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Subcellular localization of tobacco MAPKs Clegg, Alana 2004

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SUBCELLULAR LOCALIZATION OF TOBACCO MAPKS by Alana Clegg B . S c , Carleton University, 2001 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES Agricultural Science We accept this thesis as comforming to the required standard T H E U N I V E R S I T Y OF BRIT ISH C O L U M B I A Oct 2004 THE UNIVERSITY OF BRITISH COLUMBIA FACULTY OF G R A D U A T E STUDIES Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. RlfUVJrX dlBsGr Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: Degree: Year: 21)0^ Department of fifljf (CUITIKAA StiCyiCflS The University of British Columbia Vancouver, BC Canada grad.ubc.ca/forms/?formlD=THS page 1 of 1. last updated: 20-Jul-04 A B S T R A C T : Plants effect intracellular changes in response to a variety of external stimuli. Harpin, a bacterial elicitor from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola, and megaspermin, an oocyte elicitor secreted by Phythophtora megasperma H20, are both known to elicit a hypersensitive response (HR) i i i tobacco. These elicitors also induce phosphorylation of a 48 kDa protein and a 46 kDa protein that have been identified as the Salicylic A c i d Induced Protein Kinase (SIPK), and Wound Induced Protein Kinase (WIPK) mitogen activated protein kinases ( M A P K s ) respectively. Previous work has shown that transient over-expression of S IPK causes lesion formation in tobacco leaves, suggesting a possible role for S IPK in regulating tobacco H R signaling. Extracellular Regulated Kinase (ERK) , a mammalian homologue to S IPK and WIPK, has been shown to localize to different parts of the cell in response to a variety of stresses. The objective of this study was to determine the subcellular localization of the tobacco M A P K s , S IPK and WIPK, in response to challenge by harpin and megaspermin. Using both S I P K - G F P proteins and in situ immunolabeling of S IPK and WIPK , it was determined that the tobacco M A P K s were localized predominantly in the cytoplasm, with some nuclear localization. Upon challenge with harpin and/or megaspermin the phosphorylated forms of the probed M A P K s showed strong nuclear localization. In elicitor induced tobacco cultures, immunolabeling results showed strong nucleolar localization within the nucleus. These results are consistent with the model that activation of ERK-homologues by intracellular effectors leads to translocation of the activated M A P K from the cytoplasm to the nucleus, most l ikely to effect transcriptional regulation of stress-related genes. i i i Table of Contents: Tit le. . i Abstract ii Table of Contents iv List of Tables and Figures • vii Abbreviations ix Acknowledgements xi CHAPTER 1: General Introduction 1 1.1: Studying Signal Transduction 1.1.1: Signal Transduction and MAPK cascades 1 1.1.2: MAPK Structure and Function 3 1.1.3: Eukaryotic MAPK Families 6 1.1.4: Plant MAPK Families 7 1.1.5: Approaches to Analysis of MAPK Pathways 10 1.1.6: Hypersensitive Response 11 1.1.7: MAPK Elicitors 12 1.2: Studying Subcellular Localization of MAPKK and MAPK 1.2.1: Subcellular Localization 15 1.2.2: Subcellular Localization of ERK ....15 1.2.3: Methods for Determining Subcellular Localization 18 CHAPTER 2: SIPK:GFP Fusion Protein 20 2.1: Introduction to GFP 2.1.1: GFP 20 2.1.2: GFP Fusion Proteins 20 2.1.3: GFP Expression In Planta 21 2.2: Materials and Methods 23 2.2.1: Plant Growth Conditions. 23 2.2.2: Bacterial Growth Conditions. -....23 2.2.3: Restriction Digestions 23 2.2.4: Strategy for Fusion Protein Construction 24 2.2.5: Cloning of SIPK 24 2.2.6: Cloning of GFP 27 2.2.7: Construction of the Fusion Protein 28 2.2.8: Construction of the Fusion Protein Linker 31 iv 2.2.9: Yeast Vector Construction 32 2.2.10: Yeast Transformation.... 33 2.2.11: Expression in Yeast 34 2.2.12: Binary Vector Construction 34 2.2.13: Cloning of SD?K:GFP with Linker into pCAMBIA 1300 + MCS 36 2.2.14: Plant Transformation using Agroinfiltration 36 2.2.15: Infiltration of Agrobacterium for Transient Expression In Planta 37 2.3: Results 39 2.3.1: Cloning of SIPK 39 2.3.2: Cloning of GFP 39 2.3.3: Construction of the Fusion Protein 41 2.3.4: Fusion Protein Linker.. 42 2.3.5: Yeast Vector Construction 43 2.3.6: Expression in Yeast ...43 2.3.7: Binary Vector Construction 46 2.3.8: Cloning Construct into Binary Vector 47 2.3.9: Plant Transformation using Agroinfiltration 47 2.4: Discussion 50 CHAPTER 3: Immunolabelling of Tobacco MAPKs 56 3.1: General Introduction to Immunolabelling 3.1.1: Immunolabelling 56 3.1.2: Fluorochromes and Fluorescence 56 3.1.3: Fixation 56 3.1.4: Digestion/Removal of Cell Wall 57 3.1.5: Immunolabelling of SIPK and WIPK 58 3.2: Materials and Methods 3.2.1: Tobacco Seedling Growth and Treatment with Harpin and Megaspermin 59 3.2.2: Cell Culture Growth and Treatment with Harpin and Megaspermin 59 3.2.3: Fixation of Samples in Paraformaldehyde 59 3.2.4: Digestion/Removal of Cell Wall 60 3.2.5: Immunolabelling 3.2.5.1: Primary Antibodies 60 3.2.5.2: Secondary Antibodies 60 3.2.5.3: Controls 61 3.2.5.4: Immunolabelling Protocol 61 3.2.7: Quantification 63 v 3.3: Results 63 3.4: Discussion 79 Immunolabelling Troubleshooting 91 CHAPTER 4: General Discussion 94 APPENDICES 99 BIBLIOGRAPHY 104 vi LIST OF TABLES AND FIGURES Chapter 1 Figure 1.1: Atomic structure of the M A P K kinase E R K 2 at 2.3 A resolution. 4 Figure 1.2: 'Group A ' plant M A P K s , showing common domains 9 Table 1.1: Plant M A P K Activation 14 Table 1.2: Subcellular Localization of E R K and E R K homologues 17 Chapter 2 Figure 2.1: Schematic of Amplif icat ion of S IPK and mGFP5 into T O P O T A 2.1 Vector 29 Figure 2.2: Strategy of S IPK-GFP Fusion Construction and Use 30 Figure 2.3: Transfer of 35S Driven M C S f rompRT 101 to p C A M B I A 1300 35 Figure 2.4: T O P O T A 2.1 Vector Containing SIPK, and mGFP5 Fragments for Ligation 40 Figure 2.5: Colony P C R of the Fusion of S I P K G F P into the T O P O T A 2.1 Cloning Vector 41 Figure 2.6: P C R Confirmation of Insertion of Linker between S IPK and G F P in the T O P O T A 2.1 Cloning Vector 42 Figure 2.7: Insertion of S I P K G F P Fusion Sequence into the p Y E S T O P O T A 2.1 Yeast Expression Vector 43 Figure 2.8: Expression of S IPK-GFP Protein with Inserted Linker in Yeast Induced with Galactose 45 Figure 2.9: Digest Profile for the Insertion of the 35S Driven M C S from pRT 101 to p C A M B I A 1300 46 Figure 2.10: Insertion of SIPK: G F P with Linker into the Binary Vector p C A M B I A + M C S . . , 47 Figure 2.11: Agfobacterium Colony P C R of Putative S I P K G F P Fusion Protein Construct Insertion 48 Figure 2.12 a,b: Transient Expression by Agro-Infiltration of the SIPK: G F P with Linker Fusion Protein in Tobacco Leaves 49 Table 2.1: Primer Sequences .38 Table 2.2: Vectors 38 Chapter 3 Figure 3.1: Immunolabeling Control Samples of Fixed Suspension Culture Wi ld Type Tobacco Cells 65 Figure 3.2: A n t i - E R K Immunolabeled Untreated and 30 min Harpin-Treated W T Tobacco Cells 72 Figure 3.3: A n t i - E R K Comparison of Harpin and Megaspermin-Treated Cel l Culture., 73 Figure 3.4: A n t i - E R K Immunolabeled Untreated and 30 min Harpin-Treated O X Tobacco Cells 74 vi i Figure 3.5: A n t i - p E R K Immunolabeled Untreated and 30 min Harpin-Treated W T Tobacco Cells 75 Figure 3.6: An t i - pERK Immunolabeled Comparison of Harpin and Megaspermin-Treated Cultured cells 76 Figure 3.7: An t i - pERK Immunolabeled Untreated and 30 min Harpin-Treated O X : S IPK Tobacco Cells ~ 77 Figure 3.8: A n t i - F L A G Immunolabeled W T , O X - S I P K , and 30 min Harpin-Treated O X - S I P K Tobacco Cells 78 vi i i A B B R E V I A T I O N S : 2-4 D = 2-4 dichlorophenoxyacetic acid A t M P K 3 = Arabidopsis M A P K 3 A t M P K 6 = Arabidopsis M A P K 6 bp = base pair B S A = bovine serum albumin C a M V = cauliflower mosaic virus C D domain = common docking domain dATP = deoxyadenosine triphosphate dCTP = deoxycytidine triphosphate ddtbO = distilled de-ionized water D N A = deoxyribonucleic acid dNTPs = deoxynucleotide triphosphate D M S O = dimethyl sulfoxide D T T = dithiothreitol dTTP = deoxythymidine triphosphate E D domain = E R K docking domain E D T A = ethylenediaminetetracetic acid E G T A = ethylene glycol bis(2-aminoethyl ether)-N,N,N*N'-tetraacetic acid E M = electron microscopy E R K = Extracellular Regulated Kinase E R M K = Elicitor-Responsive M A P K (also PcMPK3) E V -~ empty vector F ITC = fluorescein-5-isothiocyanate F L A G = bacterial flagellin-derived epitope g = gravitational force GFP = green fluorescent protein G U S = beta-glucuronidase H A = Heme Agglutinin H E P E S = N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic) acid H P F = high pressure freezing H R = hypersensitive response h = hours J N K = c-Jun N-terminal protein kinase kb = kilobase kDa = ki loDalton L B = Luria-Bertani L M = light microscopy L U C = luciferase M A P K = Mitogen-Activated Protein Kinase (also M P K ) M A P K K = Mitogen-Activated Protein Kinase Kinase (also M K K ) M A P K K K = Mitogen-Activated Protein Kinase Kinase Kinase M C S = multiple-cloning site min = minutes M K K = see M A P K K M P K = see M A P K M S = Murashige and Skoog tissue culture medium m R N A = messenger ribonucleic Ac i d N E S = nuclear export signal N L S = nuclear localization site N m = nanometers N t M E K 2 = tobacco M E K 2 O.D.600 = optical density at 600 nm O R F = open reading frame O X = SIPK-overexpressing line P A M P = pathogen-associated molecular pattern P c M P K 3 = Parsley M A P K 3 (also E R M K ) P c M P K 6 = Parsley M A P K 6 P C R = polymerase-chain reaction pERK = phospho-ERK P K A = Protein Kinase A P R = pathogenesis related P R K K = pathogen-responsive M A P K K (alfalfa) P V P P = polyvinylpolypyrrolidone P V D F = polyvinylidine fluoride RFP = red fluorescent protein R N A = ribonucleic acid rpm = revolutions per minute R T = room temperature S A P K = Stress-Activated Protein Kinase SC medium = synthetic complete medium SDS = sodium dddecyl sulfate S I M K = Stress-Induced Protein Kinase S I M K K = Stress-Induced Protein Kinase Kinase S IPK = Salicylic acid-Induced Protein Kinase S I P K K = Salicyl ic acid-Induced Protein Kinase T A E = tris-acetate E D T A Taq = Thermus aquaticus TBST= tris-buffered saline tween T E Y = threonine, glutamate, tyosine T D Y = threonine, aspartic acid, tyrosine T X Y motif = threonine, " X " (any amino acid), tyrosine motif T M V = tobacco mosaic virus U = units U V = ultra violet Y F P = yellow fluorescent protein W I P K = Wound Induced Protein Kinase W R K Y = tryptophan, arginine, lysine, tyrosine X - G a l = 5 -bromo -4 -chloro-3- indolyl -Beta-D-galactoside A C K N O W L E D G E M E N T S : Some of the values in Table 1.0 were taken from correspondence with Dr. Marcus Samuel. A lso thanks to Dr. Marcus Samuel for use of the G S T - S I P K vector, and for help with cell culture maintenance and western analysis. Western blot of harpin activation of cultured cells was borrowed from the work of Hardy Hal l . The p C A M B I A 1300-MCS construct was obtained from Greg Lampard Thank-you to the U B C Bioimaging facility for training and use of the confocal microscope. Thank-you to my masters thesis committee; Dr. Brian El l is , Dr. Lacey Samuels and Dr. Jim Kronstad for their focused advice. Thank-you to Dr. Lacey Samuels and her lab for help with immunolabeling techniques, use of the fluorescent microscope, and for advice. A lso thanks to Geoff Wasteneys and his lab members for help with whole cell immunolabelling protocol manipulation. A special thank-you to Dr. Brian Ell is for his continual patience, guidance, and support. xi CHAPTER ONE: GENERAL INTRODUCTION 1.1: Studying Signal Transduction 1.1.1: Signal Transduction and MAPK cascades Life is modulated by signal cascades that relay information about environmental changes to affect intracellular responses which allow organisms to cope successfully with changing conditions. The necessary information is often relayed by signal transduction pathways that in eukaryotic cells can include protein kinases, phosphoprotein phosphatases, lipases, nucleotide exchange factors, ion channels, G proteins, l ipid kinases and transcription factors (Braun and Walker, 1996). Among the protein kinases, mitogen-activated protein kinases ( M A P K s ) are versatile signal transducing enzymes that are involved in multiple pathways. Their rapid and transient activation by various stresses strongly influence homeostasis, making M A P K s important proteins for study (Zhang and Klessig, 2001). . M A P K s are only one part of signaling pathways linking upstream receptors to downstream targets through upstream mitogen activated protein kinase kinase kinases ( M A P K K K s ) and mitogen activated protein kinase kinase ( M A P K K s ) (Jonak et al., 2002). M A P K signals are a phospho-transfer event between cognate upstream kinases and downstream targets (Tanoue et al., 2001). M A P K s are low abundance serine-threonine protein kinases that can mediate, transmit, and amplify cellular signals resulting in biochemical and physiological responses (Mitogen-activated protein kinase cascades in plants: a new nomenclature, 2002). M A P K / M A P K K / M A P K K K gene families are highly conserved in eukaryotes. M A P K sequences have been found to be highly conserved from primitive protozoans such as 1 Plasmodium to the more highly evolved metazoans (Kultz, 1998). Plants have more M A P K family members than animals. Genome analysis has revealed 14 family members in yeast, 25 in C. elegans, 47 in Drosophila, 47 in humans, and over 90 in Arabidopsis thaliana (Manning et al., 2002). O f the 90 or more identified arabidopsis M A P K signaling family members there are 20 M A P K s , 10 M A P K K S , and over 60 M A P K K K s (Jonak et al., 2002). This wide array of M A P K s supports recognition of a wide variety of stimuli, a versatility that is especially important for stationary organisms such as plants, which depend for their survival on efficiently sensing and responding to environmental change. In effecting appropriate responses, the plant's resources are wisely spent allowing the plant to compete for survival. The higher number of plant M A P K s might suggest plant M A P K s may play additional roles within the plant (Mizoguchi et al., 1997) or might reflect M A P K redundancy within plant species. Perhaps the M A P K number differences reveal fundamental differences in M A P K signaling mechanisms between plant and animal kingdoms. It is important to keep in mind that the record of genes revealed in genome analysis does not reveal the full diversity of the family, however, since alternative splicing of R N A transcripts from a single gene can yield different protein variants. Alternative splicing can generate dozens or even hundreds of different m R N A isoforms from a single transcript (Breitbart et al., 1987). It has been estimated that 42% of human genes may display alternative splicing (Kan et al., 2001). A notable example of experimentally identified M A P K family splice Variants is associated with the eukaryotic M A P K c-Jun N-terminal protein kinase (JNK). JNKs have been shown to be involved in mammalian embryonic development but also play a role in regulating programmed cell death. Alternative splicing of J N K transcripts creates at least ten 2 different J N K M A P K isoforms of 46-55 kDa that differ in their substrate affinities (Barr and Bogoyevitch, 2001). Alternative splicing might give more M A P K options in animal systems, explaining the lower number of identified animal M A P K s . Whether plant M A P K s routinely undergo alternative splicing has yet to be investigated, although the alternative splicing process does appear to operate in plant cells (Lazar and Goodman, 2000; Eckardt, 2002). 1.1.2: M A P K Structure and Funct ion A l l M A P K s have 11 common subdomains characteristic o f serine/threonine protein kinases (Hanks et al., 1988). Crystallographic analyses indicate that the active site of M A P K s is in an activation loop found between two of the lobes of the protein (Zhang et al., 1995). This activation loop contains a T X Y motif typical o f phosphorylation activation (Gartner et al., 1992). Ful l activation of M A P K has been shown to require the phosphorylation of both the threonine and tyrosine residues in this T X Y motif between kinase domains VI I and VIII, inducing conformational change that increases the accessibility of the substrate binding pocket (Mitogen-activated protein kinase cascades in plants: a new nomenclature, 2002) (Canagarajah et al., 1997). M A P K s are de-activated by the dephosphorylation of either one or both of the residues in the activation loop (Volmat and Pouyssegur, 2001). Figure 1.1 shows the atomic structure of the M A P K extracellular regulated kinase (ERK) 2. 3 Figure 1.1: Atomic structure of the M A P kinase E R K 2 at 2.3 A resolution. Faming Zhang, Arne Strand, David Robbins, Melanie H Cobb and Elizabeth J. Goldsmith. Nature, 367:704-711(1994) Once activated, M A P K s have the ability to activate other proteins.. The substrates of M A P K s are phosphorylated on serine or threonine residues that are immediately followed by a proline (Gonzalez et al., 1991) and the phosphorylation event must be efficient and specific enough to avoid unwanted cross-talk within M A P K cascades or with other signaling pathways (Tanoue et al., 2001). Cellular M A P K specificity is regulated by the presence of docking domains of M A P K modules, and through scaffold proteins (Tanoue et al., 2001). The C-terminus of a M A P K has the ability to bind to M A P K K s , MAPK-act ivated protein kinases, and phosphatases (Tanoue et a l , 2001) through an interaction domain called the common docking (CD) domain. The E R K docking (ED) site located on the opposite side from the active center, forms a groove with the C D domain, and site directed mutagenesis of either of the two domains caused a shift in M A P K substrate binding efficiency (Tanoue et al., 2001). Co-immunoprecipitation assays indicate that both the C D and E D contribute to protein interaction efficiency and specificity, but the C D and E D domains are not the only M A P K domains important for docking (Tanoue et al., 2001). It has been reported that multiple regions of the M A P K E R K 2 are important for determining the enzymatic specificity o f upstream kinases ( M A P K K s ) (Wilsbacher et al., 1999). Scaffold proteins are also thought to play a role in controlling M A P K specificity. Scaffold proteins are proteins that function to bring other proteins together for interaction. These proteins usually have multiple protein binding domains such as WD40 repeats. Scaffold protein interactions with M A P K s have been well characterized in yeast (Jonak et al., 2002), and function as docking proteins that can concentrate and localize M A P K components to designated subcellular locations (van Drogen and Peter, 2001). 