which lack the S22 sequence) are not observed to be transported beyond the IM (Fig. 3.5B, panels a, b). Attempts to analyze mitochondrial subfractions of plants infiltrated with R19/arm/SSVRI and R19/arm/S22 constructs were not successful due to the low levels of fusion protein in mitochondrial preparations. In a previous study, it was found that chloroplasts isolated from plants infiltrated with R/arm/SSVRI contained arm sequences but no R domain sequences (50). In addition, arm/SSVRI fusion protein constructs exclusively targeted chloroplasts. Xiang et al. (50) proposed that the R/arm/SSVRI protein was cleaved in the cytoplasm near the R/arm junction and that the exposed N-terminal arm sequence functioned as a chloroplast transit  144  peptide (TP) directly targeting the arm/SSVRI to the chloroplast stroma. In this study, we show that mitochondria isolated from plants infiltrated with R/arm/SSVRI and R/arm/S22 also contain arm sequences. However, since arm sequences cannot directly target mitochondria, we propose that they are indirectly shuttled to this location via the upstream R domain. Based on our findings, a model for mitochondrial import of the CNV R/arm/S22 is shown in Fig. 3.6.  3.3.4 The complete arm is not required for chloroplast targeting As described above, it has previously been hypothesized that the full-length CNV CP is cleaved in the cytoplasm in the arm near the R/arm junction and that sequences within the exposed N-terminal arm/SSVRI region of the CP function as a chloroplast TP (50). In this study various deletion arm mutants were also analyzed for chloroplast targeting, including one which consisted of a point mutation and others that included the complete arm but lacked the SVRI region (i.e., arm∆58-73/SSVRI, arm∆74-96, arm∆87-96, arm(P73G)/SSVRI and arm(-SSVRI). This analysis showed that an intact arm is required to target chloroplasts and that the arm(-SSVRI) construct showed reduced targeting compared with the arm/SSVRI construct, indicating that the C-terminal SVRI sequence is critical for efficient chloroplast uptake. We wanted to evaluate further the specific CP arm sequences required for import and, in addition, assess the influence of sequences from the adjacent R and S domains in chloroplast import. Nicotiana benthamiana leaves were infiltrated with R/arm/SSVRI, R/arm/S22, R6/arm/S22, arm30/S22, arm27/S22 and arm12/S22 constructs (Fig. 3.1). Confocal microscopy of leaves infiltrated with R/arm/SSVRI, R/arm/S22, R6/arm/S22, and arm27/S22  145  R  arm  S/P CNV CP  Cytoplasmic cleavage  Transport to mitochondria  (c1)  Transport to chloroplasts (+HSP70, 14-3-3)  Transport to chloroplasts  (c2)  (+HSP70, 14-3-3)  (b1) (a1)  Retrograde (b3) translocation  Cytoplasm  OM TOM  OM IM  IM  Stroma  TOC  TIM TIC  TOM MPP  Cytoplasm  TIM  (c3)  Cytoplasm  (b2) Stroma  TOC  (a2)  OM IM  OM TOM  TIC  IM  Matrix TIM TOM  SPP  HSP93?  (c4)  TIM HSP70  Cytoplasm  MPP  Stroma  Mitochondria  MPP  (a3)  Chloroplasts  Matrix MPP  Figure 3.6 A model for the translocation and import of the CNV CP to chloroplasts and mitochondria. The colouring scheme for the CP R, arm, S and P domains is shown at the top. Mitochondrial presequence-like regions in the R domain of the CP (Fig. 3.4) direct transport to the mitochondrial TOM and TIM import machinery. In (a1), the R, arm, S and P sequences enter the mitochondrial translocon. This is followed by import into the matrix (a2) which is facilitated by sequences in the S22 region of the CP, possibly via interaction of this region with HSP70, an accessory component of TIM (44). Cleavage by mitochondrial MPPs occurs at the three sites on the CP which are summarized in Fig. 3.4 and the cleaved CP products are localized to the matrix (a3) In (b1), the CP targets the mitochondrial matrix wherein MPP cleavage near the R/arm junction occurs; thereafter the cleaved mature CP product is prematurely released into the cytoplasm, possibly via retrograde translocation (b3) (17). The cleaved CP is then transported to chloroplasts via the exposed N-terminal chloroplast TP-like region as described below. In (b2), the R domain passes through TIM, accompanied by MPP cleavage at approximately 24-28 aa from the N-terminus (see Fig. 3.4). The R domain becomes associated with the inner membrane (Fig. 3.5) possibly due to the presence of a predicted amphiphilic sequence (Fig. 3.4) and/or the absence of the S22 sequences proposed to facilitate transport to the matrix. In (c1), an alternate pathway for transport of CP to chloroplasts is shown. In this model, cleavage near the R/arm junction occurs in the cytoplasm, placing the TP-like sequences of the arm region at the N-terminus. The cleaved protein in conjunction with cellular host factors such as HSP70 and 14-3-3 (see 49) is transported to the chloroplast TOC and TIC import machinery (c2). Further protein translocation and entry into the stroma (c3) may be facilitated by HSP93 which has been suggested to be a chaperone component of the chloroplast import machinery (15). Cleavage near the arm/S junction by the chloroplast SPP occurs, leaving the cleaved protein in the stroma (c4). IM=inner membrane; OM=outer membrane. 146  fusion proteins shows that each of these fusion proteins targets chloroplasts (Fig. 3.7A, panels a, b, c, d) (with R/arm/SSVRI and R/arm/S22 also targeting mitochondria as described above). Confocal analyses of leaves infiltrated with the R19/arm/SSVRI and R19/arm/S22 constructs indicated that these fusion proteins target chloroplasts; however, the GFP fluorescence of these two constructs was not as strong compared with that of the four constructs described above (Fig. 3.8).  Figure 3.9 shows isolated chloroplasts from  leaves agro-infiltrated with R/arm/SSVRI, R/arm/S22, R6/arm/S22, and arm27/S22 constructs in which GFP fluorescence can also be found in the stroma, i.e., regions of GFP fluorescence are distinct from the red fluorescing thylakoids, and R19/arm/S22 and R19/arm/S22 constructs in which GFP fluorescence appears to be found mainly in the chloroplast periphery. In addition, stromules (24) were observed from chloroplasts isolated from these agro-infiltrated leaves. The stromal location of the fusion protein is supported by further observations described below. Confocal analyses of arm30/S22 and arm12/S22 constructs showed only cytosolic localization (Fig. 3.7A, panels e, f). In  addition, leaf protein extracts of these constructs contained only full-length precursor proteins indicating that these fusion proteins do not enter chloroplasts (data not shown). As indicated by the ability of the arm27/S22 fusion protein construct to enter chloroplasts, only the C-terminal 27 aa of the 34 aa arm (along with downstream S domain sequences) are required for chloroplast targeting. It is noted that the arm30/S22 fusion protein construct did not target chloroplasts even though the arm/S22 (data not shown) and arm27/S22 constructs do. It is possible that the presence of two closely spaced prolines at  the N-terminus of the arm30/S22 fusion protein construct (see Fig. 3.1A) interfere with translocation through the chloroplast membrane.  147  A  a  R/arm/SSVRI Mock ThermoP treated lysin Trypsin GF  oc k  b  m  B  R/arm/S22  Lf Ct M S  M S M S Lf Lf  37.1  R/arm/SSVRI  30.0  R/arm/S22  32.0 29.5  b  33.7 29.5  c  32.5 29.5  d  c  a  27.5 39.1  R6/arm/S22  d  R6/arm/S22  arm27/S22 arm27/S22  e  1 2 3 4 5 6 7 8 9 10  arm30/S22  f arm12/S22 GFP  Chloroplast Merged auto-fluorescence  Figure 3.7 Analysis of the location of various R and arm-containing constructs in agroinfiltrated plants. (A) Confocal images of agro-infiltrated leaves at 48 hpi using the indicated constructs. The first column shows GFP fluorescence (green), the second chloroplast autofluorescence (red) and the third is a merged image of the GFP and chloroplast autofluorescence. (B) Western blot analysis of total protein obtained from chloroplasts and from fractions of protease-treated chloroplasts. Chloroplasts (Ct) were isolated from infiltrated leaves of the indicated constructs and treated with either thermolysin or trypsin, followed by centrifugation to separate the membrane (M) (pellet) and soluble (S) (supernatant) fractions (see text). Lanes 9 and 10 correspond to total protein extracted from GFP- and mock-infiltrated N. benthamiana leaves, respectively. The blot was probed with a GFP monoclonal antibody. The predicted sizes in kilodaltons of the full-length fusion protein of each construct and the estimated sizes (in italics) of the various cleavage products are indicated on the left. Relative protein masses were estimated by co-electrophoresis using MagicMark™XP (Invitrogen, Burlington, ON) and BenchMark™ Prestained (Invitrogen, Burlington, ON) as molecular size standards (not shown). 