5 Scaffold proteins have been identified in plants; however, no MAPK/sca f fo ld protein interactions have yet been reported. The yeast scaffold protein Ste5 was shown to interact in a conserved M A P K cascade required for mating in Sacharomyces cerevisiae. Ste5 is a zinc-finger-like protein that can form a multikinase complex with the M A P K K K Stel 1, the M A P K K Ste7, and the M A P K Fus3. Two-hybrid analysis indicates that Stel 1, Ste7, and Fus3 associate with different domains of Ste5, while K s s l associates with the same domain as Fus3, implying that Ste5 is capable of simultaneously binding a M A P K K K , M A P K K , and M A P K (Choi et al., 1994). 1.1.3: Eukaryot ic M A P K Famil ies Mammalian M A P K s can be divided into at least three main subfamilies; Jun kinases/stress-activated protein kinases ( INK /SAPKs ) , p38 M A P K s , and extracellular signal-regulated kinases (ERKs). The plant homologues of E R K 1 and 2 are the target proteins of this study, although JNKs and p38 M A P K s show similar patterns of signal , transduction. Stress-activated protein kinases (SAPK)/Jun N-terminal kinases (JNK) are mammalian M A P K s that are activated by a variety of environmental stresses, inflammatory cytokines, growth factors and G P C R agonists. Activated S A P K / J N K has been shown to translocate to the nucleus, where it regulates the activity o f several transcription factors such as c-Juh, A T F - 2 and p53 (Davis, 1999; Leppa and Bohmann, 1999). p38 M A P K s are mammalian M A P K s that are also activated by a variety of environmental stresses and inflammatory cytokines. p38 M A P K downstream targets 6 include HSP27 and M A P K A P - 2 as wel l as several transcription factors including A T F - 2 , Stat l , the Max /Myc complex, M E F - 2 , Elk-1 and (indirectly) C R E B via activation of M S K 1 (Tibbies and Woodgett, 1999). Mammalian cells contain two ERK-type M A P K s , E R K 1 and E R K 2 , which are also commonly called M A P K 42 and M A P K 44, respectively, based on their molecular weight. Mammalian E R K s are most notably involved in regulating gene transcription and cell cycle progression (Pearson et al, 2001). Some of the identified E R K regulated targets are receptor tyrosine kinases (Porter and Vaillancourt, 1998), integrins (Giancotti and Ruoslahti, 1999), and ion channels (Rane, 1999). Although the specific components of the signaling cascade vary with different stimuli, the basic architecture of the E R K activation pathway usually includes a set of adaptors (e.g. She, G R B 2 , Crk) that link the receptor to a guanine nucleotide exchange factor (e.g. Sos, C3G) that help to transduce the signal to small G T P binding proteins (Ras, Rap l ) . These, in turn, activate the core unit of the M A P K cascade composed of a M A P K K K (Raf), a M A P K K (MEK1/2) and M A P K (ERK) (Lewis et al., 1998). . In plant systems there is no evidence of p38 or J N K - M A P K homologues. Instead plants appear to have evolved a divergent and specialized set of E R K homologues. These plant E R K s have been found to play roles in the transduction of a wide variety o f stimuli, effecting physiological, hormonal and developmental responses that wi l l be discussed in greater detail below. 1.1.4: Plant MAPK Families The A. thaliana genome sequence contains 20 putative M A P K annotations. These M A P K s are divided into 4 groups (A-D) based on overall phylogenetic clustering. 7 Groups A , B , and C encompass M A P K s with a T E Y subtype of the T X Y motif and those with T D Y fall into group D (Mitogen-activated protein kinase cascades in plants: a new nomenclature, 2002). The E R K 1 and 2 plant homologues are members of Group A M A P K s , which are involved with environmental and hormonal response signaling. Figure 1.2 shows some of the group A members, and their common domains. Arabidopsis is used as a template for annotation of other plant genomes, although the homology and synteny are not perfect. Nevertheless, the high degree of sequence conservation within the M A P K family has allowed M A P K homologues in other species to be annotated by sequence similarity to arabidopsis. Plant M A P K homologues not only show considerable sequence similarity, but there is also strong evidence that they display similar signaling patterns. For example the arabidopsis and the tobacco E R K 1 and 2 homologues have both been shown to be activated by fungal elicitors (Desikan et al., 2001); (Asai et al., 2002); (Zhang et a l , 2000); (Lee et al., 2001). Table 1.1 lists specific elicitors and the respective activated E R K homologues in several plant species and humans. Many of the elicitors induce similar M A P K activation profiles in different species, indicating similar plant signaling patterns. 8 Figure 1.2: 'Group A ' plant M A P K s , showing common domains Mitogen-activated protein kinase cascades in plants: a new nomenclature (2002) Trends in Plant Science 7: 301-308 9 The known homologues of E R K 1 and 2 include A t M P K 6 and A t M P K 3 in arabidopsis, salicylic-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) in tobacco, stress-induced M A P K (SIMK) and stress-activated M A P K ( S A M K ) in alfalfa (Jonak et al., 2002), and P c M P K 6 and P c M P K 3 (Lee et al., 2004) (also called elicitor-responsive M A P K ( E R M K ) ) in parsley (Jonak et al., 2002). In non-plant studies, M A P K K s have been found to typically exhibit specificity for one M A P K , but some plant M A P K K s are able to activate multiple M A P K s (Jonak et al., 2002). For example, in tobacco, N t M E K 2 was found to activate both S IPK and W I P K (Yang et al., 2001), while the arabidopsis M K K 4 and M K K 5 can activate both M P K 3 and M P K 6 (Asai et al., 2002). Mammalian and plant M A P K K s may have different regulating roles and capabilities within their respective kinase signaling cascade. Yeast-two hybrid studies showed that S I M K interacted with S I M K K , a close homologue of A t M K K 4 / A t M K K 5 and N t M E K 2 (Kiegerl et al., 2000), but biochemical analysis showed that S I M K K activates both S I M K and M M K 3 in cells treated with an elicitor, while it activates only S I M K when the cells were challenged with salt stress (Jonak et al., 2002). In general, plant M A P K signaling cascades regulate input signals by means of cross-talking proteins to effect multiple output signals. 1.1.5: Approaches to Analysis of MAPK Pathways M A P K signaling cascades are complex networks with multiple stages of regulation. Dissection of these cascades can be achieved by in vitro methods such as protein purification, immunodetection, kinetic analysis, and biochemical assays/or in vivo techniques such as genetic disruption/mutagenesis, transcomplementatiori, two-hybrid 10 assays, and immunodetection and transgenic analysis (Tena et al., 2001). The most commonly used strategies are yeast two-hybrid studies, in vitro protein-protein interaction studies, and the use of transient expression of various combinations of M A P K pathway components in protoplasts (Jonak et al., 2002). In vivo study of M A P K activation and interactions is particularly difficult due to the low abundance of phosphorylated proteins at physiological levels. In a yeast two-hybrid system, using the tobacco M A P K S IPK as bait, L i u et al (2001) recovered M A P K K (S IPKK) as the only interactor. Although S I P K K showed both interaction and co-precipitation with SIPK, it was unable to phosphorylate S IPK in vitro (Liu et al., 2000). Studies in alfalfa showed similar precipitation and in vitro phosphorylation results between P R K K and the S IPK homologue, S I M K (Cardinale et al., 2002). These results indicate that although modular interactions can be detected, they are not good predictors of in vivo activity, and they are not necessarily the determinants of M A P K specificity. While upstream activators of both S IPK and S I M K have been identified, no downstream interactors are known. Possible reasons for this situation include a requirement for other protein partners, such as chaperone proteins and scaffold proteins as seen in yeast, or differing upstream signaling partners recognized by M A P K s or by other signaling molecules. Quick, transient activation of the substrate, or weak interaction between S I P K / S I M K and its substrate could also explain the failure to identify downstream targets. 1.1.6: Hypersensitive Response The hypersensitive response (HR) is a plant defense mechanism associated with resistance (Dangl et al., 1996). The mechanisms are not clearly understood but it is 11 known that after exposure to some plant stresses such as U V light, fungal or bacterial infection there is an accumulation o f specific pathogen resistance (PR) gene transcripts along with the formation of lesions at infection sites in the plant tissue. The lesions are somewhat isolated, or restricted from the surrounding healthy tissue (Hammond-Kosack and Jones, 1996). A long with lesion formation, a plant displaying a hypersensitive response typically develops immunity to subsequent attacks from a broad range of pathogens (Ryals et al., 1994). This broad-range pathogenic immunity is associated with disease resistance and serves to protect the plants' survival against ever-evolving pathogens. 1.1.7: MAPKs Elicitors M A P K cascades are transiently activated by a variety of input signals, including pathogen infection, elicitors, wounding, low temperature, drought, hyper and hypo osmolarity, high salinity, touch, and reactive oxygen species (Mitogen-activated protein kinase cascades in plants: a new nomenclature, 2002). A n elicitor is any applied biological agent which brings forth a response from the plant. One wel l studied pattern of M A P K induction is the pathogen-associated molecular pattern ( P A M P ) in Drosophila. P A M P s are triggers of signal transduction pathways responsible for transcriptional activation of pathogen defense genes. Conservation of the M A P K signal cascade in plants results in a native immune protection system similar to that of Drosophila against non-host disease. (Lee et al., 2004). Two P A M P - l i k e elicitors in tobacco and are Harpin and Megaspermin. , 12 Harpin is a protein from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola (harpin p s p h), which is known to elicit a H R and an accumulation of pathogen-related (PR)-gene transcripts in the non-host tobacco (Lee et al., 2001). Megaspermin is a glycoprotein ("elicitin") secreted by Phythophtora megasperma H20. There are three different forms (a, (3, y,) that are known to induce the H R response in tobacco (Bail l ieul et al., 2003). When applied to tobacco cultured cells, elicitins induce rapid protein phosphorylation, C a 2 + influx, extracellular and transient H 2 O 2 production (Bail l ieul et al., 2003). Many studies have examined M A P K signaling in plants in response to stimuli, such as challenge by bacterial or fungal attack, cold, drought, wounding, and osmotic stress. Table 1.1 lists specific treatments reported to activate E R K homologues in several species. Although all of the same stresses have not yet been tested in all the aforementioned species, it is clear that similar elicitors show similar M A P K activation patterns across species. Arabidopsis A t M P K 6 has been shown to be elicited by bacterial flagellin peptide (flg22) (Asai et a l , 2002). The Sheen group also showed that A t M K K 4 / A t M K K 5 and A t M P K 3 / A t M P K 6 were all activated by flagellin peptide (Asai et al., 2002), showing multiple M A P K activations from one elicitor. 13 Table 1.1: Plant MAPK Activation MAPK Organism Treatment Reference A t M P K 6 Arabidopsis thaliana -microbial elicitors -xylanase (fungal) - H 2 0 2 - K 0 2 -harpin (fungal) -bacterial flagellin - (Nuhse et al., 2000) - (Nuhse et al., 2000) - (Yuasae ta l . , 2001) - (Yuasa etal. , 2001) -(Desikan etal . , 2001) - (Asai et al., 2002) A t M P K 3 Arabidopsis thaliana - H 2 0 2 - flagellin (bacterial) - (Kovtun et al., 2000) - (Asai etal . , 2002) S IPK Nicotiana tabacum -salicylic acid - T M V -wounding -fungal elicitors -harpin -osmotic stress -hyperosmotic stress - U V - N O - (Zhang and'Klessig, 1997) - (Zhang and Klessig, 1998)a - (Zhang and Klessig, 1998)b - (Zhang and Klessig, 2000) - (Lee et al., 2001) - (Hoyos and Zhang, 2000) - (Cazale et al., 1999) - (M i les , 2002) - (Klessig et al., 2000) W I P K Nicotiana tabacum -wounding -osmotic stress - T M V - (Seo et al., 1995) - (Droillard et al., 2000) - (Zhang and Klessig, 1998)a S I M K Medicago truncatula (Alfalfa) -osmotic stress -salt stress -fungal elicitors - (Munn ik et al., 1999) - (Kiegerl et a l , 2000) -(Cardinale etal . , 2002) S A M K Medicago truncatula (Alfalfa) -wounding -cold -drought - (Bogreetal . , 1997) -(Jonak etal . , 1996) - (Jonak et al., 1996) Although there are many similarities among these elicitation patterns, there are also differences. In tobacco suspension cell culture, S IPK and W I P K showed phosphorylation activity in response to hypo-osmotic stress by in-gel kinase assays, and were identified by immunoblotting (Droil lard et al., 2000). However, the same group also showed that SIPK, but not WIPK , was activated by hyperosmotic stress (Cazale et al., 1999). Thus, many plant stresses affect the same downstream kinases but within different physiological contexts, how the activated kinases regulate specific cellular responses in each context is unknown. Likely regulatory factors include the duration of the M A P K 14 activation, the kinase subcellular localization, kinase accumulation and stimulus thresholds, and variability in protein-kinase/substrate interaction. 1.2: Studying SubceUular localization of MAPKK and MAPK 1.2.1: Subcellular Localization Subcellular localization refers to the location, or distribution of a given molecule within a cell. A given protein's subcellular localization can be dictated by information encoded within its sequence such as nuclear, chloroplast, mitochondrial, or ER-local izing domains. M A P K K localization is determined by its nuclear export signal (NES) in the hear N-terrriinal region (Fukuda et al., 1997), which renders it functionally a cytoplasmically localized protein. The 3D structural features of a protein, such as trans-membrane domains, can direct proteins into membranes. The distribution of charged residues can render the protein more hydrophobic or hydrophilic and thus help dictate its placement within the cell. Although many proteins have fixed positions within the cell, some proteins, upon activation, move from one part of the cell to another. 1.2.2: Subcellular Localization of ERK The mechanisms controlling the localization of M A P K s are not based on localization sequences. It is believed that M A P K is localized to the cytoplasm by specific interactions with its upstream M A P K K ( s ) , from which it dissociates upon activation of that M A P K pathway (Fukuda et al., 1997). M A P K K is thus a cytoplasmic anchoring protein for M A P K . Upon M A P K release from its cognate M A P K K in yeast, the M A P K activates a number of protein effectors located in different regions of the cell including the cytosol, cell periphery and the nucleus (Horgan and Stork, 2003). The subcellular localization of activated E R K s is important to cellular responses. ERK-mediated 15 activation occurs throughout the cell, but nuclear localization appears to be associated with longer term effects, such as involvement in cell growth and division (Marshall, 1995). It has been shown that under strong mitogens, E R K s move rapidly and persistently to the nucleus (Lenormand et al., 1993). It has also been suggested that protein kinase A (PKA) is required as wel l to import the phosphorylated form of E R K into the nucleus (Impey et al., 1998). When E R K is localized to the nucleus, it regulates gene expression by phosphorylating various transcription factors directly, such as p62 T C F / e l k - l (Seth et al., 1991) or indirectly, such as C R E B (Xing et a l , 1998). Various analytical techniques have been used to study E R K and its homologues in many different systems, using different M A P K stimuli, so it is difficult to derive a single model explaining E R K activation, localization, and use of effectors. There are, however, many activation, localization and effector similarities across these various studies. In humans, E R K 1 moves to the nucleus when stimulated by strong mitogens, and is associated with various nuclear targets, such as spindle poles and chromosome ends during mitosis (Table 1.2). However, E R K s have also been shown to localize to the mitochondria, and to autophagosomes. Plant E R K homologue studies have also revealed multiple areas for E R K subcellular localization, such as the actin cytoskeleton in growing root hair tips of alfalfa (Samaj et al., 2002). Treatment with a fungal elicitor in cultured parsley cells sent the E R K 1 homologue from the cytoplasm to the nucleus (Ligterink et al., 1997). 16 Table 1.2: Subcell ular Localization of ERK and ERK homologues Homologue Name Species/Genus Subcellular localization Inducer Reference E R K 1 Homo sapiens -cytoplasm to nucleus -mitochondria and autophagospmes -spindle poles -midbody -chromosome periphery -strong mitogens -Lewy Body disease -prophase, anaphase -cytokenesis -metaphase (Lenormand etal . , 1993) - (Zhu et al., 2002) -(Shapiro et al., 1998) -(Shapiro et al., 1998) - (Shapiro et al., 1998) A t M P K 6 Arabidopsis thaliana ? SIPK Nicotiana tabacum ? S I M K Medicago truncatula (alfalfa) -nucleus to root tip -cytoplasm to actin cytoskeleton (maybe vesicular) -root hair formation -treatment with jasplakinolide - (Samaj et al., 2.002) P c M P K 6 Petroselinium hortense (parsley) -cytoplasm to nucleus -Pep 13 -fungal elicitor - (Lee et al., 2004) - (Ligterink etal . , 1997) E R K 2 Homo sapiens -cytoplasm to nucleus -growth factor stimulation - (Horgan and Stork, 2003) A t M P K 3 Arabidopsis thaliana ? W I P K Nicotiana tabacum ? S A M K Medicago truncatula (alfalfa) ? P c P M K 3 ( E R M K ) Petroselinium hortense (parsley) -cytoplasm to nucleus Pep 13 - (Lee et al., 2004) S I P K / W I P K (undifferentiated) Nicotiana tabacum -cytoplasm and nucleus Seed germination - (Coronado etal . , 2002) 17 Compared to yeast and mammals, there has not been extensive research done in plants on subcellular localization of M A P K s , in part due to a lack of efficient tools for analysis in planta. 1.2.3: Methods of Determining Subcellular Localization There are many different methods to determine subcellular localization of proteins. Two of the most common methods include the use of reporter proteins and immunolabeling. Reporter proteins are proteins that one can detect easily, but are not normally present within the research organism. They can therefore allow one to monitor cellular events that would otherwise be difficult to observe. Some of the more commonly used reporter proteins in plant systems are beta-glucuronidase (GUS) , luciferase (LUC) and green fluorescent protein (GFP). G F P based detection allows the imaging of live cells and permits one to view dynamic changes within the cell. This is not achieved by indirect immunofluorescence. Immunofluorescence provides higher spatial resolution than fusion proteins (Arnirri, 1998), but requires specific antibodies and cellular fixation. Immunolabeling employs tagged antibodies (either primary or secondary) which can be visualized within the labeled tissue through microscopy. Immunolabeling allows the researcher to study native protein content, and localization, and yields better resolution of subcellular structures than live cell imaging. Immunolabelling is often used when fusion constructs to a given protein are either too difficult to construct, or feasibly not possible, or when there is an antibody available that is specific for their protein of interest. Some common antibody tags include covalently coupled fluorescent chemicals, gold particles (for E M ) , or enzymatic reporters such as hydrogen peroxidase. 18 The intent of this experiment was to examine the subcellular localization of the tobacco M A P K s , S IPK and WIPK, under normal and stressed conditions. Through the construction of a SIPK: G F P fusion construct for use in live cell image reporting, and immunolabelling of fixed cultured tobacco cells with an t i -MAPK antibodies, I w i l l examine the subcellular localization patterns of S J P K / W l P K . Subcellular localization of the M A P K s after elicitation with harpin and megaspermin w i l l be examined, and compared to untreated cells. 19 CHAPTER TWO: SIPK-GFP Fusion Protein 2.1: Introduction to GFP 2.1.1: GFP Green fluorescent protein (GFP) is a commonly used reporter for biological systems because it allows live, in vivo image reporting of one's protein of interest without destruction of the biological sample. This 27 kDa protein, originally isolated from Aequorea victoria, contains a chromophore that becomes excited through the absorption of photons in the 396-475 nm range, and emits green fluorescence at approximately 508 nm (Cubit, 1995; He im et al., 1994). The chromophore originates from a self-catalyzed covalent modification of the amino acid sequence Ser-Tyr-Gly at positions 65-67, which catalyze to form a/>-hydroxybenzylidene-imidazolidinone species. G F P is superior to many chromophores as it does not photobleach, a process where fluorochromes can get irreversibly damaged due to repeated excitation with an intensive light source, (van Drogen and Peter, 2004). Its small size (-27 kDa), stability and bright fluorescence make it an ideal reporter for biological systems. In addition to the native green version of G F P , site-directed mutagenesis has since been used to develop many effective G F P variants, including different chromophores. The spectral shifts of these chromophores yield different fluorescent colours, notably the red shift mutant (RFP) and yellow shift mutant (YFP) (Brandizzi et al., 2004). Use of different shift mutants of G F P permits multiple labeling within the cell, and provides an opportunity to optimize fluorescence results. 2.1.2: GFP Fusion Proteins By use of recombinant D N A manipulations, G F P can be fused to specific proteins making it possible to study subcellular localization of the chimeric protein in vivo. Both 20 C-terminal and N-terminal fusions have been used, and such flexibility is important since both the C and/or N-terminus of a given protein can possess essential binding sites or other important functions that could be affected by fusion to another protein. Similarly, attachment to G F P can potentially cause problems with proper folding or domain conformation. A recent study demonstrated that internal insertion of G F P within a chimeric protein could be used effectively in high throughput screening of plant proteins for subcellular location. 2.1.3: G F P Expression in Planta Expression of native G F P in arabidopsis was originally found to be unstable (Davis and Vierstra, 1998). Incorrect splicing was responsible for the protein instability, and optimization of the codon usage in the unstable region yielded a stable exogenous G F P , but with a weak fluorescent signal (Davis and Vierstra, 1998). mGFP5 is a specific version of the green G F P variant which has been shown to give good results in plant systems (Brandizzi et al., 2002; K i m et al., 2003; Sheahan et al., 2004). Strong expression of the green chromophore is important in plants because it is often necessary for the G F P signal to compete with the background autofluorescent signal from chloryphyll, xylem, lignin and other autofluorescent bodies present within the tissue under study. Expression of G F P in plant systems can be designed to be transient, or stable. For proteins whose over-expression may be lethal to the cell or for proteins that are rapidly degraded, transient expression is preferred. Transient expression allows fast and easy visualization o f results and is therefore often used as an indicator of gene transfer and 21 expression efficiency. Stable transgenics, on the other hand, allow for easily repeatable and long time-course experiments (Weld et al., 2001). Both transient and stable ectopically expressed G F P has been shown to be localized throughout the plant cell, with some stronger nuclear and vascular tissue fluorescence (von Arn im et al., 1998). These patterns might reflect G F P ' s small size (27 kDa) and the associated ease of diffusion into the nucleus, although it has also been suggested that GFP may contain a cryptic nuclear localization site (NLS) (von Arn im et al., 1998). Because of diffusibility, GFP-protein fusions must have a larger overall size than the size exclusion limit for the nucleus i f the construct is expected to report nuclear versus cytosolic localization (von Arn im et al., 1998). Large proteins cannot passively diffuse through the nuclear pore and must be actively transported in i f they are to carry out a function within the nucleus. G F P fusion proteins are used often when specific antibodies towards their protein are not available, or when cellular fixation processes disrupt the antibody's capability to recognize the protein. G F P is ideal for live cell imaging of the dynamics of intracellular movement, localization, and potential destruction of the protein. New constructs introduced to host species can, however, show variable expression levels of the fusion protein, which vary from native form expression levels. These different levels of expression can change the normal cellular dynamics of a cell, and give misleading imaging results. Other effects such as co-suppression can also cause undesired effects upon the existing signaling pathway which would give misleading results. Co-supression of a gene can occur when a related gene is expressed, or by the introduction of a related transgene (Pal-Bhadra et al., 1997). Homo logy-dependent gene silencing is a co-22 supression phenomenon in plants. Exogenously expressed genes introduced into a plant containing a homologous gene can cause not only the suppression of the transgene, but also suppression of the native gene (Meyer and Saedler, 1996). To monitor false localization results, GFP-fusion protein localization can be confirmed by immunolabeling, or by construct regulation by the protein's native promoters. 2.2: Materials and Methods 2.2.1: Plant Growth Conditions Nicotiana tabacum Xanthi nc seeds were sterilized using 50% JAVEX® bleach for 10 min, followed by 5 consecutive 10-min washes in sterile ddJ-^O. Seeds were plated on Vi strength M S plates and grown under controlled conditions (25/20 °C, 16-h-light/8-h-dark cycle). After 2 weeks, the seedlings were transferred to Magenta tubs containing V2 M S with no added sugar and grown under the same controlled environment conditions. 2.2.2: Bacterial Growth Conditions Bacterial cells were routinely cultured on Luria-Bertani (LB) medium solidified with 1.5% (w/v) bactoagar (Difco). Where required, media were supplemented with kanamycin (50 ug/mL) and 5-bromo-4-chloro-3-indolyl-Beta-D-galactoside (X-Gal) (40 mg/mL). Plates were incubated at 37 °C for 16-24 h. L iquid cultures of L B medium supplemented with the appropriate concentration of antibiotics were incubated aerobically with agitation for 12-18 h at 37°C. 2.2.3: Restriction Digestions D N A (1 ug) was added to 1 uL restriction enzyme, 5 uL 10X restriction buffer, and ddH20 to make 50 uL total volume, and mixed by pipetting. The digestion mix was 23 incubated at 37 °C for 1-3 h., depending on enzyme efficiency. Digestion was arrested by incubation at 50 °C for 5 min, and samples were stored on ice or at -20°C until further use. 2.2.4: Strategy for Fusion Protein Construction To construct a SIPK: G F P fusion protein that could be expressed in both yeast and plant systems, a strategy was devised using plasmids available in the lab. Both S IPK and GFP O P v F s were available on plasmids (Figure 2.1). For cloning purposes the T O P O T A 2.1 cloning vector was a good candidate to house the S IPK and G F P amplicons (Figure 2.1). To aid cloning, restriction enzyme target sequences were added to the S IPK and G F P primers. S IPK primers contained aXhol site on the forward primer and Noil site on the reverse primer. Similarly, a Notlsite was added to the G F P forward primer and an Xbal site added to the G F P reverse primer (Figure 2.1). The. NotI site on both fragments would link S IPK and G F P together. To keep GFP in frame when fused to SIPK, and extra base pair was added to the G F P forward primer. T O P O T A 2.1 cloning vector containing S IPK in the orientation that placed the 3' end of S IPK next to the Xbal site in the T O P O T A 2.1 cloning vector multiple cloning site (MCS) (Figure 2.2) could be digested with Not I and Xbal to accept the G F P digested fragment with NotI m&Xbal sticky ends. Once the two fragments were fused in the T O P O T A 2.1 cloning vector, the fragment could be digested out, or amplified with S IPK forward and G F P reverse primers to be integrated either in a yeast expression vector, or a binary vector for expression in planta (Figure 2.2). 2.2.5: Cloning of SIPK Cloning of the S IPK O R F into the T O P O T A 2.1 cloning vector strategy is outlined in Figure 2.1. S IPK was amplified by P C R from a GST-fusion expression plasmid 24 (provided by Marcus Samuel) using the S IPK primers listed in Table 2.1. The P C R amplification of S IPK was performed using H i F i Platinum Taq (Invitrogen) and Xhol SIPK forward and NotI reverse primers (Table 2.1). A 50 uL P C R reaction mixture included 5 uL 10X P C R amplification buffer, 2.0 uL M g S 0 4 , 2.5 uL 10 m M dNTPs, 2 (j.L of l O m M each of the forward and reverse primer, 1 u.L Platinum H i F i Taq, and ddH20 to make 50 pL. The P C R amplification program performed on a Biometra T-gradient thermocycler was set at 95 °C for 5 min, followed by 32 cycles of 95 °C for 30 sec , 55 °C for 30 sec, and 68 °C for 1 min, with a final elongation cycle at 68 °C for 10 min. The S IPK amplicon was ligated into the T O P O T A 2.1 vector as directed by the manufacturer. P C R product (2 pL) was mixed with 1 pL salt solution, 1 pL T O P O T A 2.1 vector and ddHaO in a total volume to 5 uL. The reaction was mixed gently, incubated at room temperature (RT) for 5 min, and 2 uL of the ligation product was used to transformlOO pL CaC^-competent D H 5 a E. coli cells. The transformation mixture was cooled on ice for 20 min, followed by heat shock treatment at 42 °C for 45 sec, then immediately placed on ice for 2 min L B medium (500 uL) was added to the transformation mix which was allowed to recover for 1 h with aerobic agitation at 120 rpm. 50 pL of the transformation mix was then plated on L B medium plates solidified with 1.5% agar (Difco) containing 50 pg/mL ampicil l in, and 40mg/mL X-ga l and incubated at 37 °C overnight. Putative transformants were initially identified by blue/white LacZ screening followed by colony P C R analysis. Candidate colonies were inoculated by sterile toothpick transfer into the P C R amplification reaction consisting of 5 uL 10X P C R amplification buffer, 2 uL 50 m M M g C l 2 , 2 pL 10 m M dNTPs, 1 uL each 25 of forward and reverse 10 m M S IPK primers, 1 uL Taq D N A polymerase, and d d H 2 0 to make 50 pL. The amplification program was performed on a Biometra T-gradient thermocycler as detailed above, and the products were examined by gel electrophoresis (0.8% agarose, I X T A E buffer). Plasmids were isolated from positive transformants using Wizard Prep Purification Ki t (Promega). Transformant cells were incubated aerobically overnight in 5mL L B medium supplemented with 50 ug/uL ampicil l in, harvested by centrifugation at 10,000 g for 2 min, and re-suspended in 300 uL 'Ce l l Resuspension Solution'. 'Ce l l Lysis Solution' (300 uL) was added, and the solution inverted 4 times, fol lowed by addition of 10 uL 'Alkal ine solution', and 10 min incubation at RT. 'Neutralization Solution' (300 pL) was added, mixed by inversion, then centrifuged at 10,000 g for 10 min to pellet cell debris and chromosomal D N A . The supernatant was then transferred to a Wizard kit column in a 2 mL collection tube, and spun at 10,000 g for 1 min. The elutate was discarded, 750 uL 'Wash Solution' was applied to the column, and the column was spun at 10,000 g for 2 min. The eluate was discarded, and a second wash with 250 pL 'Wash Solution' was applied to the column as described above. The column was spun at 10,000 g for 2 min and the column was then transferred to a fresh microfuge tube. The plasmid D N A was eluted from the column by the addition of 50 pL ddFbO followed by centrifugation at 10,000 g for 2 min. The eluate containing the plasmid D N A was stored on ice, or at -20 °C until further use. Plasmid concentration and quality was determined by comparison to a D N A mass ladder on a 0.8% agarose gel. Plasmid D N A from putative transformants was then analyzed by restriction digestion with NotI and Xbal. The digestion mix consisted of 5 uL 10X React®3 buffer, 1.5 pL 26 NotI restriction enzyme (Invitrogen), 1 [iLXbal restriction enzyme (Invitrogen), 1 pg plasmid D N A , and ddHaO to make 50 pL. The mix was incubated 4 h at 37 °C with occasional mixing, followed by centrifugation. One of the NotI sites was located on the insert, and a second NotI site, followed by an Xbal site, was located on the host T O P O T A 2.1 vector. Proper orientation of the insert was expected to show a digestion profile of -5 .0 kb vector plus the insert fragment and a small (-lOObp) fragment of the M C S . Improper orientation of the insert would give an -3 .9 kb vector product, and a -1 .2 kb insert fragment. 2.2.6: C lon ing of G F P The strategy for cloning of G F P into the T O P O T A 2.1 cloning vector strategy is shown in Figure 2.1. The D N A coding sequence for GFP was PCR-ampl i f ied from the plasmid p C A M B I A 1302, which contains the mGFP5 version of G F P . P C R amplification of G F P was performed using H i F i Platinum Taq (Invitrogen). A 50 pL P C R reaction consisting of 5 pL 10X buffer, 2.0 pL M g S 0 4 , 2.5 pL 10 m M dNTPs, 2 pL of lOmM each of the forward NotI GFP and reverse Xbal G F P primers (Table 2.1), 1 pL Platinum H i F i Taq, and ddH20 to 50 pL. The P C R amplification program on a Biometra T-gradient thermocycler consisted of 95 °C for 5 min, followed by 32 cycles of 95 °C for 30 sec, 55 °C for 30 sec, and 68 °C for 1 min, with final elongation cycle of 68 °C for 10 min. The G F P amplicon was digested witWXbal and NotI, and purified using a QIAquick® Spin Ki t (Q IAGEN) . Buffer P B was combined with the digested P C R amplicon in a 5:1 ratio, mixed gently, and applied to a QIAquick spin column in a 2 mL collection tube. The column was centrifuged at 10,000 g for 1 min. The flow-through was discarded, and 0.75 m L buffer P E was added to the column. The column was 27 centrifuged at 10,000 g for 1 min. The flow-through was discarded and the column centrifuged an additional min at 10,000 g. The column was transferred to a new microfuge tube, 50 pL dcfflbO was applied to the column, and the column was held for 3 min at R T before centrifugation for 1 min at 10,000 g. The eluate was stored on ice or at -20°C until further use. 2.2.7: Construct ion of the Fusion Protein The T O P O T A 2.1 vectors containing GFP and S IPK were both digested with NotI and Xbal to produce a linearized S I P K - T O P O T A 2.1 vector and a G F P fragment. Both fragments were excised from a 0.8% agarose gel in I X T A E buffer using a QIA®quick gel purification Kit . Gel fragments were weighed in micro fuge tubes, and 3 volumes buffer Q G was added to each gel fragment. The mixtures were incubated at 50°C for 10 min (or until the gel dissolved), and 1 gel-volume 100%> isopropahol was added to the sample. The solutions were gently mixed before transfer to QIAquick columns in 2 m l collection tubes and centrifuged at 10,000 g for 1 min. The flow-through was discarded and 750 pL buffer P E was added to the column. The column was centrifuged at 10,000 g for 1 min. The flow through was discarded and the column centrifuged an additional min. at 10,000 g. The column was placed in a new micro fuge tube, 50 pL ddFkO was applied to the column, and the column was incubated for 3 min at R T before centrifugation for 1 min at 10,000 g. The eluate was stored on ice or at -20 °C until further use. 28 Figure 2.1: Schematic of Amplification of SIPK and mGFP5 into TOPO TA 2.1 Vector Figure 2.1 depicts the amplification of SIPK and mGFP5 ORFs, and their insertion into the TOPO TA 2.1 vector. 29 SIPK G F P -Transfer to binary vector for expression in planta -Transfer to yeast vector for expression in yeast Figure 2.2: Strategy of SIPK-GFP Fusion Construction and Use Figure 2.2 shows the fusion of SIPK and G F P , as well as the addition of a linker fragment in the fusion zone. The S IPK :GFP fusion with linker can then be transferred to a yeast or plant binary vector for expression. 3 0 The concentration and quality o f the fragments were determined by comparison to a D N A mass ladder on a 0.8% agarose gel in preparation of ligation. A total of 1 ug total D N A (vector and insert) in a 3:1 insert to vector ratio was added to a 15 uL total volume reaction consisting of 3 pL 5 X ligase buffer, 1 pL ligase, 0.5uL 5mM A T P , and ddFkO to 15 L. The reaction was incubated overnight at 4 °C. The ligation product was transformed into CaCk-competent DH5a , plated on ampicil l in supplemented L B agar as described in section 2.2.2. Putative transformants were analyzed by colony P C R using S IPKF and G F P R primers, and colonies showing the expected ~2kb fragment were sent for sequencing to ensure the fusion protein sequence was contained and that there were no mismatches or frameshifts. 