148  R19/arm/S22  R19/arm/SSVRI  GFP  Chloroplast autofluorescence  Merge  Figure 3.8 Confocal images of N. benthamiana leaves agro-infiltrated with CNV R19/arm/S22 and R19/arm/SSVRI constructs at 2 dpi. Note that GFP fluorescence of the R19/arm/S22 fusion protein construct colocalizes with chloroplasts whereas the GFP fluorescence is found mainly around the chloroplasts isolated from R19/arm/SSVRI infiltrated leaves. For each panel, the first column shows GFP fluorescence (green), the second column shows chloroplast fluorescence (grey) and the third column shows a merged image. [The (red) MitoTracker channel is not shown].  149  A  B  R/arm/SSVRI  R/arm/S22  R6/arm/S22  arm 27 /S22  R19 /arm /SSVRI  R19 /arm /S22  GFP  Chloroplast autofloresence  Merge  GFP  Merge  Figure 3.9 Confocal microscopy of chloroplasts isolated from plants agro-infiltrated with R/arm/SSVRI, R/arm/S22, R6/arm/S22, arm27/S22, R19/arm/S22 and R19/arm/S22 constructs was conducted to determine GFP localization. (A) For the first four constructs, GFP appears to co-localize with the chloroplast stroma whereas GFP is found mainly in the chloroplast periphery and as patches in the last two constructs. Red fluorescent thylakoids are indicated by the black arrow. For each panel, the leftcolumn shows GFP fluorescence (green), the middle-column shows chloroplast fluorescence (red) and the right-column shows a merged image. (B) Occasionally, stromules, apparent extensions of the stroma (24) were observed (white arrows) and areas of no GFP localization (grey arrows) were observed in isolated chloroplasts which may be due to starch granules. For each panel in B, the left-column shows GFP fluorescence (green) and the right-column shows a merged image of the GFP fluorescence and red chloroplast fluorescence (the chloroplast fluorescent channel is not shown). 150  The location of the R/arm/SSVRI, R/arm/S22, R6/arm/S22, and arm27/S22 fusion proteins in chloroplasts was evaluated. Chloroplasts purified from infiltrated leaves were either mock incubated or incubated with thermolysin or trypsin to assess the location of fusion proteins in chloroplast subfractions (Fig. 3.7B). Thermolysin digests only those proteins associated with the chloroplast outer membrane, whereas trypsin digests proteins present on the outer membrane as well as within the chloroplast intermembrane space. Proteins present in the stromal fraction or the thylakoids are not digested by either thermolysin or trypsin (6). Following protease treatment, chloroplasts were lysed and the membrane and soluble fractions were separated. Proteins from these fractions were then analyzed by Western blotting using a GFP monoclonal antibody for detection. As previously observed by Xiang et al. (50), very low levels of the full-length 37.1K fusion protein and two cleavage products of approximately 31K and 27K (revised to 30K and 27.5K in this thesis) were found in total leaf extracts of R/arm/SSVRI (Fig. 3.7B, lane 1, panel a; Fig. 3.4, lane 1, panel c) and these two cleavage products are also found in purified chloroplasts (Fig. 3.7B, lane 2, panel a) (see below). In R/arm/S22 infiltrated leaves, trace amounts of the full-length 39.1K protein as well as a prominent 29.5K cleavage product are found in total leaf extracts (Fig. 3.7B, lane 1, panel b). In contrast, only the prominent 29.5K cleavage product and very low levels of an intermediate-sized 32K product were detected in purified chloroplast extracts (Fig. 3.7B, lanes 2, 4, 6, panel b). Examination of the chloroplast membrane and soluble fractions of R/arm/SSVRI and R/arm/S22 shows that the 30K and 27.5K proteins and the 32K and 29.5K protein, respectively, are detected almost exclusively in the soluble fractions (Fig. 3.7B, lane 4, panels a, b). The 32K and 30K proteins are resistant to thermolysin treatment (Fig. 3.7B,  151  lane 6, panels a), confirming that they are not associated with the chloroplast outer membrane. However, the 32K species is sensitive to trypsin digestion indicating that it is present in the intermembrane space. The 30K cleavage product is only partially trypsin sensitive and therefore some of it may be in the chloroplast stroma. The 27.5K cleavage product of R/arm/SSVRI is trypsin resistant (Fig. 3.7B, lane 8, panel a), indicating that this species is present only in the stroma. Soluble chloroplast fractions of R/arm/S22 consistently show only the 29.5K fully cleaved product which is trypsin resistant (Fig. 3.7B, lane 8, panels b), indicating that this species are present in the stroma. It is possible that the trace amounts of the 32K cleavage product of R/arm/S22 in chloroplast purifications are due to the greater import efficiency of this construct. Total leaf extracts of R6/arm/S22 contain low levels of the predicted full-length 33.7K protein and an abundant 29.5K cleavage product (Fig. 3.7B, lane 1, panel c). Similarly, total leaf extracts of arm27/S22 contain low levels of the full-length 32.5K protein and an abundant 29.5K cleavage product (Fig. 3.7B, lane 1, panel d). Purified chloroplasts of each of these constructs contain only the 29.5K cleavage product (Fig. 3.7B, lane 2, panels c, d). Analysis of fractionated chloroplasts purified from leaves infiltrated with R6/arm/S22 or arm27/S22 shows that the 29.5K species of both constructs are thermolysin and trypsin resistant and present predominantly in the soluble fraction. Therefore the 29.5K species is a component of the chloroplast stroma (Fig. 3.7B, lane 8, panels c, d). The estimated molecular sizes of the stromal species suggest that they are each a result of specific cleavage near the arm/S junction and likely by the action of the stromal processing peptidase (SPP). The presence of predominantly one protein species in the stroma of agro-infiltrated plants suggests these constructs are efficiently processed  152  upon chloroplast import. The presence of little or no intermediate products from R/arm/S22, R6/arm/S22 and arm27/S22 (as compared with R/arm/SSVRI) suggests that the additional 18 residues from the N-terminus of the S domain facilitate transport to the chloroplast stroma (see below). HSP70 is known to assist in the transport of proteins into the chloroplast stroma (15); therefore, similar to the model proposed above for uptake of R/arm/S22 to the matrix, it is possible that chloroplast stromal proteins, such as HSP70, may facilitate passage across the chloroplast inner membrane. Analysis of the R/arm/S region of the CNV CP by TargetP (41) predicts a chloroplast TP-like region originating immediately downstream of the R/arm junction (data not shown) with a point of origination given the highest probability factor (prob = 0.615) at the Ala residue (aa 65) that precedes the initiating Phe residue (prob = 0.514) in construct arm27/S22. This reinforces our present and previous (50) conclusion that the arm functions as an efficient chloroplast TP. In addition, TargetP predicts that stromal  cleavage of the TP-like region occurs between the Phe (F) and Gln (Q) residues present 6 and 5 amino acids upstream from the arm/S junction (Fig. 3.1; data not shown). This is also consistent with our Western blot analyses where stromal cleavage is predicted to occur near the arm/S junction.  