2.2.8: Construction of the Fusion Protein Linker After the SIPK: G F P fusion construct was completed and transferred into a binary vector, it was discovered that expression was not detectable by western analysis or by R T - P C R (data not shown), and it was postulated that the fusion protein was not folding properly. A linker fragment between the two proteins was constructed to widen the space between the two proteins to allow both to fold properly. An~100bp linker fragment was constructed by amplification of a 3 X H A tag from pKS/3S H A (Table 2.2) using the linker F and R primers with added NotI sites (Table 2.1) and P C R amplification conditions as previously described in section 2.2.5. Both the 3 X H A tag fragment and the T O P O T A 2.1 S IPK :GFP fusion vector were digested with NotI. 1000U ( l p L ) CIP (New England Biolabs) was added to 30 uL linearized fusion protein vector and incubated at 37 °C for 1 h to dephosphorylate the vector ends. D M S O was added to the linker fragment (to make 10% D M S O final concentration) to allow for better 31 agarose gel separation of the small linker fragment from primer dimers and undigested fragments of amplified 3 X H A tag linker sequence. The linker fragment was gel-purified on a 2% agarose gel in I X T . A . E buffer. The digested fusion protein vector was gel-purified on a 0.8% agarose gel as described in section 2.2.5. Concentration and quality of the linker and vector were determined by comparison to a mass ladder on a 0.8% agarose gel in preparation for ligation. The linker was ligated into the linearized, dephosphorylated fusion protein vector at a 3:1 insert to vector ratio. The ligation product was transformed into CaCk-competent DH5d , and plated on L B plates supplemented with ampicill in as previously described in section 2.2.2. Putative transformants were analyzed by colony P C R using S IPK F and G F P R primers (Table 2.1) and run on a 0.8% agarose gel as previously described in section 2.2.5. The fusion protein containing the linker fragment amplicon was run next to control SIPK: G F P fusion amplicons which did not contain the added linker. Amplicons exhibiting a fragment 100 bp larger than those without the linker were sent for sequencing. 2.2.9: Yeast Vector Construct ion The vector p Y E S 2.1 was used for the cloning and expression of the fusion construct in yeast. P C R amplification of the T O P O T A 2.1 cloning vector containing the SIPK-l inker-GFP was performed using H i F i Platinum Taq (Invitrogen) using 10 vcMXhoI SIPK forward and 10 mM Xbal GF? reverse primers (Table 2.1) as previously described in section 2.2.5. The amplification product was ligated into the p Y E S 2.1 vector as directed by the manufacturer, and as previously described for cloning into T O P O 2.1 T A cloning vector 32 (section 2.2.5). Candidate transformant plasmids were isolated using Wizard Prep Purification K i t and analyzed by colony P C R as previously described in 2.2.5 with the G A L 1 F and G F P R primers (Table 2.1) to confirm orientation of the insert. The desired orientation would read 5' to 3' of the 3 X H A tag. 2.2.10: Yeast Transformation Yeast Extract/Peptone/Dextrose (YPD) medium (10 mL) was inoculated with a colony of I N V S c l yeast cells and grown overnight at 30 °C with shaking at 150 rpm. Overnight cultures were diluted to an ODeoo of 0.4, pelleted at 250 rpm for 10 min, and re-suspended in 40 m L I X T E . Cells were pelleted at 2500 rpm for 10 mm, and suspended once more in 2 mL I X L iAc /0 .5X T E and incubated at R T for 10 min. Yeast suspension (100 pL) was mixed with l p g plasmid D N A , 100 pg sheared herring sperm D N A , 700 pL I X L i A c / 40% PEG-3350/ I X T E and incubated at 30 °C for 30 min. D M S O (88 uL) was added to the suspension, heat shocked at 42 °C for 7 min, then centrifuged for 10 sec. at 18, 000 rpm. The supernatant was removed, and the pellet was re-suspended in 1 mL I X T E and then re-pelleted as above. The washed pellet was re-suspended in 50 pL 1 X T E and plated on selective media containing no uracil (selected for by uracil phototrophy). The construct was isolated using a plasmid isolation kit (Q IAGEN) as directed by the manufacturer with the exception of an added glass bead step in the lysis step. Approximately 300 pL glass beads were added to l m L re-suspended yeast cells. Cells were shaken on at max speed for 3 intervals of 10 min at 4 °C. Colony P C R was used to confirm the presence of the fusion protein construct in p Y E S 2.1 as previously described in section 2.2.5 with S IPKF and G F P R primers (Table 2.1). 33 2.2.11: Expression in Yeast For construct expression, overnight cultures of positive transformants were grown in SC medium without uracil. The pYES TOPO 2.1 TA vector has zURAS gene, which produces uracil, acting as a selective marker in uracil-deficient media. Cultures were appropriately diluted to obtain 50 mL of O.D6oo= 4, and culture dilutions were centrifuged at 8,000 g for 10 min. The supernatant was discarded, and cells were re-suspended in 50 mL induction medium containing 2% galactose. Cells were harvested for time course expression samples at 0, 3, 8 and 16 h., and viewed on a Leica D M R r fluorescence microscope equipped with an FITC filter set (excitation 465-495 nm). The fluorescence microscope was equipped with a Q I C A M along with the program Openlab 3.0® for the viewing and interpretation of captured images. 2.2.12: Binary Vector construction The binary vector pCAMBIA 1300 vector did not have a 35S-driven promoter for the MCS. The 35S promoter and MCS from pRTlOl were therefore transferred to p C A M B I A 1300 as shown in Figure 2.3 The vector pCAMBIA 1300-MCS contained the restriction sites PstI, SphI, and Hindlll. pCAMBIA 1300-MCS, andpRTlOl (Table 2.2) were digested separately with Hindlllas previously described in section 2.2.3. Both digestion mixtures were run separately on a 0.8% agarose gel, and the Hindlll liberated 35S-MCS fragment from the pRTlOl vector and the linearized pCAMBIA 1300-MCS were gel-purified as previously described in section 2.2.5. The concentrations of the fragments were determined by comparison to a mass ladder on a 0.8% gel in preparation for ligation. The fragments were ligated in a 3:1 insert to vector ratio, and used to transform CaCk-competent DH5a 34 : Hint Sphl p*i; Xb*I Ban HI S M I 4-E(0RS Sit I / Eco K / Region between Hindlll sites added to p C A M B I A M C S hygromycin (RJ Xko I (6S33 CaMV35S polyA" T-Border (left) Sat n (6317) kanamycin (R) &:XI(S716) CaMV35S promoter Xko I (7929) pBR322 ori \ pBR322 bom M f I (3392) pVS1 rep Figure 2.3: Transfer of 35S Driven MCS from pRT 101 to pCAMBIA 1300 Figure 2.3 depicts the MCS portion between Hindlll sites in the vector pRTlOl that will be digested and inserted into a pCAMBIA 1300 vector which has had a large portion of its MCS removed. The new construct is entitled pCAMBIA + MCS 35 as previously described in section 2.2.5. Putative transformants were analyzed by restriction endonuclease digestion using Hindlll, Xhol, Xbal, BamHI, Sail, and PstI to identify the orientation of the 35-S driven M C S , and any residual restriction sites that may have been present in p C A M B I A - M C S . The new construct was named p C A M B I A 1300 + M C S . 2.2.13: Cloning SD?K:GFP with Linker into pCAMBIA 1300 + MCS The S I P K : G F P fusion with 100 bp linker and p C A M B I A 1300 + M C S were digested separately with Xbal and Xhol as previously described in section 2.2.3. The linearized p C A M B I A 1300 + M C S vector and 2 kb SIPK: G F P with linker fragment were gel-purified and their concentrations determined in comparison to a mass ladder bii a 0.8% gel in preparation for ligation as previously described in section 2.2.5. The two fragments were ligated in a 3:1 insert to vector ratio, transformed into D H 5 a cells, and plated on L B media containing 50 pg/mL kanamycin. Putative transformants were analyzed by colony P C R as previously described in section 2.2.5 using 35S forward, and GFP reverse primers (Table 2.1). 2.2.14: Plant Transformation using Agroinfiltration SIPK: G F P with 100 bp linker in p C A M B I A 1300+MCS, along with the controls of a 35S driven G F P in p C A M B I A ( p C A M B I A 1302), and an empty vector ( p C A M B I A 1300 + M C S ) were transformed into either Agrobacterium transfumerens strain G V 3101, or strain E H A 101. Vector D N A (1 pg) was placed on 200 pL CaCb.competent Agrobacterium. The tubes were immediately placed in liquid nitrogen, followed by incubation at 37 °C for 5 min. L B (1 mL) was added to the transformation mixture, and the tubes were incubated at 28 °C with shaking at 180 rpm for 3 to 4 h. Cells were 36 pelleted at 12,000 g for 2 min, and re-suspended in 200 uL L B . Samples were plated on selective L B plates as previously described in section 2.2.2 for DH5a . Four-week-old Xanthi tobacco plants grown in a sterile environment on XA strength M S medium were routinely used as infiltration hosts. Leaf discs cut from leaves of infiltration hosts with sterile scalpels were dipped for 1 min into Agrobacterium cultures grown to an ODeoo of 0.8. Leaf discs were then rinsed with fresh M S medium, placed on parafilm-sealed plates of shoot induction medium (see Appendix 1) with no antibiotics, and incubated at R T in the dark for 2 days. Leaf discs were then transferred to shoot induction medium supplemented with antibiotics (Appendix 1) and grown at 25/20 °C, 16-h-light/8-h-dark cycle. New shoots were transferred to fresh medium and grown under the same conditions. When shoots were 2-3 leaves in size, they were transferred to root induction medium (Appendix 1). 2.2.15: Infiltration of Agrobacterium for Transient Expression in planta Agrobacterium strains containing the constructs S IPK :GFP with linker in p C A M B I A 1300+MCS, p C A M B I A 1302, and the empty vector control p C A M B I A 1300 + M C S were grown to an O D 6 0 0 = 0.8. The cultures were transferred to V2 M S medium, and infiltrated into host tobacco leaves. Tissue for protein and R T - P C R analysis was harvested and frozen in l iquid nitrogen after 2 days, and plants were monitored for lesion formation for 3-5 days. 37 Table 2.1: Primer Sequences Primer Name Primer Sequence XhoISWKF (forward) 5'- C A C T C G A G A T G G A T G G T T C T G G T C A G C A G A C G G A C A - 3 ' NotI S I P K R (reverse) 5'- C A G C G G C C G C C C A T A T G C T G G T A T T C A G G A T T A A A T - 3 ' NotI GFPF (forward) 5'- C A G C G G C C G C A T G G T A G A T C T G A C T A G T A A A G G A G A - 3 ' Xbal G F P R (reverse) 5 ' - C A T C T A G A T C A C A C G T G G T G G T G G T G G T G G T G G C T A - 3 ' NotI LinkerF (forward) 5 ' - G G C G G C G G C C G C G G A T C C A C T A G T T A C C C A T A C G A T G T T C C T G A C T A T G - 3 ' NotI L inkerR (reverse) 5'- A A G C G G C C A G C G T A A T C T G G A A C G T C - 3 ' 35S Forward (forward) 5 ' - A T G A C G C A C A A T C C C A C T - 3 ' G A L 1 (forward) (Invitrogen, provided with p Y E S 2.1 kit) Table 2.2: Vectors Plasmid Name Source p G E X - S I P K Marcus Samuel T O P O T A 2.1 Cloning Vector Invitrogen T O P O 2.1 + S I P K G F P New intermediate construct p K S / 3 X H A Oncogene 17:1097 T O P O 2.1 + SIPK- l inker-GFP New intermediate construct p C A M B I A 1300-MCS Greg Lampard: p C A M B I A 1300 with deleted M C S p C A M B I A 1302 (35S GFP) C A M B I A vectors pRT 101 Nucleic Acids Res. 15 (14), 5890 (1987) p C A M B I A + M C S New construct from p C A M B I A - M C S and pRT 101 M C S between Hindlll sites S I PK : GFP Fusion Vector with Linker New construct in p C A M B I A + M C S p Y E S 2.1 T A Cloning Vector Invitrogen p Y E S 2.1 + SIPK- l inker-GFP New construct from insertion of fusion protein into p Y E S 2.1 p Y E S 2.1 + G F P Greg Lampard: G F P from p C A M B I A 1302 inserted into p Y E S 2.1 2.3: R E S U L T S 2.3.1: C lon ing of S I P K S IPK was successfully cloned as confirmed both by digestion of the T O P O T A 2.1 vector with Xbal and NotI producing ~ 1.2kb fragment, and also by sequencing showing no mismatched base pairs to the genbank database sequence of S IPK (gi:27374989). 2.3.2: C lon ing of G F P G F P was successfully cloned as confirmed by digestion of the T O P O T A 2.1 vector with NotI and Xhol producing -750 bp fragment, and also by sequencing, which showed no mismatched base pairs to the genbank sequence (gi:7638073). Figure 2.4 shows the purified S I P K - T O P O vector digested with Xbal and NotI in anticipation of the mGFP5 fragment, and the mGFP5 fragment, also digested with NotI and Xbal. The clean bands show the expected sizes for the fragments, and no indication of degradation. 39 1 2 3 4 5 8144 bp 5090 bp 1636 bp ^ 1018 bp <*- 506.5 bp Figure 2.4: T O P O T A 2.1 vector containing S I P K , and mGFP5 Fragments for ligation Figure 2.4 shows the results of the SIPK containing TOPO TA 2.1 vector digested with NotI and Xbal endonucleases (lanes 1-3). The approximately 5000 bp single band shown indicates a linearized SIPK-TOPO vector. Lane 4 shows a ~ 750 bp mGFP5 fragment digested from an mGFP5 containing TOPO vector digested with the endonucleases NotI and Xbal. Lane 5 shows the lkbp vector from Invitrogen. Fragments were run on a 0.8% agarose gel in 1 X T.A.E buffer at 80V for 45 min. 4 0 2.3.3: Construction of Fusion Protein The fusion protein construct was shown to be correct by digestion with appropriate restriction enzymes producing the proper ~2 kb fragment (figure 2.5), and by sequencing over the fusion site. 3054 bp 2036 bp 1636 bp Figure 2.5: Colony PCR of the Fusion of SIPK:GFP into the TOPO TA 2.1 Cloning Vector Figure 2.5 shows positive insertion of mGFP5 into the TOPO-SIPK vector as determined by colony PCR. Lane 1 shows the Invitrogen lkb ladder, and lanes 2-5 show separate colony PCR amplicons approximately 2000 bp in length indicative of SIPK fused to mGFP5. Fragments were run on a 0.8% agarose gel in 1 X T.A.E buffer at 80V for 45 min. 41 2.3.4: Fusion Protein Linker Insertion of the linker was successful as shown by colony PCR over the entire fusion protein in comparison to the fusion protein without the linker insertion (figure 2.6). There was a noticeable 100 bp difference between colonies containing the linker, and colonies without the linker. The insertion of the linker was further analyzed by sequence analysis. It was noticed by sequencing of 6 separate ligation events that all linkers were inserted in the opposite orientation to that originally desired. Due to time constraints and an inability to explain these events, the construct with the opposite orientation of the linker sequence was chosen for further work. The protein translation of the sequence is as follows: A A A S V L Y N A I Y I G S S I V R D V I G I A R I V R N I V W V T S G S A A A A 1 2 3 4 5 6 3054 bp 2036 bp 1636 bp Figure 2.6: PCR Confirmation of Insertion of Linker between SIPK and GFP in the TOPO 2.1 cloning vector Figure 2.6 shows the results of the insertion of the linker fragment between the SIPK and the mGFP5 fragments. Amplicons showing positive insertion of the - 100 bp fragment between the SIPK:GFP fusion would run at approximately 2100 bp (Lanes 2-5) when compared the control SIPK-mGFP5 fusion without the linker at -2000 bp (lane 6) amplified with SIPK F and GFP R primers and run on a 0.8% agarose gel in IX T . A . E buffer at 80V for 45 min. Lane 1 shows the 1 kbp ladder (Invitrogen). 42 2.3.5: Yeast Vector Construction Insertion of the SIPK:GFP with linker fusion sequence into the pYES vector was confirmed by growth on SC-uracil medium, and by colony PCR using the GAL1 forward and GFP reverse primers to confirm orientation of the insert (Figure 2.7). Three positive insertion colonies were chosen to transform from DH5a into the yeast strain INVSc 1. 1 2 3 4 5 2036 bp 1606 bp Figure 2.7: Insertion of SIPK:GFP Fusion Sequence into the pYES TOPO TA 2.1 Yeast Expression Vector Figure 2.7 shows the results of the insertion of the SIPK-mGFP5 fusion into pYES TOPO vector. Lanes 1-4 show separate putative transformants of the fusion protein as determined by colony PCR using pGAL forward and GFP reverse primers used to amplify the ~2kbp fragment. Improper orientation of the insert would yield no amplicon with the same reaction conditions (as in lane 4). Fragments were run on a 0.8% agarose gel in 1 X T.A.E buffer at 80V for 45 min. 2.3.6: Expression in Yeast Expression of the construct was confirmed by fluorescence microscopy (Figure 2.8). Of the intervals tested, overnight expression produced the highest expression of the 43 fusion protein, whereas the control vector containing G F P was shown to express strongly after 3 h of induction. The fusion protein does not appear to be expressed in every cell within the culture, whereas the G F P construct appears to express strongly seemingly in all of cells observed. 44 G F P Contro l (4 hr) EXPOSURE: 0.1 s e c 3 s e c Figure 2.8: Expression of SIPK-GFP Protein with Inserted Linker in Yeast Induced with Galactose Figure 2.8 shows the expression of the SIPK:GFP construct in yeast under the GAL1 promoter over a period of 16 hours. The GFP control was expressed under the same conditions. 45 . B J ; X I ( S 7 1 « ) CaMV35S p r o m o t e r Hindlll CaMV35S p o l y A ' T - B o r d e r (left) So? 1 1 ( 6 3 if) k a n a m y c i n (R) Xbal, BamHI, Smal, | - MCS Kpnl, SstI, EcoRI, p B R 3 2 2 ori \ p B R 3 2 2 b o m . " f c I ( 3 3 9 2 ) p V S 1 rep \h,il Y 3 5 S Hindlll P r o m o t e r Digest profi le: Hindlll- 8914, 711 bp Xhol- 7060, 1447, 1094 bp (opposite orientation would be 7233, 1284, 1094bp) Xbal- 9625 bp BamHI- 9625 bp Sail- 9625 bp PstI- 9625 bp L J J 8 1 4 4 b p " " j | 2036 bp ~ 1636 bp 1018 bp 506 bp all 8144 bp 8144 bp — 2036 bp — 1636 bp M| 1018 bp 2036 bp 1636 bp 506 bp 1018 bp Figure 2.9: Digest Profile for the Insertion of the 35S Driven MCS from pRT 101 to pCAMBIA 1300 Figure 2.9 shows digestion profiles of the of the new pCAMBIA 1300 + MCS vector with various restriction enzymes, and the resultant fragments of those digestions as seen on a 0.8% agarose gel in IX TAE buffer at 80V for 45 min. Digestion with the endonucleases Xhol, Xbal, PstI, BamHI, Sal.I and Xhol all give the appropriate sized fragments. 2.3.7: Binary Vector Construction The insertion of the 35S driven MCS from pRTlOl between Hindlll sites into pCAMBIA 1300's Hindlll site was successful as seen by the fragments produced with digestion using the restriction enzymes Hindlll, Xhol, Xbal, BamHI, Sail, and PstI. A l l 46 digestions gave the expected sized fragments (figure 2.9), indicating that the fragment was inserted in the desired orientation, that only one copy of the fragment was inserted into the vector, and that the tested restriction sites housed within the MCS digested predictably to give the desired fragment sizes. 2.3.8: Cloning Construct into Binary Vector: The insertion of the approximately 2 kb sized SIPK-linker-mGFP5 fusion was successful as confirmed by amplification of the fusion construct by colony PCR using SIPK forward and GFP reverse primers, and by sequencing of the construct. 