3.3.5 The N–terminal 22 aa sequence in the CNV CP S domain influences the efficiency of transport of CNV CP GFP fusion proteins into mitochondria and chloroplasts  As mentioned above, the SVRI sequence at the N-terminus of the S domain was shown to be essential for efficient chloroplast targeting directed by CNV CP arm  153  constructs (50). As described above in Fig. 3.7, the GFP fusion proteins containing the S22 sequence rather than only the SSVRI sequence are more efficient in generating their respective fully cleaved 29.5K and 27.5K stromally located fusion protein products. This was also observed in mitochondrially imported R/arm/SSVRI and R/arm/S22 constructs where uptake and cleavage to the fully mature cleavage product is influenced by the S22 sequence (see above; Fig. 3.5). To further investigate the role of the N-terminal sequences of the S domain in mitochondrial and chloroplast import, three GFP fusion protein constructs (i.e., R/arm/SSVRI, R19/arm/SSVRI and arm/SSVRI, respectively) that contain only the SVRI residues of the S domain were directly compared at 24 and 48 hpi to corresponding constructs that contain an additional 18 N-terminal residues from the S domain (i.e., R/arm/S22, R19/arm/S22 and arm/S22). Figure 3.10 shows that two main 30K and 27.5K cleavage products are evident in total leaf extracts of plants infiltrated with construct R/arm/SSVRI at 24 and 48 hpi (lanes 1, 2) whereas in the “side by side” comparison the corresponding R/arm/S22 construct contains predominantly a single 29.5K cleavage product (lanes 3 and 4) and very little of the intermediate product even at 24 hpi. The absence of the larger of the two cleavage products in plants infiltrated with R/arm/S22 suggests that chloroplast and mitochondrial transport is more efficient with this fusion protein than with the corresponding R/arm/SSVRI protein. A similar pattern of facilitated maturation to the fully cleaved protein is observed upon comparison of R19/arm/SSVRI and R19/arm/S22. Figure 3.10 (lanes 5, 6) shows little or no cleaved products in plants infiltrated with R19/arm/SSVRI at either 24 hpi or 48 hpi, whereas the fully cleaved 29.5K product of R19/arm/S22 is readily observable at 24 hpi but largely disappears by 48 hpi (Fig. 3.10, lanes 7). It is noted that expression levels of the  154  R/arm/SSVRI R/arm/S22 R19 /arm/SSVRI R19 /arm/S22 arm/SSVRI 24  48  24  48  24  48  24  48  24  48  arm/S22 24  GFP mock  48  39.1 37.1 35.1 33.1 31.1  32.0 30.0 29.5 27.5  1  2  3  4  5  6  7  8  9  10  11  12  13  14  Figure 3.10 Western blot analyses of total protein extracted from leaves agro-infiltrated with the indicated three sets of fusion protein constructs containing the R/arm, R19/arm and arm regions and either SVRI or the first 22 amino acids of the N-terminus of the S domain. Total proteins were isolated from infiltrated leaves at 24 and 48 hpi as indicated. Lanes 13 and 14 correspond to total protein extracted from GFP- and mock-infiltrated N. benthamiana leaves, respectively. Shown below is the blot stained with 0.2% Ponceau S to demonstrate the loading of equivalent protein masses (except for the GFP lane which was intentionally underloaded to avoid overexposure) as determined by the quantity of the large subunit of ribulose bis-phosphate carboxylase (Rubisco). The numbers on the left correspond to the predicted sizes (in kilodaltons) of the full-length fusion protein and the numbers on the right to the estimated sizes (in kilodaltons) of the cleavage products (shown in italics).  155  R19/arm/SSVRI construct consistently decreased over time (data not shown). Similarly, and as previously found (50) leaf extracts of plants infiltrated with arm/SSVRI contain both the full-length and fully cleaved protein products at 24 and 48 hpi (Fig. 3.10, lanes 9, 10), whereas the fully cleaved protein is the predominant species observed in plants infiltrated with arm/S22 (Fig. 3.10, lanes 11, 12). Although, it cannot be ruled out that the close proximity of the GFP to the proposed arm/S cleavage site may hinder efficient cleavage in the -SSVRI constructs, these findings provide evidence that the presence of the additional 18 N-terminal aa of the S domain in the fusion protein constructs is associated with more efficient uptake and/or cleavage in their respective chloroplast and mitochondrial compartments.  3.3.6 Mitochondrial targeting of R/arm/SSVRI and R/arm/S22 appears to precede chloroplast targeting  The experiments thus far described show that constructs R/arm/SSVRI and R/arm/S22 can target both mitochondria and chloroplasts, wherein the R domain contains determinants specific for mitochondrial localization and the arm region determinants for chloroplast targeting. The dual targeting capability of these constructs was further analyzed by confocal microscopy and Western blot analysis of mitochondrial and chloroplast specific proteins. Confocal microscopy of plants agro-infiltrated with R/arm/SSVRI and R/arm/S22 GFP constructs showed that mitochondria became labelled with GFP prior to that of chloroplasts (data not shown). To investigate the timing of targeting further, mitochondria and chloroplasts were purified from equal weights of infiltrated leaves at 24 and 48 hpi and total protein from these organelles was analyzed by  156  SDS-PAGE, followed by Western blotting using a GFP monoclonal antibody. At 24 hpi, the R/arm/SSVRI fusion protein and cleavage products are readily detected in purified mitochondria (Fig. 3.11, lane 3), but they are not detected in purified chloroplasts (Fig. 3.11, lane 2). However, at 48 hpi the relative levels of R/arm/SSVRI protein in chloroplasts and mitochondria are approximately equal (Fig. 3.11, compare lanes 5 with 6). Similar results were obtained with construct R/arm/S22 where at 24 hpi, fusion protein is readily detected in purified mitochondria but only present at low levels in chloroplasts (Fig. 3.11, compare lanes 8 with 9). However at 48 hpi, the relative levels of R/arm/S22 are approximately equivalent in these organelles (Fig. 3.11, compare lanes 11 with 12). Thus it appears that targeting to mitochondria may initially precede targeting to chloroplasts. In previous work, using an R/arm/SSVRI construct, we suggested that cleavage of the arm region near the R/arm junction occurs in the cytoplasm and that the N-terminal region of the resulting cleavage product directs chloroplast targeting (50). However, our present observation is that both R/arm/SSVRI and R/arm/S22 constructs also target mitochondria and that mitochondrial targeting appears to precede chloroplast targeting. It is possible that because chloroplast targeting requires a prior cleavage to expose the Nterminal TP-like region, it lags behind mitochondrial targeting. In some mitochondrial precursor proteins that are processed by the MPP, a portion of the cleaved product is redirected into the cytosol (e.g., fumarase) (17). Therefore, it is also possible that cleavage at the R/arm junction may occur while the fusion protein is in transit through the mitochondrial translocons and that the cleaved protein is released back into the cytoplasm. Additionally, preprotein saturation of the mitochondrial translocons may lead  157  R/arm/SSVRI 24 Lf  48  Ct Mi Lf  39.1K 37.1K 32.0K 30.0K 29.5K 27.5K  R/arm/S22  Ct  2  Mi Lf  3  4  5  Mi Lf *  6  7  8  48 48  48  Ct  *  * 1  24  GFP mock Ct Mi Lf Lf *  *  9 10 11 12 13 14  Figure 3.11 Western blot analysis of total protein extracted from chloroplasts and mitochondria isolated from R/arm/SSVRI and R/arm/S22 infiltrated leaves at 24 and 48 hpi. Lanes 1-6 correspond to samples obtained from R/arm/SSVRI infiltrated leaves and lanes 7-12 from R/arm/S22 infiltrated leaves. Time points are indicated in hpi above the respective lanes. Lanes 13 and 14 correspond to total protein extracted from GFP- and mock-infiltrated N. benthamiana leaves, respectively. The symbols used are described in Figs. 3.4 and 3.5. The numbers on the left correspond to the predicted sizes (in kilodaltons) of the full-length fusion protein and the cleavage products (shown in italics). The asterisks indicate non-specific bands.  158  to excess preproteins in the cytosol available for cleavage. Based on our findings, a model for chloroplast import of the CNV R/arm/S22 is shown in Fig. 3.6. Further research is required to provide additional support for this model.  