1 2 3 4 5 6 3054 bp 2036 bp 1636 bp 1018 bp Figure 2.10: Insertion of SIPK: GFP with linker into the Binary vector pCAMBIA+MCS Figure 2.10 shows the expected amplicon from colony PCR of putative SIPK:GFP with linker containing transformants. Lane 1 shows the Invitrogen lkbp ladder. Lanes 2-6 show colonies A-E, with A-D being positive transformants showing an approximate 2kbp fragment amplified with SIPKF and GFPR primers. Fragments were run on a 0.8% agarose gel in 1 X T.A.E buffer at 80V for 45 min. 2.3.9: Plant Transformation using Agro-infiltration The construct was successfully inserted into Agrobacterium as shown by figure 2.11. Infiltration of the Agrobacterium into the plant tissue was observed visually (Figure 2.12). 47 Lesions were seen from infiltration of the over-expressing SIPK control construct OX-SIPK. No lesion formation was seen, as expected, from the 35S driven GFP pCAMBIA vector (1302) or the empty vector construct pCAMBIA 1300+MCS in GV3101. The fusion construct, S IPKGFP with inserted linker, did not show significant lesion formation GV3101 (Figure 2.12A). When the fusion construct was transferred to the Agrobacterium strain E H A 101, and transfected into tobacco leaves, significant lesion formation was apparent (Figure 2.12B), signifying that SIPK is active and functional despite the addition. 1 2 3 4 5 3054 bp 2036 bp 1636 bp 1018 bp Figure 2.11: Agrobacterium Colony PCR of Putative SIPK:GFP Fusion Protein Construct Insertion Figure 2.11 shows colony PCR results of putative Agrobacterium colonies. PCR products were amplified with SIPKF and GFP R primers. Lane 1 shows the Invitrogen lkbp ladder. Lanes 2-5 show putative vector containing colonies showing a - 2kbp amplicon. Fragments were run on a 0.8% agarose gel in 1 X T.A.E buffer at 80V for 45 min. 48 Figure 2 .12A ,B: Transient Expression by Agro-Infiltration of the SIPK:GFP with Linker fusion protein in Tobacco Leaves Figure 2.12 shows the results of agroinfiltration of GV3101 (A) and EHA101(B) Agrobacterium cultures containing the SIPK:GFP construct, as well as control cultures containing the empty vector p C A M B I A 1300 +MCS, and a 35S driven GFP in p C A M B I A 1300 + MCS. Lesion formation is indicative of a hypersensitive response. 49 2.4: D I S C U S S I O N : Although M A P K - G F P fusions have been reported in yeast and animal systems, there have not yet been any reported in plants. Perhaps M A P K : G F P fusions are difficult to express successfully due to post-translational problems. Improper folding in multiple portions of the protein could compromise cellular recognition of the protein, causing it to be targeted for degradation. Aside from many areas of the protein being important for proper folding, multiple portions of the protein are important for activation/inactivation of the protein of interest. These regions have a function in the protein's-ability to activate other proteins. Compromising essential binding or recognition regions of proteins can alter cellular expression levels, activation/deactivation processes, and subcellular localization of the proteins. Both the C - and N - termini are known to often contain important functional regions including signal peptides. The C - and N - termini are also the regions most often used to fuse to reporter proteins. Many fusion constructs are not functional due to compromised C - or N - termini functions. Some N-terminal fusion complications can include obscuring of the endoplasmic reticulum, mitochondrial or chloroplast targeting sequences located therein, thus generating localization artifacts (Tian et a l , 2004). In the same regard, some C-terminal fusions have been shown to give misleading results for a variety of proteins, including C e s A l or P in l when compared to other fusions of the same protein (Tian et al., 2004). A second reason for the lack of reported M A P K : G F P fusions in planta might be the low expression levels o f endogenous M A P K s , resulting in too few expressed G F P molecules to visualize effectively. Plant systems have a specific hindrance to the visualizafion of G F P from the natural auto fluorescence given off mainly by chloroplasts, 50 as well as from other light refracting complexes. Special filter sets that block out, or cancel out, unwanted background information are used for fluorescence microscopy to research G F P localization in plants. If expression of G F P is low, it would be difficult to detect the G F P signal from background noise without special filters and a sufficiently sensitive detection system. Tagged proteins are most often expressed under a strong constitutive rather than the native promoter, which can cause the protein to be expressed under conditions in which it would not normally be expressed, or in cells which it would not normally be expressed (Tian et al., 2004). In this experiment, the plant 35S promoter was used instead of using the native S IPK promoter. The choice of promoter was based primarily on time constraints to find, test, and use the native promoter, and the availability of the 35S promoter for experimental use. Although this project was not completed to the stage of visualization of the fusion construct in planta, there has been valuable information gained about the fusion construct. Primarily, it has been determined that the two portions of the fusion protein, S IPK and G F P , retain activity when fused together in a C-teifninal fusion with a linker inserted between them. B y sequencing the construct it was determined that the fusion kept G F P in frame for proper translation. It was also determined that there were no mismatched base pairs within the fusion construct sequence. From expression of the construct in yeast, it was determined that the G F P portion of the fusion protein was expressing, as seen by visualization of the fluorochrome under a fluorescence microscope. From agro-infiltration of tobacco leaves it was determined that S IPK is expressed, and that it has retained its ability to cause lesion formation in agro-infiltrated tobacco leaves. 51 The fusion protein's expression in yeast was found to be optimal after overnight induction with galactose. Due to the low expression of the fusion protein within the culture, even after an overnight incubation, it is possible to hypothesize that in yeast, high expression of the SIPK might only be permitted to be expressed within certain time frames, such as during cell division, or perhaps under induction with a certain stress. E R K 1 has already been determined to be active in regulation of growth and differentiation (Porter and Vaillancourt, 1998). The localization pattern of the fusion protein SIPK: GFP seen in yeast cells expressing higher levels showed bright spots within the cell, with large bright central spots, the actual subcellular localization of the fusion construct was unclear in yeast cells, due to their small size, and the continual motion present in live yeast cells. Further study could have been done on the expression and subcellular localization of the fusion construct in yeast, but the subcellular localization focus was on SIPKAVTPK's expression in tobacco cells, not yeast. Expression of the construct in yeast was a quick and easy tool to confirm GFP expression when fused to SIPK. Both the control GFP and the fusion construct showed expression of GFP in yeast. Despite being driven by the same G A L 1 promoter, the expression of GFP was observed to be much higher and stronger than the expression of the GFP: SIPK fusion protein. High expression of GFP is a good indicator that the expressed protein is not targeted for degradation, and also indicates that most likely the expression of the foreign construct does not have any easily observable effects on cellular processes. The GFP portion of the fusion protein, despite low expression compared to the control GFP construct, was expressed in yeast cells under the G A L 1 promoter. The activity of 52 SIPK when fused to G F P was tested using agro infiltration. Transient expression of active S IPK from agro-infiltration is known to cause lesions in young leaves (Zhang and Klessig, 2001). Lesion formation from agro-infiltrated S IPK might be attributed to S IPK becoming activated once infiltrated from the wounding stress caused by agro-infiltration. High levels of activated S IPK might trigger cell death, and lesion formation. From my agro-infiltration results, lesion formation was observed in both the control O X : S IPK-infiltrated construct, and the fusion construct. The 35S-driven G F P and empty vector controls did not induce lesion formation (Figure 2.12b). O f the Agrobacterium strains available in the lab, quite a bit of difficulty was had working with the GV3101. Although good results have been obtained with this strain in arabidopsis, it did not work well in these experiments using Xanthi tobacco. The expression from the binary vector p C A M B I A 1300 + M C S in GV3101 was either too low to be detected, or transfer of the T - D N A was not successful. Both of these hypotheses would be supported from the lack of lesion formation in transient expression of the fusion protein, and from the lack of a strong signal in both R T - P C R and western analysis (data not shown). With the information that both the SIPK and G F P portions of the construct are functional, some preliminary conclusions can be drawn about the fusion construct as a whole. The linker insertion appears to have kept enough distance between the two proteins to allow for proper folding of both S IPK and GFP. Act iv i ty of S IPK from agro-infiltration results indicates essential activation regions of S IPK have not been compromised by its fusion to GFP. 53 To observe this fusion construct in planta, both transient and transgenic expression of the construct could be observed. Transient expression of the construct was examined by both cross-sectioning of agro-infiltrated tissue, and by particle bombardment of leaf tissue (data not shown). No obvious expression of the construct was observed in planta, due to high background autofluorescence within the tissue, although with appropriate filters different results may have been found. If expression of the fusion construct in planta is similar to its expression in yeast, low expression levels would be expected. Tissue autofluorescence coupled with low expression levels demonstrated transient expression analysis to be an insufficient method for observing subcellular localization of the S I P K : G F P fusion protein in tobacco. Transgenic analysis of the SIPK: G F P construct was attempted, but was not completed within the time parameters of this experiment. It would be expected that either transgenic callus/cell culture or the roots of tobacco seedlings would allow clear visualization of the construct without interference from strong background autofluorescence. By use of epi-fluorescence microscopy and confocal/fluorescence microscopy, the subcellular locations of the fusion protein could be tracked. Information about the subcellular location of 35S driven S IPK under various conditions could be observed. Changes in subcellular localization could be observed live, allowing translocation times to be determined, as wel l as the duration of location upon activation. Stable transgenics bearing the S I P K G F P fusion could also be used to study upstream and downstream SIPK interactors through crosses with transgenics of suspected interactors, for example crossing with a W I P K R N A i or W I P K Y F P stable lines. Phosphatase: Y F P stable lines could be used to investigate Bogre's hypothesis that denovo synthesis of phosphatases 54 are involved in M A P K inactivation in plants following M A P K induction by wounding or treatment with elicitors (Bogre et al., 1997); (Suzuki and Shinshi, 1995), though Y F P / G F P interactions are not as efficient of a pair as C F P and Y F P using F R E T analysis. Knock out and overexpression lines of upstream double kinases could be used to examine Lee's hypothesis that M A P K K activity is required for sustained M A P K activity (Lee et al., 2004). In conclusion, the SIPK: G F P fusion construct has been shown to give observable expression in yeast and tobacco leaves, indicating that the fusion construct design and assembly were successful. There are many possible experiments in which SIPK: G F P stably transformed tobacco lines could be employed. This construct thus has the potential to be a valuable tool for future studies. 55 CHAPTER 3: Immunolabeling of Tobacco MAPKs 3.1: General Introduction to Immunolabeling 3.1.1: Immunolabeling Immunolabeling is the use of labeled antibody to show the localization of specific macromolecules in fixed tissue sections and cells. Both tissue section and whole cell immunolabeling provides higher spatial resolution than in vivo reporter proteins such as GFP. This technique is therefore frequently used in both plant and animal systems. Commonly used antibody labels include covalently-coupled fluorescent molecules, gold particles, and enzymatic reporters such as peroxidase. Macromolecules decorated by immunolabeling can be visualized using electron microscopy (EM) , light microscopy (LM) or fluorescence microscopy, depending on the choice of label. 3.1.2: Fluorochromes and Fluorescence The basic principle of fluorescence is that a compound w i l l absorb light energy at one wavelength and emit part of the absorbed energy as light of a longer wavelength. Fluorescent compounds (fluorochromes) can be detected in very low concentrations. Common fluorochromes that can be covalently bound to antibodies are fluorescein, rhodamine, Texas Red, the A lexa series o f fluors, Cy3, and Cy5. Excitation and emission wavelengths, along with fluorochrome stability, are the major factors that determine which fluorochrome is appropriate for a given experiment. 3.1.3: Fixation The aim of fixation is to put into stasis the subcellular activities and dynamic internal movements of the cell while retaining a physical state that resembles, as closely as possible, the l iving state of the cell. Some frequently used reagents for this purpose are 56 formaldehyde, osmium tetroxide and glutaraldehyde (O'Brian, 1981 pp 4.17). Different fixatives have specific strengths and weaknesses in terms of preserving certain cellular structures, and the choice of a fixative is dependent on the goal of the study. Aldehyde fixatives act by reacting with amino side-chains on proteins. The proteins are thereby immobilized, and are resistant to further deterioration. Di-aldehydes, such as glutaraldehyde, create a very strong cross-linked protein matrix since they have two aldehyde groups available for cross-linking. Strong matrices are critical for detailed ultrastructure analysis of the sample. Mono-aldehydes, such as paraformaldehyde, create a weaker matrix, which can be useful in immunolabeling since a too strongly cross-linked matrix can hinder antibody recognition of the protein (O'Brien, 1981). Since chemical fixation always modifies cellular structures to a greater or lesser degree, alternative approaches have been actively sought. High Pressure Freezing (HPF) is a method that results in excellent preservation of structure. HPF involves instantaneous freezing of the sample at high pressure to prevent ice crystal formation. Frozen cell water is then slowly substituted with a solvent such as acetone at freezing temperatures (O'Brien, 1981 pp 4.29). While high pressure freezing is a very effective method for preserving cellular and subcellular structures, the requirement for extremely rapid freezing means that only small quantities can be processed at a time. The sample size is therefore small, which can make it difficult to locate a cell displaying the desired traits. ' ' '. 3.1.4: Digestion/Removal of Cell Wall Plant cell walls can cause immunolabeling problems by reducing permeability of the cell to reagents, as well as by emitting autofluorescence in fluorescence microscopy. Cell 57 wall autofluorescence predominantly stems from lignin, an aromatic polymer that forms the most prominent non-saccharide component of the cell wal l . Because of the problems associated with cell walls, it can sometimes be desirable to digest or remove the cell wall. Digestion or removal o f the cell wal l can be obtained by gentle digestion with a mixture of hydrolytic enzymes such as cellulase and pectolyase (O'Br ien, 1981 pp3.12). Cel l wal l digestion can be performed on either live or fixed cells, but live cells become extremely fragile in the process. Fluorescence of lignified cell walls, cuticles, and suberized walls can also be reduced by fixation in osmium (O'Br ien, 1981 pp 2.28), but osmium disrupts antigenicity. The removal, or weakening of the cell wal l by digestion hot only reduces autofluorescence, but also improves cellular permeability. 3.1.5: Immunolabel ing of S I P K and W D ? K Previous investigation of harpin and megaspermin-treated tobacco cultured cells in our laboratory revealed that both the 44 and the 48 kDa phosphorylated proteins, S IPK and WIPK , were activated within 30 min of treatment with 40 pg/mL harpin or 40 pg/mL megaspermin (Appendix 2). These results were used as the basis for designing my protocol. Those previous investigations also revealed that the anti-/?ERK antibody gave little or no non-specific binding to other tobacco proteins on western blots when the antibodies are used at appropriate concentrations. A recent immunolabeling study of maturing tobacco pollen used anti-/?ERK to detect phosphorylated S IPK and/or W I P K in fixed cells (Coronado et al., 2002). Both fluorescent chemical-tagged antibodies (fluorescence microscopy) and gold-tagged antibodies (TEM) showed that these low abundance kinases are located in both the cytoplasm arid nucleus within developing 58 pollen grains (Coronado et al., 2002). These reports indicate that the ant i -pERK antibodies should be capable of reporting the distribution of pSIPK/ /?WIPK in tobacco cells. 3.2: Materials and Methods 3.2.1.: Tobacco Seedling Growth and Treatment with Elicitor Tobacco (Nicotiana tabacum cv Xanthi nc.) plants (WT and O X : SIPK) were grown for 2 weeks on agar-solidified half-strength Murashige and Skoog (MS) medium (1962) containing 50 mg/L kanamycin (to select for transgenic plants) under controlled conditions (25/20C, 16-h-light/8-h-dark cycle). Plantlets were floated in 40 pg/mL harpin or 40 pg/mL megaspermin for 30 min. Root tips were immediately excised and fixed in fresh 4% paraformaldehyde in half-strength M S medium. 3.2.2: Cell Culture Growth Conditions and Treatment with Elicitor N. tabacum cv Xanthi nc. suspension cell cultures were established and maintained in Murashige and Skoog medium (see Appendix 1) supplemented with lmg /L 2,4-D and 0.1 mg/L kinetin (50mL volume in 250 mL Erlenmeyer flasks), and sub-cultured weekly. The cultures were shaken at 120 rpm (gyratory shaker) in the dark at 25 °C. Cultures (3 days after sub-culture) were treated with 40 pg/mL harpin or with 40 pg/mL megaspermin for 30 min. After exposure, the cells were permitted to settle by gravity, washed with fresh M S medium and fixed in fresh 4% paraformaldehyde in half-strength M S medium. 