3.3.7 Dual targeting of R/arm/SSVRI and R/arm/S22  Many cytoplasmically synthesized proteins are dually targeted to chloroplasts and mitochondria (for examples see 17, 29, 39). In these cases, dual targeting is achieved by one of two main means: by the presence of distinct N-terminal sequences on the same protein (which results from either transcriptional or translational processes) or by a single N-terminal sequence which contains targeting signals for both mitochondria and chloroplasts (29, 32). In this study, a distinctive mechanism is responsible for dual targeting of CNV R/arm/S sequences. The arm sequence within the R/arm/S region directs import to chloroplasts due to the TP-like function of the arm; additionally, this region is also directed to mitochondria due to the mitochondrial presequence-like function of the R domain. Interestingly, the SPP and the MPP can both efficiently cleave at a similar site near the arm/S border of R/arm/S22 (Fig. 3.11, compare lanes 11 with 12). Note that cleavage near the arm/S junction of the R/arm/SSVRI construct is not as evident in mitochondria as it is in chloroplasts, further reinforcing the importance of the S22 sequence in mitochondrial import (Fig. 3.11, compare lanes 5 with 6).  159  3.3.8 CNV CP is associated with both chloroplasts and mitochondria during CNV infection  Purified chloroplasts and mitochondria from CNV-infected N. benthamiana leaves were examined for the presence of CNV CP by Western blot analysis using a polyclonal antibody raised to the CNV S/P domain (50). Similar to that shown by Xiang et al. (50), CNV CP-specific proteins are detected in purified chloroplast preparations.  The CNV CP species include the full-length 41K protein, as well as a prominent smallersized protein with an estimated size of approximately 35K and a faint species of approximately 33K (Fig. 3.12, lane 2). These species are similar in size to the two previously observed in chloroplasts isolated from CNV-infected leaves (50). A large amount of full-length CP and a less abundant 35K cleavage product were also found in protein extracted from purified mitochondria, indicating that the CNV CP enters mitochondria during infection wherein protein cleavage occurs (Fig. 3.12, lane 4). Thus it appears that CNV CP sequences are associated with both mitochondria and chloroplasts in CNV-infected plants. Based on the predicted size, the 35K cleavage product associated with mitochondria and chloroplasts would result from cleavage near the R/arm junction and the 33K protein in chloroplasts from cleavage within the arm region immediately upstream of the arm/S junction. These results are consistent with the findings obtained with the R, arm and S constructs used in these studies. It is possible that the full-length CNV CP species detected in purified chloroplasts and mitochondria are due to contamination with virions from the cell cytosol during the purification procedure. Xiang et al. (50) showed that the full-length CP associated with purified chloroplasts was sensitive to thermolysin treatment and thereby could either be  160  Ct  Mi  Lf  d d d cte ck ck cte ock fecte in mo infe mo infe m  41K (CP) 35K 33K  1  2  3  4  5  6  Figure 3.12 Subcellular location of CNV CP in CNV-infected leaves. Chloroplasts and mitochondria were isolated from CNV-inoculated leaves at 3 (days post-inoculation) dpi. Total protein from purified chloroplasts (Ct), mitochondria (Mi) and leaf tissue (Lf) were electrophoresed through an SDS-PAGE gel and subjected to Western blot analysis. The blot was probed with a CNV polyclonal antibody raised to the CNV CP S/P domain. The size of the CNV CP and the relative masses of the main CP cleavage products, as estimated by co-electrophoresis using MagicMark™XP (Invitrogen, Burlington, ON) and BenchMark™ Prestained (Invitrogen, Burlington, ON) as protein molecular size standards, are indicated on the left.  161  due to contamination of chloroplast preparations with CNV particles or CP, or to a loose association of particles or CP with the chloroplast OM. It is assumed that the chloroplast and mitochondrial purification methods used in these experiments do not result in artifactual co-purification with CNV particles. However, confirmation of this would require further experimental analyses. Nevertheless, the finding that both chloroplasts and mitochondria contain specific CP cleavage products suggests that at least some CNV CP enters and is cleaved within these organelles during the course of infection. Xiang et al. (50) suggested that the observed association of the CNV CP with chloroplasts supports their role in particle disassembly or assembly during CNV infection. Our finding that the CP also associates with mitochondria suggests that mitochondria may also be involved in the disassembly/assembly process. It is generally believed that the replication and assembly processes of plant viruses are closely associated, and presumably occur in similar cellular sites. CNV replication has been found to occur on peroxisomes (28). Peroxisomes are naturally found in close association with chloroplasts, particularly during cellular photorespiration (33). A close association is found between mitochondria and chloroplasts. Chloroplasts may gain a means to reduce excess energy by-products that may damage metabolic functions through photorespiration; mitochondria are able to dissipate excessive photosynthetic redox equivalents and help sustain repair and recovery by providing ATP (13, 27, 34). 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J Virol 80:7952-7964.  169  4 CHAPTER FOUR: Concluding Chapter 4.1 General Discussion Molecular studies of the CNV CP N-terminus were conducted to assess the importance of specific regions in virus particle structure and to evaluate their significance with respect to subcellular localization. These studies were undertaken to further understand the role of the CNV CP in the infection process and to gain insight into possible sites for CNV assembly and disassembly. Moreover, it is anticipated that these studies will contribute to a greater understanding of the role of viral CPs in the infection cycle and thereby, assist in the development of new strategies for the management of virus-induced disease. Overall, the studies presented in this thesis show that the N-terminal regions of the CNV CP have multifunctional roles during infection. Specifically, the 58 aa R domain is shown to be involved in targeting CP to mitochondria. This adds to its previously described role in binding viral RNA in the virus particle interior (3), forming an internal shell within the virus particle (9), and contributing to T = 3 icosahedral symmetry (5). It is interesting to speculate that mitochondria may serve as sites for R domain anchoring during the initiation of particle assembly whereby subsequent additions of CP subunits, possibly directed by the N-terminal R domain mitochondrial presequences, and RNA result in the closely associated R domain/RNA complex found within the particle interior. The results described in Chapter 2 showed that that the 34 aa arm region of the CNV CP is involved in particle stability, stable zoospore attachment and fungal transmission. These results are consistent with the known roles of the arm in the assembly of stable particles and CP dimer formation. The arm is also known to be 170  extruded from the shell during particle “breathing” (1), a process believed to be part of the virus disassembly process. The results described in Chapter 3 showed that the arm (along with 22 aa of the S N-terminus) plays a major role in targeting the CP to chloroplasts, and as shown in the model described in Fig. 1.8 (Section 1.5.3), suggest that chloroplasts may serve as sites for virus particle disassembly. The subsequent release of the viral RNA would be available for replication. Together, these results demonstrate that specific structural regions of the CNV CP have specific functions within cells. Although not directly examined in this thesis, this relationship between structure and function may relate to the role of the R and arm regions in virus disassembly and/or assembly in association with chloroplasts and mitochondria. Role of the CNV CP arm in virus structure. The first objective was to investigate the importance of the CP arm region in particle structure. The arm region consists of a relatively short, but highly basic sequence that connects the R to the S domain. The ε and β regions of the arm can be distinguished based on their overall net charge and whether they become part of a structurally ordered (C) or disordered (A or B) subunit. The ε region is highly basic and is therefore likely involved in virion RNA interactions, neutralization of negative charges from virion RNA (3) and possibly conformational switching required for T = 3 particle formation. The β region interdigitates at the particle three-fold axis forming the internal scaffold-like β-annulus proposed for virus particle stability. The disordered arms and possibly the R domain of the A or B subunit have been hypothesized to extrude from the quasi three-fold axis when the virus particle expands (13). Particle expansion has been shown to be important for fungal transmission and is hypothesized to occur upon interaction with the zoospore outer membrane (6, 14).  171  It is also likely that expansion occurs during the initial stages of the virus disassembly process (14). Deletion mutagenesis was used to determine the importance of the two arm regions in particle structure. Deletion mutants lacking the ε region did not produce particles in infected plants, indicating that this region is essential for particle assembly and/or accumulation. The ε region of the arm extends along the inner edge of the S domain and most of the viral RNA can be found in a layer immediately beneath the shell (9, 17). Based on its highly basic characteristics, it is proposed that deletion of the ε region impairs the ability of the CP to interact with viral RNA during particle assembly. Mutational analysis of the basic residues within the ε region may help substantiate the importance of electrostatic interactions of virion RNA in particle structure and the assembly process. Arm mutants that lack the β region were capable of accumulating particles in infected leaves of N. benthamiana, although the virion yield was reduced compared to that of WT CNV. This indicates that the β region of the arm plays an important, but not essential role in particle assembly and/or stability. Insufficient cooperative ordering of the arms of C/C subunits in forming the particle β-annulus might be expected to affect particle stability and thereby reduce particle accumulation. Previous work in the Rochon lab has shown that there are specific residues of the CNV CP that are involved in virus transmission by zoospores of O. bornavanus (7, 12). In this thesis, virus transmissibility was lost when β(-) arm mutant particles were used in fungus transmission assays, indicating that the β-annulus is important for CNV transmission. As stated above, the extrusion of N-terminal regions of arm regions of the  172  A and B subunits the CP may be required for virus transmission (6). Therefore, the lack of transmissibility may reflect the importance of the β region in the extrusion of the arm or in zoospore attachment upon extrusion. As stated in Hui and Rochon (4), the lack of transmission of β(-) particles may also be attributed to the importance of the β region in the formation of the β-annulus and thus particle stability. During virus particle “breathing”, specific regions of the CP are extruded and extended beyond the particle surface which may possibly lead to particle uncoating. Xiang et al. (19) showed that an R/arm/SSVRI GFP fusion protein targets chloroplasts. Upon chloroplast targeting, the fusion protein is imported into the stroma upon where it is processed by the SPP. It was proposed that CP targeting to chloroplasts may indicate a role for chloroplasts in the particle disassembly process, whereby interaction of the extruded arm region of the virus CP and the chloroplast import machinery facilitates uncoating (see Section 1.5.3, Fig. 1.8). To assess this possibility, a local lesion assay was conducted to determine if β(-) arm mutant particles are impaired in the ability to initiate infection in leaves of a local lesion host, Chenopodium quinoa. If the interaction between the arm region and chloroplasts is required for virus uncoating, it is expected that fewer lesions would develop in leaves inoculated with the β(-) arm mutant particles. Significantly fewer lesions were produced by the β(-) mutant particles when compared with CNV WT (see Appendix A, Fig. 5.1). However, when virion RNA was used as the inoculum, no significant differences in the number of local lesions were found (p≤0.05). Therefore, the decrease in the number of local lesions produced by β(-) arm mutant particles is likely due to an inability to initiate infection which may be attributed to an  173  uncoating defect upon inoculation. This observation is consistent with the low virion yield recovered from β(-) arm mutant-infected plants. Unlike WT CNV, β(-) mutant particles disassembled when subjected to particle swelling conditions and increased temperatures. This suggests that the β-annular structure at the CNV particle three-fold axis may serve an important role in maintaining particle stability and thus would account for the decreased particle accumulation. It has been suggested that hexamer formation at the three-fold axis of Cowpea chlorotic mosaic virus (CCMV) (18) and Sesbania mosaic virus (SeMV) (15) are not required for assembly initiation. Although it is not known if the formation of the three-fold axis of CNV is involved in initiating assembly, it is possible that lower particle accumulation observed with the β(-) mutants could be due to inefficient initiation of assembly. In vitro particle assembly experiments using CNV CP and CP mutants (e.g., β- and ε-) would further provide greater insight into their roles in particle assembly. Difficulties in past attempts in the Rochon lab to purify CNV CP or to express the soluble CP in a bacterial system have precluded initiation of these studies. During zoospore attachment, the means by which the interiorly residing CP regions become extruded are not known. Virus particle expansion is generally believed to be the result of loss of virus calcium ions from the quasi three-fold axis; this results in the exposure of negative charges which repel each other and cause the particle to expand. Preliminary studies to determine the role of calcium in virus transmission have been conducted but were not conclusive (Appendix B).  174  The virus particle assembly and structural studies described in this thesis represent one of the few in vivo studies that have been conducted to illustrate the structural role of the CP in particle assembly in plants. CNV CP N-terminal residues in subcellular localization. Subcellular membranes are known to be sites that viruses exploit during the infection process (for references see Section 1.4.). Work in this thesis and partly in Xiang et al. (19) has shown that the Nterminal regions of the CNV CP target chloroplasts and mitochondria. In addition, evidence is presented that the CNV CP can also be found associated with these organelles in infected plants. The presence of organellar targeting signals in the CNV CP provides another example of how viruses are able to exploit host cellular machineries to serve viral functions (see section 1.1). Xiang et al. (19) and the work described in appendix A show that defects in the arm region of the CP affect the initiation of infection and perhaps particle uncoating. Our current hypothesis is that during infection, the virus recruits subcellular membrane systems as sites of particle disassembly (and possibly assembly as well) (19). As the virus particle “breathes”, the R/arm regions of the A and B subunits extrude beyond the shell surface whereby exposure of these regions leads to mitochondrial and chloroplast targeting. Cleavage near the R/arm junction, possibly following targeting of the R/arm region to mitochondria, exposes a chloroplast TP-like region identified in the arm (see below). Cytosolic chaperones such as Hsp70 and 14-3-3 are postulated to interact with the CNV chloroplast TP and to translocate the virus particle to the chloroplast surface. It is postulated that chloroplast-bound particles then undergo disassembly, partly as a result of particle destabilization upon CP import into chloroplasts (19).  