3.2.3: Fixation of Samples in Paraformaldehyde Paraformaldehyde (0.4g) was added to 50 mL lA M S p H 5.5. In the fumehood, the covered mixture was heated to around 60 °C, but not permitted to boil. A few drops of 59 concentrated N a O H were added to help dissolve the paraformaldehyde. Once dissolved, the mixture was cooled to RT, and the p H adjusted to 5.5. Approximately l m L of settled tobacco suspension cells or 12 root tips were added to the paraformaldehyde solution, which was then rotated slowly for 1 hour. The tissue sample was then permitted to settle, the supernatant removed, and the samples were re-suspended in fresh Vi M S buffer. 3.2.4: Digestion/ Removal of Cell Wall Both root and cell culture samples were digested in 0.1% cellulase (Yakult Honsha Co. Ltd), 0.5% maceroenzyme (Yakult Honsha Co. Ltd), and 0.25% pectinylase (Yakult Honsha Co. Ltd) in I X T B S T containing 0:1% saponin from quillaja bark (Sigma) with rotation at 100 oscillations/min for 20 min. Saponin was used to permeabilize cell membranes to aid immunolabelling solutions to pass through cellular membranes. 3.2.5: Immunolabeling 3.2.5.1: Primary Antibodies The primary antibodies used in this experiment were anti phospho-ERK (anti-/?ERK) (1:200 dilution, Ce l l Signalling), ant i -ERK (1:200 dilution, Santa Cruz), an t i -FLAG (1:500 dilution)(Sigma), a negative control rabbit pre-irhmune serum (1:200 dilution), and a positive control anti-histone antibody (1:500 dilution)(Chemicon). A l l antibodies were made up in 4 % B S A in T B S T containing 0.1% saponin. 3.2.5.2: Secondary Antibodies The primary antibodies used in these experiments were detected with anti-rabbit IgG:Alexa 5 9 4-conjugated secondary antibody (Molecular Probes). A l e x a 5 9 4 is a Texas Red-equivalent fluorochrome with emission at 594 nm. The secondary was used at a 1:300 dilution in 4 % B S A in T B S T . 60 Stored conjugates sometimes develop precipitates, and were therefore routinely centrifuged (8,000g for 15 sec) before use to remove any particulate matter. 3.2.5.3: Controls Anti-histone antibody was used as a positive control because histone is an abundant protein in tobacco cells, and is restricted to the nucleus. Use of this nuclear-localized antibody gives a good indication of what a nuclear localization signal pattern should look like. The positive control also confirmed whether a given protocol was working. Several negative controls were used incthese experiments. Samples treated with no primary or secondary antibody gave information about natural background autofluorescence in the sample. These samples were treated with blocking solution instead of either the primary or secondary antibody at the appropriate steps. Samples treated with secondary antibody, but no primary antibody, revealed the extent of non-specific binding of the secondary antibody. Use of pre-immune serum as the primary antibody reveals any non-specific binding associated with rabbit anti-serum. 3.2.5.4: Immunolabel ing Protocol Fixed tissue samples were treated with 50mM E G T A for one hour to chelate any interfering metal ions. Samples were treated with 1% Triton-X 100 with shaking at 100 oscillations/min for 20 min, followed by treatment with ice cold methanol with shaking at 100 oscillations/min for 10 min. Samples were incubated in blocking solution (bovine serum albumin (BSA) (4% w/v) in I X T B S T containing 0.1% saponin and I m M glycine prepared fresh and filtered) overnight, rinsed with I X T B S T + 0.1% saponin, and blotted dry with Whatman #2 filter paper. 61 Samples were applied to individual wells of poly-L-lysine-coated 8 mm wel l slides. Each slide contained 8 individual wells. For observation of cells , a 20 uL droplet of culture was permitted to settle in each wel l for 20 min, and excess culture medium was removed by pipetting. Roots were attached by gently squashing the root onto the slide by pressing with the thumb directly on the coverslip over the root. The coverslip was then removed by sliding it to the side, taking care not to mangle the root tip. Care was taken to keep specimens as moist as possible for immunolabeling, since air drying can produce structural artifacts within a given tissue, mainly on the cell surface, as well as negative effects from oxidation (O'Br ien, 1981. pp3.21). Moist chambers were created for the slides by putting two I X TBST-soaked filter paper wedges inside a Petri dish. Primary antibody (5 pL/well) was applied to the individual wells (or blocking solution to appropriate controls) and the slides were then incubated 1 h in the covered chambers at RT. Following incubation with the primary antibody, slides were rinsed with I X T B S T containing 0.1% saponin, and then let sit in four consecutive baths of fresh I X T B S T containing 0.1% saponin for 10 min each before being blotted dry with filter paper, and placed back in the moist chambers. Secondary antibody (5 pL/well) was then applied to the individual wells (or blocking solution for appropriate controls), which were incubated for one hour in the covered chambers at RT. Slides were then rinsed with I X T B S T followed by four consecutive 10 min washes in fresh I X T B S T containing 0.1% saponin. Following treatment with secondary antibody, samples were treated with 5 pL/wel l 30 n M D A P I in T B S T containing 0.1% saponin for 30 min. The slides were then rinsed with I X T B S T , followed by four consecutive 10-min washes in fresh I X T B S T 62 containing 0.1% saponin, briefly rinsed in fresh distilled water to remove any excess salts from buffers that might crystallize over time under the coverslip. Samples were blotted dry with filter paper. Mounting medium consisting of 33% aqueous glycerol was added and cover slips were applied. The mounted slides were sealed with clear nail polish, and left in the dark at 4 °C until use. 3.2.5.5: Quant i f icat ion A Leica D M R fluorescence microscope equipped with a Texas Red #2 (rhodamine) filter (emission 645-75 nm) was used to visualize fluorescence. The microscope was equipped with a digital Q-irriaging Q I C A M image capture system running under Openlab 3.0 software for viewing and recording of immunolabeling results. Exposure times were set at 3 sec (unless otherwise rioted) so comparisons could be drawn between control and experimental samples. 3.3: Results Fixed cells were examined under three different microscope conditions; bright field, fluorescence with inserted D A P I filter, and fluorescence with inserted rhodamine filter. Light microscopy reveals the basic cellular shape, gives an indication of pre-fixation viability o f cells, and reveals some basic cellular structures (Figure 3. la,d,g,j). Although most cultured cells had an oval shape, some cells displayed an elongated shape, or were clumped together with other cells. The pre-fixation viability could be assessed by the degree to which the cell membrane remained tightly pressed to the cell wall. Dead cells displayed extensive detachment of the membrane. Some of the basic structures visible within the cells with light microscopy are the nucleus, vacuoles, cytoplasm, and cell wal l . 63 DAPI staining (Figure 3. lb,e,h,k) verified the location of the nucleus within the cell, indicated whether there was more than one cell present (based on the number of nuclei present) and sometimes revealed the mitotic stage of the cells. Cultured tobacco cells showed a low level of auto fluorescence in the rhodamine channel (Figure 3.1c), but this was not significant compared to the intensity o f fluorescently labeled structures seen in the anti-histone immunolabeled positive control (Figure 3.11). The positive control anti-histone antibody showed strong labeling of the nuclei, similar to the D A P I labeling (Figure 3.1k). The secondary antibody used in this experiment, Alexa 5 9 4 - tagged anti-rabbit antibody, did bind nbri-specifically to some internal cellular structures, such as the cell wall/membrane, and within the nucleus (Figure 3. If), but the fluorescent signal was negligible in comparison to the positive control (Figure 3.11). The pre-immune serum-stained cells showed a moderate amount of non-specific binding to cell membranes/walls and nuclei, as wel l as to some unidentified internal cellular structures (Figure 3. l i ) : However, pre-immune-labeled cells still showed a much lower fluorescence signal than the positive control (Figure 3.11). 64 Light DAPI Rhodamine Channel Controls (3 sec exposure except for anti-histone, 200 ms) Figure 3.1: Immunolabeling Control Samples of Fixed Suspension Culture Wild Type Tobacco Cells Figure 3.1 shows the light image, the D A P I stained image, and the rhodamine channel fluorescence of 4 different immunolabeling controls; untreated negative control, no primary with applied secondary A l e x a 5 ' anti-rabbit negative control, a rabbit pre-immune serum with secondary negative control, and anti-histone with secondary positive control. 65 As wel l as wild-type tobacco suspension cell culture, I had available a transgenic cell culture line in which S IPK is ectopically over-expressed. The S IPK transgene has been modified to include a F L A G epitope at the C-terminus of the S IPK protein. This cell culture line was included in my study because it was hypothesized that over-expression of S IPK might give a stronger S IPK subcellular localization signal. The advantage of having a F L A G - t a g on the ectopic S IPK was that it would allow me to use an t i -FLAG immunolabeling to visualize S IPK subcellular localization, as wel l as avoiding any potential non-specific binding an anti-SIPK antibody might have to similar M A P K s or other proteins. However, the O X : S I P K cultured cells displayed some physiological differences from W T (Appendix 3.0). O X : S I P K cultured cells were generally smaller than WT , had a somewhat higher level of native autofluorescence, and were predominantly spherical. These differences emphasize that the O X : S IPK cells cannot be directly compared to W T cells, but might still provide valuable information about general M A P K localization patterns as wel l as specific information about S I P K localization patterns in a S IPK over-expressing system. Both W T and O X : S IPK cultured cells were immunolabeled with three different antibodies (aside from controls): ant i -ERK, ant i -pERK and ant i -FLAG. A n t i - E R K antibody was used to show general M A P K distribution patterns within W T and O X : S IPK tobacco cells. Ant i - /?ERK antibody provides specificity, since it is a monoclonal antibody directed at the phosphorylated T E Y sequence of 42-48 kDa M A P K s , which in tobacco are S IPK and WIPK. Phosphorylated S IPK and W I P K localization patterns could then be compared to overall M A P K cellular distribution in W T and O X : S IPK tobacco cells. The third antibody, an t i -FLAG, would provide information about S IPK 's 66 localization patterns in OX: SIPK tobacco cells, and could be compared to anti-ERK and anti-/?ERK immunolabeled OX:SIPK cells. Each of these antibodies showed some discrete localization pattern that differed from the non-specific binding pattern associated with the pre-immune serum negative control in cultured cells. For every treatment group, approximately 100 cells were observed, and observed trends recorded. Images presented here from each treatment group represent typical patterns. Untreated cultured cells probed with anti-ERK primary antibody (Figure 3.2 Cii) showed predominantly cytoplasmic localization of the signal. Although a few untreated cells did show some nuclear accumulation of label, the majority of cells displayed an even distribution of the label throughout the cytoplasm, extending from the nucleus through the cell to the outer periphery of the cell. By contrast, most harpin-treated cells probed with anti-ERK primary antibody (Figure 3.2 Ciii) displayed increased nuclear localization of the signal, and within this population a considerable number of cells displayed strong accumulation of label in a central region of the nucleus, believed to be the nucleolus. A minority of harpin-treated cells probed with anti-ERK displayed cytoplasmic localization of label with no real accumulation in the nucleus or nucleolus. Confocal projections of both untreated and treated anti-ERK-probed cells yielded a pattern similar to the results found with fluorescence microscopy (Figure 3.2: 2b,c), but the confocal analysis revealed a cleaner picture of the presence and absence of label within the cell, and provided images of higher resolution than basic fluorescence microscopy. 67 It should be noted that some of the bright punctate spots seen in this figure are not indicative of large labeled structures, but are an artifact of projecting multiple layers of images that contain smaller bright spots. These large spots were not present in all confocal projections of cells. Cells treated with megaspermin showed no noticeable differences from those treated with harpin when probed with anti-ERK (Figure 3.3). Megaspermin-treated samples echoed a nuclear localization in the majority of cells, with considerable nucleolar accumulation. Anti-ERK immunolabeled OX-SIPK cultured cells closely resembled the WT labeled cultured cell localization patterns. The majority of untreated cells showed cytoplasmic localization of signal (Figure 3.4 Cii , 2a). Harpin-treated OX: SIPK cells showed nuclear localization of label (Figure 3.4 Cii i , 2b), but did not display the distinctive nucleolar accumulation seen in harpin and megaspermin-treated WT cells (Figure 3.2 Cii i , 2c). Few sampled cells displayed cytoplasmic localization in treated OX: SIPK cells. Cells immunolabeled with the anti-pERK primary antibody displayed a more spatially restricted distribution of label. There was a stronger nuclear accumulation of label in anti-pERK probed WT cells than in anti-ERK probed WT cells, but overall the distribution of anti-pERK label in the majority of untreated cells remained predominantly cytoplasmic. WT cells harpin-treated (Figure 3.5 Ciii) showed strong nuclear localization of the anti-pERK signal, but with very distinct nucleolar localization. The great majority of harpin-treated cells showed strong visible nuclear localization of the antibody signal, with a strong nucleolar signal within the nucleus. Nucleolar localization was also observed in untreated cells, but at a very low frequency, and label accumulation did not 68 exhibit the sharp edged localization pattern seen in the harpin-treated cells (Figure 3.5 Ci i) . The nucleolar localization of signal in harpin-treated W T cultured cells is again observed in the confocal images (Figure 3.5: 2c), and also revealed that within the nucleolus there is a central region that shows no label. O f the cells that were examined, smaller nucleoli showed no internal black spot, whereas larger nucleoli show a distinct unlabeled central region. Confocal images of W T cultured cells that had not been exposed to harpin showed strong cytoplasmic localization extending from the nucleus to the cell periphery (Figure 3.3: 2b). , W T cells treated with megaspermin and probed with anti-/?ERK primary antibody appeared indistinguishable from harpin-treated cells in terms b f p E R K subcellular localization (Figure 3.6). O X - S I P K cells immunolabeled with the ant i -pERK primary antibody showed different localization patterns to those from W T anti-/?ERK labeled cultured cells. Approximately half of untreated O X - S I P K cells sampled already exhibited nuclear localization of signal, with the reminder exhibiting cytoplasmic localization (Figure 3.7 Ci i) . These results were also reflected in confocal projection images (Figure 3.7: 2b). The majority o f harpin-treated O X : S IPK cells probed with ant i -pERK displayed nuclear localization of signal, similar to W T immunolabeled cells, but only a few cells displayed prominent nucleolar localization (Figure 3.7 C i i i , Figure 3.7: 2c). Although nucleolar labeling in O X : S IPK cells was less pronounced than that observed in W T labeled cells, it was not totally absent. 69 The an t i -FLAG polyclonal antibody from S I G M A was recommended by Brett Finlay's lab for immunolabeling purposes, based on their experience with both the monoclonal and polyclonal reagents. In their immunolabeling experiments, they had found less non-specific binding with the polyclonal form than with the monoclonal form. Western analysis of tobacco cell extracts with the polyclonal an t i -FLAG antibody did show non-specific binding when concentrations of the antibody were high (data not shown). A dilution of 1:500 was used to achieve minimal non-specific binding during my immunolabeling experiments (Figure 3.8 Cii). As a result, signal intensities in anti-F L A G labeled cells appear lower than in ant i -ERK and ant i -pERK probed cells. Non-specific binding of the an t i -FLAG antibody in W T cells produced a diffuse signal throughout the cell, with higher amounts in the cell wall/membrane and the nucleus (Figure 3.8 Cii). O X : S I P K cells expressing the FLAG-tagged S IPK and not treated with an elicitor showed accumulation of label in both the cytoplasm and nucleus (Figure 3.8 Ci i i ) . The signal within the cytoplasm appears to be associated with cytoplasmic strands extending toward the nucleus. In contrast to the ant i -ERK and ant i -pERK, the nuclear signal appears to be largely excluded from the nucleolus. Among the 100 cells examined, the fluorescent signal was observed approximately half of the time to have a stronger cytoplasmic localization, and half o f the time to display a stronger nuclear localization. The harpin-treated O X : S I P K cells showed an t i -FLAG immunolabeling signal patterns similar to those seen in untreated O X cells, but with slightly less cytoplasmic signal (Figure 3.7 C iv). The majority of the cells observed displayed higher nuclear localization, with a minority of the cells showing cytoplasmic localization (Figure 3.7). The label was diffusely localized throughout the nucleus. The confocal projection 70 images of harpin-treated O X - S I P K an t i -FLAG immunolabeled cells (Figure 3.7: 2) reflect the fluorescent microscope image patterns, but have better resolution. Localization patterns show higher immunolabeling signal in the anti-FLAG-treated O X cells (Figure 3.7: 2c,d) than in the anti-FLAG-treated W T control (Figure 3.7: 2b) or the untreated O X cells (Figure 3.7: 2a). Distinct punctate cytoplasmic labeling with general nuclear labeling is present in untreated O X cells (Figure 3.7: 2c), reflecting the patterns found with fluorescence microscopy. The treated O X - S I P K cells examined by confocal microscopy showed less cytoplasmic labeling and higher nuclear labeling (Figure 3.7: 2d), similar to results obtained from fluorescence microscopy. 71 WT pre-immune WT anti-ERK WT anti-ERK (control) untreated harpin treated A B C 2 A=LM. B=DAPI. C=Rhodamine (fluorescent) 3 sec exposure. D=Rhodamine (fluorescent confocal projection) Figure 3.2: Anti-ERK Immunolabeled Untreated and 30 min Harpin-Treated WT Tobacco Cells Figure 3.2 shows the light (li-iii), the fluorescence DAPI channel (2i-iii), and the fluorescence rhodamine channel images (3i-iii) and confocal projections (2a-c) from anti-ERK primary and Alexa594-tagged secondary immunolabeled WT tobacco cultured cells. Harpin treatment concentration was 40 ug/mL. Controls for this experiment were WT cells treated with rabbit pre-immune serum (column 1). 