175  In addition to chloroplasts, mitochondria may also serve a role in virus particle disassembly. The results presented in this thesis work show that the N-terminus of the R domain of the CNV CP possesses features similar to those found in the presequences of known mitochondrial precursor proteins and that this domain is sufficient to target mitochondria. However, mitochondria may also play a role in virus particle assembly whereby the mitochondrial localization signal of the R domain allows CP subunits to anchor onto mitochondria and initiate the virus assembly process. A unique form of dual-targeting has been discovered in this thesis where it is shown that the N-terminal region of the CNV CP is found in association with both chloroplasts and mitochondria. The literature provides many examples where an identical protein can be found in more than one subcellular compartment (10, 16). Two general mechanisms for dual-targeting have been documented for proteins destined to both chloroplasts and mitochondria (10). One mechanism is the result of alternative transcriptional or translational processing or exon splicing of a single gene such that two proteins each with distinct targeting sequences are produced. The other type consists of a single undelineated “ambiguous” targeting signal that can be recognized by both organelles. In this thesis, it is shown that the CNV CP arm region can associate with mitochondria due to the upstream mitochondrial presequence-like R domain. The arm can also target chloroplasts directly (following proteolytic removal of the R domain) due to its possession of TP-like sequences (8). The work in this thesis suggests at least two possible modes of dual-targeting of CNV CP sequences: 1) chloroplasts and mitochondria are targeted independently due to exposure of their respective targeting signals; or 2) mitochondria are targeted initially due to the N-terminal R domain  176  whereupon proteolytic processing occurs following partial import of the CP N-terminus; subsequent retrograde translocation of the newly exposed N-terminal arm (i.e., chloroplast TP) leads to chloroplast targeting (see Chapter 3, sections 3.3.8 and 3.3.7 and Fig 10). The mode of dual-targeting of CNV CP sequences may be assessed by conducting protein import studies using isolated chloroplasts and/or mitochondria (see below). The data in this thesis suggest that the chloroplast TP-like region initiates immediately downstream of the N-terminus of the arm region; however, the precise aa sequence is not known. It may be possible to determine this sequence by purification of the preprotein followed by mass spectrophotometry or Edman degradation (2). However, attempts to obtain sufficient quantities of the preprotein have so far been unsuccessful (D. Rochon, personal communication). Similar subcellular targeting studies focusing on the CP N-terminal region of two related viruses [the Tombusvirus (TBSV) and the Aureusvirus (CLSV)] are being conducted in the Rochon lab. Similar to CNV, it has been found that the TBSV R domain GFP-fusion protein construct targets mitochondria, the R/arm/S18 GFP construct targets both mitochondria and chloroplasts and the arm region targets only chloroplasts (D. Rochon, unpublished data). However, in CLSV, the R/arm/S18 GFP fusion protein construct only targeted chloroplasts and the R domain GFP fusion proteins were found only in the cytosol (Rochon et al., manuscript submitted) . These findings reinforce the importance of the CP R/arm regions in subcellular targeting among members of the Tombusviridae family.  177  Further work to assess the interaction between the CNV CP and mitochondria or chloroplasts may be carried out by conducting in vitro binding assays using purified CNV particles and isolated organelles. One major source of difficulty in the interpretation of the results would be to assure that the virus detected was specifically bound to chloroplasts and not due to contaminating residual unbound particles. The specificity of virus particle-chloroplast binding could be assessed by using WT and mutant particles in: 1) comparative binding assays; 2) competitive binding assays; or 3) competitive binding assays using bacterially expressed R/arm protein. Additionally, since known TOC receptors are able to recognize presequences in a GTP-dependent manner, determination of whether binding of the CNV chloroplast-TP region to chloroplasts is GTP-dependent could be assessed by conducting binding assays with or without GTP. Experiments could also be conducted to study virus particle disassembly in association with isolated chloroplasts. Chloroplasts and mitochondria are traditionally considered to be autonomous organelles; however, they are generally found in close association (along with other membrane-bound organelles such as peroxisomes) where an exchange and/or sharing of metabolic substrates (such as glycerate, malate and ATP) occurs (11). The close proximity and biochemical interplay that occurs among these organelles could provide an opportunistic situation that viruses can exploit. It is generally believed that the replication and assembly processes of viruses are closely coupled (see Section 1.2). It can be hypothesized that these closely associated processes are coordinated via interacting organelles (e.g., peroxisomes, mitochondria or chloroplasts). This raises the question of how CNV replication which occurs on peroxisomes can be coordinated with  178  particle assembly if assembly were to occur in association with mitochondria or chloroplasts. Further research is required to determine if indeed the assembly or disassembly process of CNV occurs in association with mitochondria and/or chloroplasts. One approach to study this association would be to monitor the release of RNA from labelled R and/or arm mutant particles in infected plants. The hypothesis is that a labelled R and/or arm mutant particle which contains a mutation within the CP region known to be important for mitochondrial and/or chloroplast targeting will not uncoat or uncoat with difficulty as a result of its failure to target one or both organelles. A measure of virus uncoating will be reflected by the amount of free viral RNA released relative to total viral RNA from R/arm mutant-infected plants in comparison to that found in CNV-infected plants. Electron microscopy of immunogold labelled-particles can be used to determine particle association with chloroplasts and/or mitochondria. Difficulties in this method for analyzing virus-organelle associations in past attempts (D. Rochon, unpublished data) may be attributed to the transient nature of disassembly and the inability to observe disassembled particles by electron microscopy.  4.2 References 1.  Broo, K., J. Wei, D. Marshall, F. Brown, T. J. Smith, J. E. Johnson, A. Schneemann, and G. Siuzdak. 2001. Viral capsid mobility: a dynamic conduit for inactivation. Proc Natl Acad Sci U S A 98:2274-2277.  2.  Edman, P. 1970. Sequence determination. Mol Biol Biochem Biophys 8:11-55.  179  3.  Harrison, S. C. 1983. Virus structure: High-resolution perspectives. Advances in Virus Research 28:175-240.  4.  Hui, E., and D. Rochon. 2006. Evaluation of the roles of specific regions of the Cucumber necrosis virus coat protein arm in particle accumulation and fungus transmission. J Virol 80:5968-5975.  5.  Kakani, K., R. Reade, U. Katpally, T. Smith, and D. Rochon. 2008. Induction of particle polymorphism by Cucumber necrosis virus coat protein mutants in vivo. J Virol 82:1547-1557.  6.  Kakani, K., R. Reade, and D. Rochon. 2004. Evidence that vector transmission of a plant virus requires conformational change in virus particles. J Mol Biol 338:507-517.  7.  Kakani, N. K. 2004. Molecular and biochemical characterization of viral and vector components required for Cucumber necrosis virus transmission. Ph.D thesis. University of British Columbia, Vancouver, BC.  8.  Karniely, S., and O. Pines. 2005. Single translation--dual destination: mechanisms of dual protein targeting in eukaryotes. EMBO Rep 6:420-425.  9.  Katpally, U., K. Kakani, R. Reade, K. Dryden, D. Rochon, and T. J. Smith. 2007. Structures of T=1 and T=3 particles of Cucumber necrosis virus: evidence of internal scaffolding. J Mol Biol 365:502-512.  10.  Peeters, N., and I. Small. 2001. Dual targeting to mitochondria and chloroplasts. Biochim Biophys Acta 1541:54-63.  180  11.  Raghavendra, A. S., and K. Padmasree. 2003. Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation. Trends Plant Sci 8:546-553.  12.  Robbins, M. A. 2000. Molecular characterization of the interaction between Cucumber necrosis virus and zoospores of the fungal vector Olipidum bornovanus. Ph.D. thesis. University of British Columbia, Vancouver, BC.  13.  Robinson, I. K., and S. C. Harrison. 1982. Structure of the expanded state of Tomato bushy stunt virus. Nature 297:563-568.  14.  Rochon, D., K. Kakani, M. Robbins, and R. Reade. 2004. Molecular aspects of plant virus transmission by olpidium and plasmodiophorid vectors. Annu Rev Phytopathol 42:211-241.  15.  Satheshkumar, P. S., G. L. Lokesh, M. R. Murthy, and H. S. Savithri. 2005. The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids. J Mol Biol 353:447-458.  16.  Silva-Filho, M. C. 2003. One ticket for multiple destinations: dual targeting of proteins to distinct subcellular locations. Curr Opin Plant Biol 6:589-595.  17.  Timmins, P. A., D. Wild, and J. Witz. 1994. The three-dimensional distribution of RNA and protein in the interior of Tomato bushy stunt virus: a neutron lowresolution single-crystal diffraction study. Structure 2:1191-1201.  18.  Willits, D., X. Zhao, N. Olson, T. S. Baker, A. Zlotnick, J. E. Johnson, T. Douglas, and M. J. Young. 2003. Effects of the Cowpea chlorotic mottle bromovirus beta-hexamer structure on virion assembly. Virology 306:280-288.  181  19.  Xiang, Y., K. Kakani, R. Reade, E. Hui, and D. Rochon. 2006. A 38-amino-acid sequence encompassing the arm domain of the Cucumber necrosis virus coat protein functions as a chloroplast transit Peptide in infected plants. J Virol 80:7952-7964.  182  APPENDICES Appendix A: Local lesion analysis of two CNV CP arm mutants  Average lesions per le a  300 250 200 150 100 50 0 WT  2ng/leaf  0.667ng/leaf  0.22ng/leaf  Virion concentrations  C p(B-)'  Average lesions per leaf  300 250 200 150 100 50 0 WT Cp(B-)'  25ng/leaf  12.5ng/leaf  6.125ng/leaf  Virion RNA concentrations  Figure A.1 Local lesion analysis of two CNV CP arm mutants. (A) Equal concentrations of wild-type (WT) CNV or coat protein β-mutant [Cp(β-)] particles were inoculated onto leaves of Chenopodium quinoa, and the number of local lesions was determined at 4 days post-inoculations (dpi). Two independent experiments were conducted, using two plants for each treatment and four leaves per plant. (B) As in panel A except that equal concentrations of purified virion RNA were used. Pair-wise t-tests for each concentration for each treatment were conducted. Significantly fewer local lesions were produced by Cp(β-) particles than by WT CNV at concentrations greater than or equal to 0.667 ng/leaf (p≤0.05). No significant differences in the number of local lesions were found when virion RNA was used as the inoculum (p≤0.05). 183  Appendix B: The effect of calcium on interactions between Cucumber necrosis virus and Olpidium bornavanus zoospores B.1 Introduction In vitro virus studies have shown that calcium ions dissociate from virus particles in an alkaline pH environment and that this is concomitant with an increase in particle size (i.e., swelling) due to a shifting of CP structural components (8). The conformational change of the CNV particle required for efficient attachment to zoospores of its fungal vector appears to be similar to the particle change during the swelling process (3). It has been proposed that the swelling of CNV particle during zoospore attachment results in the extrusion of the interiorly residing N-terminal R/arm regions of the CP beyond the capsid shell and subsequent insertion into the zoospore plasmalemma (9). It is well-known that various chytrid zoospores require calcium for motility, germination and sporulation (1, 11). Together, the observations have resulted in the hypothesis proposed here that calcium is released by CNV and is subsequently sequestered by zoospores during attachment of virus to its vector and that this local increase in calcium ion concentration may affect zoospore motility while promoting CNV disassembly. This could, therefore, be mutually beneficial for virus transmission and zoospore infection of roots. This appendix describes a preliminary study conducted to assess the role of virus particle calcium ions in zoospore-mediated virus transmission. Confocal analysis was conducted to visually assess fluorescently-labelled calcium uptake by zoospores. Transmission analyses were conducted to assess the ability of the zoospores to transmit virus in the presence of exogenously added calcium (as an alternative calcium source for 184  zoospores). In addition, two calcium membrane blockers (to inhibit calcium uptake by zoospores) were used to determine if CNV transmission is associated with uptake of calcium by zoospores.  B.2 Materials and methods B.2.1 Virus purification Purified wild type CNV particles were obtained by a differential centrifugation method previously described (4) (see Section 2.2.1).  B.2.2 Maintenance of O. bornavanus cultures Olpidium bornavanus was maintained on cucumber roots (Cucumis sativus cv. Poinette 76) as described in Robbins et al. (7) (see Section 2.2.9).  B.2.3 Confocal microscopy Five hundred nanograms of CNV particles or a solution of 50 mM of calcium chloride were incubated in the dark with 1 μM Fluo-4 (Invitrogen, Molecular Probes, Carlsbad, CA) in a volume of 500 μl in 50 mM NaPO4 (pH 7.6) for 20 min at RT. These mixtures were then added to a 500 μl mixture of O. bornovanus zoospores in 50 mM NaPO4 (pH 7.6). Fluo-4 is a dye which will fluoresce when bound to calcium ions. Fluo4 uptake by zoospores was observed using a Leica SP2-AOBS confocal microscope with a 63X water immersion objective. An excitation wavelength of 480 nm was used.  185  B.2.4 Fungus transmission assay Purified CNV virions were tested for transmission by O. bornovanus zoospores as previously described (4). Virus transmissibility was analyzed using the following treatments of zoospores: (i) addition of calcium chloride to final concentrations of 0, 25, 50, 100, 200 and 500 mM; and (ii) addition of calcium channel blockers, using either lanthanum chloride with concentrations of 0, 2, 3 and 4 μM or Verapamil® with concentrations of 0, 10, 20 and 30 μM. Calcium chloride and the calcium channel blockers were added to the zoospore suspension prior to the addition of virus. After a period of 10 min, CNV was added and incubated with zoospores for 15 min prior to inoculation of cucumber seedlings. Calcium chloride treated zoospores were inoculated onto eight plants and Verapamil® treated zoospores were inoculated onto 10 plants per experiment; these experiments were replicated twice for each treatment. Treatments with lanthanum chloride were conducted using two replicates of eight plants each and one replicate of nine plants.  