72 harpin-treated megaspermin-treated A=LM, B =Rhodamine (fluorescent), 2 sec exposure Figure 3.3: Anti-ERK Comparison of Harpin and Megaspermin-Treated Cell Culture Figure 3.3 shows the comparison of WT cells probed with anti-ERK treated with 40 ug/mL harpin or megaspermin for 30 min. WT pre-immune OX: SIPK anti-ERK OX:SIPK anti-ERK (control) untreated harpin treated A B C 2 A=LM, B=DAPI. C & D=Rhodamine (fluorescent), 2 sec exposure Figure 3.4: Anti-ERK Immunolabeled Untreated and 30 min Harpin-Treated OX Tobacco Cells Figure 3.4 shows the light (li-iii), the fluorescence DAPI channel (2i-iii), and the fluorescence rhodamine channel images (3i-iii) and confocal projections (2a-c) from anti-ERK primary and Alexa594-tagged secondary immunolabeled OX-SIPK tobacco cultured cells. Harpin concentration was 40 ug/mL. Controls for this experiment were cells treated with rabbit pre-immune serum (column 1). 74 WT pre-immune WT anti-pERK WT anti-pERK (control) untreated harpin treated A B C 2 A=LM, B=DAPI, C=Rhodamine (fluorescent) 3 sec exposure, 2a-c=Rhodamine (fluorescent confocal projection) Figure 3.5: Anti-pERK Immunolabeled Untreated and 30 min Harpin-Treated WT Tobacco Cells Figure 3.5 shows the light (li-iii), the fluorescence DAPI channel (2i-iii), and the fluorescence rhodamine channel images (3i-iii) and confocal projections (2a-c) from anti-pERK primary and Alexa^9"1-tagged secondary immunolabeled WT tobacco cultured cells. Harpin concentration was 40 ug/mL. Controls for this experiment were cells treated with rabbit pre-immune serum (column 1). 75 harpin-treated megaspermin-treated A=LM. B =Rhodamine (fluorescent), 2 sec exposure Figure 3.6: Anti-pERK Immunolabeled Comparison of Harpin and Megaspermin-Treated Cultured cells Figure 3.6 shows the comparison of 40 ug/mL harpin or megaspermin treated (30 min exposure) WT cells probed with anti-pERK. 76 WT pre-immune OX:SIPK anti-pERK (control) untreated OX:SIPK anti-pERK harpin treated A B C 2 A=LM, B=DAPI. C=Rhodamine (fluorescent) 2 sec exposure, D=Rhodamine (fluorescent confocal projection) Figure 3.7: Anti-pERK Immunolabeled Untreated and 30 min Harpin-Treated OX:SIPK Tobacco Cells Figure 3.7 shows the light (li-iii), the fluorescence DAPI channel (2i-iii), and the fluorescence rhodamine channel images (3i-iii) and confocal projections (2a-c) from anti-pERK primary and Alexa594-tagged secondary immunolabeled OX-SIPK tobacco cultured cells. Harpin concentration was at 40 ug/mL. Controls for this experiment were cells treated with rabbit pre-immune serum (column 1). 77 OX:SIPK anti-FLAG anti-FLAG anti-FLAG OX:SIPK control WT (control) OX:SIPK untreated harpin treated A=LM. B=DAPI, C=Rhodamine (fluorescent) 2 sec exposure, D=Rhodamine (fluorescent confocal projection) Figure 3.8: Anti-FLAG Immunolabeled WT, OX-SIPK, and 30 min Harpin-Treated OX-SIPK Tobacco Cells Figure 3.8 shows the light (li-iv), the fluorescence DAPI channel (2i-iv), and the fluorescence rhodamine channel images (3i-iv) and confocal projections (2a-d) from anti-FLAG primary and Alexa594-tagged secondary immunolabeled tobacco cultured cells. Controls for this experiment were untreated OX-SIPK cells (column 1), and WT cells immunolabeled with anti-FLAG (column 2) to show any non-specific binding of the FLAG antibody. Harpin treatment concentration was 40 ug/mL. 7 8 3.4: Discussion: This study of subcellular localization of plant M A P K s in response to stress was carried out primarily in cultured cells as opposed to using whole plant material. Plant suspension cultures have been shown to reflect whole plant metabolism in many studies, including responses to fungal elicitors (Romeis et a l , 1999). From the perspective of fluorescence microscopy, cell culture was a good experimental medium due to the lack of developed chloroplasts, and low autofluorescence. These characteristics, together with the relative uniformity o f tobacco suspension cultured cells, and their ease of fixation and ease of digestion of the cell wal l , made them a superior system for immunolabeling in planta. The choice of paraformaldehyde as a fixative was based on its rapid rate of infiltration, its ability to penetrate cuticularized cell walls and coagulants within the fixative mixture, and the excellent preservation o f cellular structure that it provides (O'Br ien, 1981 pp4.22). Fluorochrome-tagged immunolabeling was employed for these experiments because fluorescent compounds can be detected in very low concentrations, and M A P K s , especially phosphorylated M A P K s , are expected to be in low abundance within the cell. The A l e x a 5 9 4 secondary antibody was chosen based on fluorochrome stability, suitable emission wavelength, and the intensity of its fluorescence. The A lexa fluors are known to resist fading, and are not affected by p H variation within the cell. The A l e x a 5 9 4 fluor has emission that can be detected in the rhodamine channel (594 nm), which is convenient since Rhodamine and far red channels capture the least amount of autofluorescence from cultured tobacco cells, (data not shown). The Alexa 5 9 4 - labeled secondary antibody was found to yield a strong fluorescent signal in immunolabeled 79 tissue, as seen in the positive control anti-histone immunolabeled cell (Figure 3.11), and little to no non-specific binding when no primary antibody was applied (Figure 3. If). Labeling with the pre-immune serum is an important control that reveals any non-specific binding reactions. Preliminary experiments explored various measures to reduce non-specific binding, such as longer blocking solution treatments, shorter primary antibody incubation times, and decreased primary antibody concentrations. Based on these results, an optimized protocol was developed. Using this protocol, the pre-immune serum generated some diffuse non-specific binding throughout the cell (Figure 3. Ii), although in comparison to the positive control this was minimal. From the low levels of non-specific binding observed on a western gel, it was hoped there would be little to no non-specific binding in whole cell immunolabeling as well. As it turned out, the levels of non-specific binding were higher than hoped, but were still considered acceptable for immunolabeling. When compared to the modest levels of staining subsequently observed in the anti-FLAG, anti-/?ERK, and anti-ERK labeled samples, this non-specific binding does appear more significant (Figures 3.2-3.6), but the patterns of non-specific binding of the pre-immune serum were always diffuse, rather than punctuate; i.e. they never showed a significant amount of accumulated label in one area. Pre-immune serum non-specific binding was therefore readily distinguishable from antibody labeled signal. The choice of the anti-histone antibody as a positive control was based on the prediction that my protein of interest would show a higher "nuclear localization upon treatment. The anti-histone antibody proved to give a clean and consistent nuclear localization pattern, and was a valuable resource for showing the effectiveness of the immunolabeling method (Figure 3.11). 80 A n t i - E R K immunolabeling of cultured W T tobacco cells showed a significant amount of cytoplasmic labeling in untreated cells, with a high nuclear, and particularly nucleolar, accumulation of label (Figure 3.2). Pre-treatment of the cells with either harpin or megaspermin resulted in a shift in labeling intensity that could be interpreted as a re-localization of cytoplasmic S I P K / W I P K to the nucleus/nucleolus. This is consistent with numerous studies in other organisms showing that E R K and E R K homologues have a general cytoplasmic localization in untreated cells, and move at least partially to the nucleus upon induction with certain elicitors. Both harpin and megaspermin were shown to induce this change in localization pattern, as seen by probing with ant i -ERK. It should be noted that the use of ant i -ERK does not allow me to distinguish between S IPK and W I P K signals. However, it is known that the basal level of S IPK in untreated tobacco is ~10-fold higher that of W I P K (Zhang and Klessig, 1998), which would imply that the majority o f signal observed in these tobacco cells is associated with SIPK. Since the activation of M A P K s by stress is known to be rapid (<10 min), it is reasonable to ask whether the infiltration rate for paraformaldehyde was faster than the ability of the cultured cells to detect the chemical stress. It was hypothesized that i f this fixation was too slow, paraformaldehyde infiltration might trigger premature M A P K activation in untreated cells. To address this question, high pressure-frozen samples were compared to paraforrnaldehyde-treated samples. N o obvious differences were found between dextran-fixed and paraformaldehyde-fixed samples, but the H P F sample sizes were too small for a thorough comparison (Appendix 5). A n t i - E R K was used to probe both W T and O X : SIPK-treated and untreated cultured cells. In an over-expressing system, it was believed there would be higher levels of 81 cellular SIPK, and thus more label present. The stable over-expression of both SIPK and WIPK in transgenic tobacco had been previously shown to not affect the activity of the kinases (Yang et al., 2001), but some physiological differences were noted in the OX-SIPK line. In cell culture, OX: SIPK cells were found to be smaller in size, sometimes half that of WT, and the culture consisted mainly of isodiametric cells, whereas cells in WT culture have multiple shapes and sizes ranging from round to oval, and from small (50pm diameter) to large size (100pm diameter) (Appendix 3). OX:SIPK plants were found to be slightly shorter overall when compared to similarly grown WT tobacco, and the overexpressing line had denser foliage and floral buds (unpublished data, Marcus Samuel). The OX: SIPK transgenic plants are also more sensitive to growth on high salt concentrations, or higher concentrations of some growth hormones, (unpublished data, Marcus Samuel). Since overexpression of SIPK thus has a number of observable pleiotropic consequences, it is perhaps not surprising to observe different E R K localization patterns in OX: SIPK cells as compared to WT cells. Higher levels of nuclear localized signal were observed in both treated and untreated OX:SIPK cells (Figure 3.4). Perhaps OX-SIPK cells are more sensitive to the stress associated with the handling of the cells, or with the fixation process, and thus displayed more of a constitutively active phenotype, even in untreated cells. In a recent study of parsley M A P K (PcMPK6) subcellular localization, Lee et al. found that 20% of cells in a deliberately stressed culture population did not show any different subcellular localization (2004). Their hypothesis was that a certain number of cells will typically remain unaffected by a given stress, a response pattern that may help maintain stability. In an analogous fashion, it is 82 also possible that in untreated over-expressing S IPK cultured cells there is a higher frequency of cells within the population that act stressed at all times. There are several possible explanations for the differences in localization patterns observed between W T and O X : S IPK cells. First, overexpressing S IPK may cause a shift in M A P K signaling patterns that does not reflect native signaling patterns. A n accumulation of S IPK within the cell could also cause competition for interaction with N t M E K 2 in the cytoplasm, causing diminished activated W I P K signal. Overexpressing S IPK could also cause a variety of effects outside the M A P K signaling pathway that might affect the subcellular localization of the kinase. For example, over-expressed S IPK could be targeted for degradation. Higher levels of the kinase could trigger some cellular S IPK threshold level that activates processes that would not be active under native S IPK levels. Perhaps overexpressed S IPK draws in other interacting proteins in an inappropriate manner and thus confounds the signaling pathway. Although ant i -ERK antibodies have been shown to recognize both S IPK and W I P K in western gel analysis, this is not as specific an antibody as the ant i-pERK. The ant i -pERK antibody recognizes only those proteins that are doubly phosphorylated on the - T X Y -motif. The two proteins recognized by ant i -pERK antibodies in stressed tobacco tissue were previously determined to be S IPK (46 kDa) and W I P K (44 kDa). Untreated wi ld type cells probed with ant i -pERK antibodies showed a general cytoplasmic localization of signal, with higher concentration in the nucleus. Phosphorylation of M A P K s does indicate activation of the M A P K s . Since ant i -pERK only recognizes the activated form of S IPK and WIPK , one might not expect much label to be present in non-induced cells. From western gel analysis S IPK and W I P K are 83 known to have increased activation in response to stress, but there is still a relatively low basal level o f activated M A P K s present in non-induced cells. This basal level of activated M A P K s is what is observed in untreated anti-/?ERK-probed W T cultured cells. These activated M A P K s might have functions outside of stress signaling, or may be responding to a different stress than the applied one. Activated M A P K s are known to show a nuclear localization trend in response to a variety of inducers. Higher basal levels of nuclear localized ant i -pERK in non-induced W T cells (as compared to ant i -ERK probed cells) reflect the localization of active M A P K s in untreated cells. Non-induced O X : S IPK cells, much like the non-induced W T cells when probed with ant i-pERK, had a higher basal level of nuclear label accumulation as well. There were more cells displaying higher nuclear accumulation of label in O X : S I P K cells than in W T cells. In O X : S I P K cells, the higher nuclear accumulation of ant i -pERK label in untreated cells could reflect increased levels o f S IPK signaling that may (or may not) be involved in stress signaling. The higher nuclear label localization does suggest that O X : S IPK cells are either more sensitive to handling than the W T cells, or that there are higher levels of signaling activity constitutively underway in untreated O X : S IPK cells, or both. Both harpin and megaspermin-treated W T and O X : S IPK cultured cells probed with ant i -pERK showed particularly strong signal localization patterns in the interior of the nucleus. The distinct round body within the nucleus, which is presumably the nucleolus, showed a higher level of label accumulation than was seen with any other antibody treatment, including anti-pERK-probed O X : S I P K cells. The nucleolar accumulation of S I P K / W I P K appears to be a novel immunolabeling result. Cel l fractionation of human nuclei, and protein analysis o f the subcellular 84 fractions, found E R K to indeed be present in the cytoplasm and nucleus, but to be distinctly absent from the nucleolus (Scherl et al., 2002). It should be noted, however, that these tests were performed on non-induced cells. It is possible that nucleolar accumulation of S I P K / W I P K might be uniquely associated with tobacco cell culture systems. In an attempt to answer this question, whole roots from tobacco seedlings were fixed, immunolabeled and analyzed by immunolabeling for comparison to results from cultured cells (Appendix 4). Although results were not very good due to extremely low resolution and lack of full penetration of fixative, antibodies, and washes through this thick tissue, some conclusions could still be drawn. A higher nuclear accumulation of label was still observed in treated cells, and within the nuclei some distinct spots appeared brighter than other parts of the nucleus. However, the sharp-edged, extremely distinct nucleolar accumulation pattern seen in cultured cells was not observed in tobacco seedling roots! The major function of the nucleolus is thought to be the assembly o f ribosomes. However, it is also involved in various other tasks such as signal recognition particle assembly, cell cycle regulation, control of aging, modification of small nuclear R N A ' s and modification of telomerase functions (Olson, 2004). Recent studies have found that the pattern of proteins identified in fractions from the nucleolus do not map wel l onto the presumed functions of the nucleolus. After finding high sustained levels of p53 in the nucleolus of human cells following treatment with 11 different stress-related treatments, Rubbi and Mi l le r proposed that the nucleolus may act as a major cellular stress sensor that helps regulate p53 activity (2003). Elevated p53 levels can inhibit mammalian cell growth and lead to cell death (Olson, 2004). It is unknown what the stress signal 85 perceived by the nucleolus might be. It is also unknown how the cell distinguishes between stresses and decides whether to repair the damage, or to trigger cell death. There are many functions S I P K / W I P K could have in the nucleolus. Some functions might include roles in the cell death process, or regulation of transcription factors. W I P K activity, either alone or with S IPK has been proposed to be involved in induction of cell death in cultured tobacco cells by fungal elicitor treatments (Zhang et al., 2000), so the presence of either M A P K in the nucleus, or nucleolus, following induction with harpin and/or megaspermin could be related to controlling the commitment to the cell death process. S I P K / W I P K might therefore have roles in plants that resemble the roles of mammalian p38 rather than the roles played by E R K . M A P K involvement in activation regulation of transcription factors has also been extensively documented. In humans, E R K has been shown to phosphorylate transcription factors such as p 6 2 T C F / e l k - l . A recent microarray study of tobacco cell culture treated with harpin or megaspermin showed that expression of a range of transcription factors was significantly affected by elicitation, including members of the W R K Y , S C A R E C R O W , T I N Y , L I M , b H L H , SWI1, and H E families (unpublished data, Hardy Hall). The close plant homologues of S IPK and W I P K in arabidopsis, A t M P K 6 and A t M P K 3 , have been found to interact with the transcription factors W R K Y 2 2 and W R K Y 2 9 (Jonak et al., 2002) adding further support to the idea of a possible S IPK/WIPK: W R K Y interaction. A different nuclear labelling pattern was seen in treated O X : S IPK cells than in W T cells probed with anti-/?ERK. There was significantly less nucleolar label in harpin-treated O X : S I P K cells than in W T cells. As with the anti-ERK-labelled O X : S I P K cells, 86 some of these differences may reflect constitutive (pleiotropic) differences in physiology and biochemistry between these genotypes. One possible explanation is that the strong nucleolar signal might be specifically indicative of WIPK accumulation, since in an overexpressing SIPK line, WIPK is underexpressed, and the nucleolar signal would be reduced. The commercially available anti-pERK antibodies are raised against a synthetic phospho-peptide based on the rat ERK1 /2 sequence, and since E R K homologues have differing degrees of sequence similarity, these antibodies might have a higher affinity for one kinase over another. Such a binding preference could affect the immunohistochemical results and give "unbalanced" M A P K localization patterns in wild type cells. This, together with shifts in the relative abundance of SIPK and WIPK, could offer an additional explanation for some of the localization differences between WT and OX anti-pERK immunolabeled cells. One way to test this possibility would be to immunolabel transgenic cultured cells derived from a SIPK deficient genotype. SIPK elimination has been shown to lead to prolonged hyper-activation of WIPK in stressed cells, a pattern which would perhaps give a dear signal of WIPK localization. Unfortunately, the RNAi-SIPK transgenic tobacco lines available to me were not found to be a good candidate for such an experiment due to drastic physiological differences compared to WT (Appendix 3). The RNAi-SIPK cultured cells contained high levels of unidentified phenolics that produced strong autofluorescence. Large members of round grape-like structures accumulated in the cells, and the culture had a mixed composition of isodiametric and elongated cells. 