B.3 Results and discussion Virus particle expansion which is believed important for fungus transmission of CNV (3) occurs when calcium ions are removed in vitro from the virus surface. However, the fate of CNV calcium ions and the means by which they are removed during fungus transmission is unknown. I hypothesize that CNV surface calcium ions are sequestered by zoospores of O. bornovanus during virus attachment. Calcium uptake by zoospores was visualized using confocal microscopy and Fluo-4, a dye that fluoresces when bound to calcium ions. As shown in Fig. B.1,  186  (A)  (B)  No calcium treatment control  50 mM Calcium  Increasing fluorescence intensity for (B)  500 ng CNV particles  Fluo-4 fluoresence  Fluorescence intensity image  Figure B.1 Confocal images of O. bornovanus zoospores treated with Fluo-4 only (in NaPO4 buffer), Fluo-4 labelled calcium or Fluo-4 labelled CNV particles. (A) Confocal images of zoospores incubated with no calcium treatment, 50 mM of calcium chloride or 500 ng of CNV. (B) Confocal images of (A) showing the relative fluorescent intensities for each treatment. The range of fluorescent intensities is indicated on the right.  187  fluorescence can be observed within zoospores incubated with both Fluo-4 labelled calcium and CNV virions treated with Fluo-4 when compared with Fluo-4 only treatment. In these two treatments (with calcium or with CNV), Fluo-4 fluorescence is observed with increasing intensity toward the interior of the zoospore body (Fig. B.1). Preliminary observations of the fluorescence intensities found in zoospores subjected to the two treatments appear to be similar, suggesting that it is possible that CNV calcium ions are being taken up zoospores. Verification of this finding requires further experimental analyses. This would also indicate that it may be possible to use confocal microscopy to visually monitor calcium uptake by zoospores. Calcium was added to zoospores to assess its role as a competitive inhibitor of CNV transmission. My hypothesis is that excess available calcium would outcompete uptake of CNV calcium by the zoospores. Lack of calcium removal from the viral surface would result in impaired virus transmissibility. Zoospores of O. bornovanus exposed to calcium concentrations of less than 100 mM showed no difference in transmission efficiency compared with 0 mM calcium (Fig. B.2). At calcium concentrations greater than 100 mM, a decrease in virus transmission efficiency was observed. It is noted that at the higher calcium concentrations, cucumbers seedlings displayed symptoms of leaf wilt and chlorosis (data not shown). Thus this method of examining the effects of calcium on zoospore transmission efficiency may have complicating factors that make it difficult to draw conclusions. Lanthanides are effective competitors of calcium uptake at the cellular level since the lengths of their ionic radii are similar to that of calcium, which enables them to compete with calcium ions in physiological systems (6). Lanthanides competitively  188  Transmission efficiency (%)  15/16  100 90  15/16  13/16  80 70 60  6/16  50 40 30 20 10 0  01  2 25  3 50  4 100  0/8  0/8  5 200  6 500  Calcium concentrations (mM) Figure B.2 Effect of calcium on transmission efficiency of CNV particles. WT CNV particles (1 μg) were incubated with the indicated calcium concentrations of CaCl2 along with 10 ml of zoospores (1 X 106 zoospores/ml). After 15 min, the mixture was poured into pots containing cucumber seedlings. Five days later, roots of the seedlings were tested for the presence of CNV by DAS-ELISA using polyclonal antisera raised to CNV particles. The percentage of pots showing transmission for each virus is indicated on the y-axis. The numbers on the columns indicate the number of pots showing transmission versus the number of pots inoculated with virus/zoospore mixtures (+S.E.). The data represent a compilation of two separate experiments with two replicates per experiment.  189  block many types of voltage-gated calcium channels in cells from many animal tissues and organs (10). Lanthanide ions have been shown to be antagonists of type T calcium channels which are a subtype of voltage-gated channels (5). Lanthanum chloride was added to virus-zoospore suspensions to assess whether calcium ions are important for virus transmission. It was found that at increasing concentrations of lanthanum chloride, virus transmission efficiency decreased (Fig. B.3., A). However, the basis for decreased transmission could be due to a loss in zoospore motility. Verapamil® showed similar transmission efficiencies at all concentrations used (Fig. B.3., B). Verapamil® is a distinct type of channel blocker and thus may not be as competitively competent as lanthanum chloride to block the calcium channels of the zoospore membrane (1). If virus particle swelling relies on zoospores to sequester CNV calcium ions during vector attachment, decreased transmission efficiency may be due to an inability to take up these virus-associated calcium ions by zoospore in the presence of channel blocker, lanthanide chloride. Alternatively, calcium channels blocked by lanthanide chloride are known to alter swimming patterns of fungi (2) and may have negatively affected virus transmission. Further experimental analyses would be required to determine the role of virus calcium ions in virus transmission. Determination of the mechanism associated with particle swelling is significant to our understanding of virus transmission and will contribute to the continuing goal in the Rochon lab to study virus-vector interactions.  190  % Transmission efficiency  (A) Lanthanum chloride (μM) 100  24/25  90 80 70 60  13/25  50 40 30 20  3/25  10  1/25  0 2 01 2 33 44 Lanthanum chloride concentrations (μM)  (B) Verapamil (μM) % Transmission efficiency  100 90  16/20  17/20  18/20  19/20  80 70 60 50  1  40 30 20 10 0 1  0  2  3  4  10 20 30 Verapamil® concentrations (μM)  Figure B.3 Effect of calcium channel blockers on transmission efficiency of CNV particles. Virus transmission is as described in Fig. B.2., except that different concentrations of channel blockers, Lanthanum chloride (A) and Verapamil® (B) were added to the virus-zoospore mixtures. The percentage of pots showing transmission is indicated on the y-axis. The numbers on the columns indicate the number of pots showing transmission versus the number of pots inoculated with virus/zoospore mixtures (+S.E.). The data represent a compilation of three separate Lanthanum experiments and of two separate Verapamil® experiments.  191  B.4 References 1.  Donaldson, S. P., and J. W. Deacon. 1993. Changes in motility of Phythium zoospores induced by calcium and calcium-modulating drugs. Mycol Res 97:877883.  2.  Hill, A. E., D. E. Grayson and J. W. Deacon. 1998. Suppressed germination and early death of Phytophthora infestans sporangia caused by pectin, inorganic phosphate, ion chelators and calcium-modulating treatments. Eur J Plant Pathol 104:367-376.  3.  Kakani, K., R. Reade, and D. Rochon. 2004. Evidence that vector transmission of a plant virus requires conformational change in virus particles. J Mol Biol 338:507-517.  4.  Kakani, K., J. Y. Sgro, and D. Rochon. 2001. Identification of specific Cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment. J Virol 75:5576-5583.  5.  Mlinar, B., and J. Enyeart. 1993. Block of current through T-type calcium channels by trivalent metal cations and nickel in neural rat and human cells. J Physiol 469:639-652.  6.  Palasz, A., and P. Czekaj. 2000. Toxicological and cytophysiological aspects of lanthanides action. Acta Biochim Pol 47:1107-1114.  7.  Robbins, M. A., R. D. Reade, and D. M. Rochon. 1997. A Cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain. Virology 234:138-146.  192  8.  Robinson, I. K., and S. C. Harrison. 1982. Structure of the expanded state of Tomato bushy stunt virus. Nature 297:563-568.  9.  Rochon, D., K. Kakani, M. Robbins, and R. Reade. 2004. Molecular aspects of plant virus transmission by olpidium and plasmodiophorid vectors. Annu Rev Phytopathol 42:211-241.  10.  Wadkins, T., J. Benz, and W. Briner. 1998. The effect of lanthanum administration during neural tube formation on the emergence of swimming behavior. Metal Ions Biol Med 5:168-171.  11.  Warburton, A. J., and J. W. Deacon. 1998. Transmembrane Ca2+ fluxes associated with zoospore encystment and cyst germination by the phytopathogen Phytophthora parasitica. Fungal Genet Biol 25:54-62.  193  

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