87 By comparing the ant i -ERK and ant i -pERK localization results, I was able to draw preliminary conclusions, but it was not clear that all nuclear-localized S I P K / W I P K represents the phosphorylated forms of these M A P K s . Phosphorylation of M A P K s does induce conformational changes that increases their catalytic activity, but phosphorylation does not dictate E R K ' s ability to enter the nucleus. As previously reported in humans by use of an E R K : G F P fusion protein, E R K : GFP accumulated in the nucleus regardless of the phosphorylation state (Matsubayashi et al., 2001). M y results do, however, suggest that there are higher levels of cytoplasmic unphosphorylated S I P K / W I P K when comparing non-induced levels of both ant i -ERK and ant i -pERK probed cells. There are higher phosphorylated S I P K / W I P K basal levels within the nucleus in ant i -pERK probed cells than in an t i -ERK probed cells. Double-labelling using ant i -ERK and ant i -pERK could have been tested, but would have been difficult as both were rabbit antibodies. Although this study indicated that subcellular localization patterns observed in O X -S IPK and W T cells were not identical, there were many similar localization trends observed. Nuclear signal accumulation in harpin-treated cultured cells was observed in all genotypes, and with all three tested antibodies, an t i -FLAG, ant i -pERK, and ant i -ERK. The most notable differences between the O X - S I P K and W T lines were the higher percentage of nuclear localized signal in untreated cells in the overexpressing line, and the lack of strong nucleolar localization signal in harpin-treated O X : S I P K samples. The specificity o f the an t i -FLAG antibody was important to this immunolabeling study, since it tracked just S IPK 's localization. However, non-specific binding in W T cells with an t i -FLAG was higher than non-specific binding from the pre-immune seriim in W T cells, indicating that the an t i -FLAG polyclonal antibody from Sigma was not ideal for cultured 88 cells. With anti-FLAG antibody concentrations set lower than the other two experimental probes (1:500 dilution vs 1:200 dilution) the observed fluorescent signal was lower, and the overall signal/noise ratio was more significant. Despite noise difficulties, untreated cells showed a general cytoplasmic distribution, with an increase in nuclear accumulation upon treatment with harpin, similar to the results observed with anti-ERK and anti-pERK probed cells. There was very little nucleolar accumulation of the F L A G epitope, which was effectively absent from the nucleolus. Based on the results from anti-FLAG-probed cells, the localization of basal SIPK in OX: SIPK-cultured cells appears to generally be throughout the cell. Upon treatment with harpin, enhanced nuclear accumulation of SIPK is observed in OX: SIPK cells. The results obtained with anti-FLAG-probed OX: SIPK-treated and untreated cells mirror those from the anti-ERK-probed treated and untreated cells. It is therefore possible to draw the conclusion that the majority of the signal observed in anti-ERK-probed OX: SIPK cells is likely to be SIPK. Since anti-FLAG-probed OX: SIPK cells did not show nucleolar accumulation of label, it appears SIPK does not accumulate significantly in the nucleolus in these cells. From these results, it can be hypothesized that SIPK does not significantly accumulate in the nucleolus in WT cultured cells. To address this question, SIPK-specific antibodies could be used on fixed WT cultured cells, or SIPK: GFP fusion proteins could be tracked through live cell imaging. By putting together the information that anti-pERK antibody recognizes SIPK and WIPK in tobacco cells, and that anti-pERK probed harpin/megaspermin-treated cells show a strong nucleolar accumulation, one can deduce that one, or both, of the MAPKs 89 have a function in the nucleolus following elicitation. Since my data suggests that it is probably not S IPK localizing to the nucleolus, there is a good hypothesis generated that W I P K specifically has a nucleolar function. M A P K s , once activated, must find their target. To allow it to function in the regulation of multiple pathways, M A P K must have the ability to recognize a number of substrates. A l l M A P K s recognize similar phospho-acceptor sites of target proteins, (serine and threonine followed by a proline residue). Gene expression in eukaryotes is ultimately controlled at the transcriptional level by transcription factors. Many different stimuli that affect gene expression also lead to M A P K activation indicating that transcription factor function is highly dependent on phosphorylation and dephosphorylation cycles that are catalyzed by protein kinases and protein phosphatases (Hunter and Karin, 1992). A likely model for activated M A P K s is that they modify transcription factors by activation or deactivation through phosphorylation, and thus cause changes in transcription of target genes. The results of my experiment support the hypothesis that the tobacco M A P K s S IPK and W I P K are predominantly cytoplasmic, although basal levels can be found in almost all other regions of the cell in non-induced cells. Upon activation, SIPK, and most likely WIPK , move in part to the nucleus. It is hypothesized through deductive reasoning based on immunolabeling results that W I P K concentrates in the nucleolus. This shift in M A P K localization may be part of the dynamic by which they exert a regulatory role over transcription factors involved in pathogen regulation and cell death processes. 90 Quantification of results using imaging software to compare the intensities of label found in various parts of the cell may have better characterized shifts in subcellular distribution and complimented observed localization trends. Whole cell immunolabeling in tobacco cultured cells is an effective means of determining subcellular localization, and the specific methods used to immunolabel tobacco cultured cells were shown to be robust. Both negative and positive controls (Figure 2.1) showed expected results, and the experimental antibodies revealed discrete information about M A P K cellular distribution in untreated and harpin-treated cultured tobacco cells. Immunolabeling Troubleshooting The three main technical problems that arose in this study were autofluorescence from contaminants and from the tissue itself, non-specific binding of antibodies, and low abundance of immunolabeling signal within the tissue. These problems can be further discussed for insight on improvement of the methods. Autofluorescence: Immunolabeling using fluorescent probes often presents difficulty with artifactual fluorescence from natural sources within the tissue. Each tissue set offers its own difficulties, and it is important to minimize their effect, by choosing appropriate fixatives, fluorescent probes, filters, tissue types, and chemical treatments designed to minimize autofluorescence. Glycine ( ImM) can be used to bind free aldehydes (Eystathioy et al., 2002)and reduce autofluorescence. Autofluorescence from the cell wal l was seen in my material, but was minimized by digestion of the cell wal l and treating with glycine. This autofluorescence was most l ikely due to secondary structures within the cell wall. In plant tissues, some of 91 the most well known natural autofluorescent chemicals are chlorophyll, carotenoids, lignin, tannins, and phenolics (O'Brien, 1981)pp 2.27). Plant chloroplasts are another major source of auto fluorescence within the plant. .Chloroplasts occur predominantly in leaves, in the cortex of stems, and generally in tissues exposed to light. They are involved in photosynthesis, synthesis of lipids, nitrate reduction, and the temporary build up of starch (Cutter, 1978). Plant mesophyll cells contain 30-500 chloroplasts (Cutter, 1978). Chloroplasts emit red autofluorescence which can often be separated from other fluorescence during fluorescence microscopy by using appropriate filter sets (Kohler et al., 1997), but leaf and stem tissue is not the preferred tissue for immunolabeling due to the levels of chlorophyll and lignin/phenolic autofluorescence present. Non-specific binding: A second problem that arose in this study was non-specific binding. Although antibodies are supposed to recognize only one substrate, and are often purified multiple times to ensure purity, there is often non-specific binding to the experimental tissue. This problem can be accentuated by using too high a concentration of antibody, treating your sample with appropriate blocking solutions can help reduce non-specific binding, and optimization of antibody concentrations can aid in improving substrate specificity. Weak Signal: A third problem present in this study was the low abundance of immunolabeling signal within the tissue. Some explanations for the low signal could be a low abundance of native S IPK and WIPK , a low abundance of the phosphorylated form of the M A P K s , a low level of exposed epitopes within the fixed tissue or a lowered specificity of the antibody for the protein in its fixed form. Low levels of S IPK and W I P K are observed by western gel analysis where there appears to be a moderate to low 92 level of the phosphorylated protein in the non-induced, and higher amounts present in the harpin induced state. The low levels of phosphorylated M A P K s found in my experiments were consistent with the fluorescent immunolabeling and T E M data reported by Coronado et al. (2002) using the same antibody on tobacco. They found very low levels of the phosphorylated protein in the cytoplasm and nucleus, but were unable to demonstrate increased nuclear localization in mature pollen (Coronado et al., 2002). The levels of protein may appear higher in these experiments as whole cell immunolabeling was employed, whereas the Coronado group used thin sections of material. 93 CHAPTER 4: General Discussion Protein kinases, and in particular M A P K s , have an essential role in eukaryotic stress signaling pathways, as they control signaling of defense mechanisms including transcription factor activation and systemic responses. M A P K s also function as negative regulators or desensitizers of defense responses and cell death pathways (Asai et al., 2002); (Romeis, 2001). There are numerous reports of induced M A P K activity in tobacco tissues by both biotic and abiotic stresses. The most frequently studied tobacco M A P K s are S IPK and WIPK. These two kinases respond to a range of stimuli, but in particular, they are known to respond to treatment with harpin and megaspermin (Appendix Two). The objective of this study was to determine the subcellular localization of the plant M A P K s S IPK and W I P K in response to pathogen attack by harpin and megaspermin. Although E R K subcellular localization has been wel l studied in mammalian systems, E R K plant homologues have been studied to a lesser degree. The apparent involvement of the tobacco E R K homologues S IPK and W I P K in stress signaling, and probable involvement in cell death makes their study exceedingly important in both agricultural, . and biomedical circles. Tobacco was chosen as the biological system for these experiments due to its extensive use and study of in our laboratory, and due to its ease of transformation, and its availability in plant and cell culture form. Observation of l iving cells and tissues is an important tool for understanding the dynamic aspects of plant structure and function. It is equally as important as the study of embedded or sectioned material. This study used both methods to look at M A P K subcellular localization patterns. A G F P : S IPK fusion 94 construct was created for study in planta, and the antibodies ant i -ERK, ant i -pERK and an t i -FLAG were used to probe M A P K s in paraformaldehyde fixed cultured cells. The SEPK: G F P construct design and assembly was shown to yield functional G F P in yeast and S IPK in planta, but due to time constraints the construct was never studied in stable transgenic lines. In stable lines, SIPK's translation, localization, change of location in response to stimuli, and duration of localizations could have been studied using live cell imaging. The fusion construct is, however, an excellent tool for future M A P K research. The immunolabeling studies of fixed tobacco cultured cells allowed for the imaging of M A P K subcellular localization in untreated, harpin-treated, and megaspermin-treated cells. With all three antibodies used to probe the cells, it was revealed that there was a general cytoplasmic localization of M A P K within the cell, and a higher localization of label in nuclei of harpin and megaspermin-treated cells. There were variations of label distribution between cells probed with ant i -ERK, anti-p E R K , and an t i -FLAG, and between W T and over-expressing S IPK (OX:SIPK)-treated cultured cells. Differences between antibody treatment groups reflected both the antibodies themselves and the subcellular localization of their respective targets. The use of ant i -ERK, which is known to recognize S IPK and W I P K in all activation states, provided information about the general distribution of M A P K s in tobacco in untreated, harpin-treated, and megaspermin-treated cultured cells. The majority o f non-induced cells revealed a cytoplasmic localization, and upon induction, there was a shift in localization to the nucleus. In W T cells, there was also nucleolar localization of label observed. This trend was not observed in transgenic O X : S I P K cultured cells. 95 The use of ant i -pERK, which is known to be specific to the phosphorylated forms of S IPK and WIPK , revealed subcellular localization patterns of activated M A P K s within untreated and treated cultured cells. Although the majority of untreated cells revealed a general cytoplasmic localization of label, there was a higher basal level of nuclear localized M A P K in anti-/?ERK-treated cultured cells. As with the ant i -ERK probed harpin and megaspermin-treated cells, there was a strong re-localization of label to the nucleus, and more specifically to the nucleolus in anti-pERK-treated wt cultured cells. Treated O X : S IPK cultured cells did show a high nuclear accumulation of label, but did not show the nucleolar accumulation observed in W T cells. A n t i - F L A G was used to probe FLAG-tagged O X : S I P K transgenic cultured cells. The O X : S IPK cultured cells were a valuable tool for tracking S IPK 's subcellular localization apart from that of WIPK. In O X : S IPK cultured tobacco cells, S I P K was found to have a general cytoplasmic localization, with higher nuclear accumulation of label in harpin-treated cells. There was not a strong nucleolar accumulation observed in harpin-treated O X : S I P K an t i -FLAG probed cells. M A P K relocation is believed to coincide with a function in the new location. S I P K / W I P K ' s probable role in the nucleus and nucleolus, discussed in greater detail in Chapter 3, is most l ikely to phosphorylate transcription factors involved in stress perception and response elicitation. Some tools that could be used to identify the downstream targets of S IPK and W I P K include phospho-protein profiles in various transgenic genotypes of S IPK and W I P K , and using proteornic tools such as I C A T , 2D gel electrophoresis combined with mass spectrometry and microarray profiling. Preliminary microarray profiling of harpin and megaspermin-treated cultured cells has 96 revealed many putative interactors with M A P K s , many of which are transcription factors (unpublished data, Hardy Hall). Activation of these transcription factors upon elicitation with harpin or megaspermin could coincide with my subcellular localization results. The M A P K localization differences observed between O X : S IPK and W T cells are strongly believed to be secondary effects from over-expressing S IPK under the 35S promoter in cultured cells. It was hoped that a line over-expressing S IPK would merely magnify the observable S IPK signal, but after examination of immunolabeling results, it was apparent that there are many other factors involved that change native M A P K localization patterns in the over-expressing line. These possible factors, discussed in Chapter 3, either diminish, or eliminate a strong nucleolar signal in OX:SIPK-cul tured cells that was otherwise observed under the same conditions of WT-cultured cells. M y hypothesis from deductive reasoning is that the observed nucleolar signal stems from W I P K re-localization, and that overexpression of S IPK either suppresses W I P K , or competes with WIPK , thus altering W I P K ' s subcellular re-localization under elicited stress with either harpin or megaspermin. In light of the different immunolabeling patterns seen in O X : S IPK cultured cells when compared to WT , it is possible that the transgenic SIPK: G F P might have behaved in a similar fashion to O X : S IPK immunolabeled cells, giving a false impression of tobacco M A P K subcellular localization patterns. The 35S induced constructs produce un-natural amounts of the protein in regions where it might not otherwise be expressed. For a better look at native S IPK subcellular localization the upstream promoter of S I P K would need to be identified, amplified, and used in a binary vector to control SEPK's expression. 97 Although cultured cell immunolabelling experiments were not fully mirrored in seedling roots, there was an observed increase in nuclear label in harpin and megaspermin-treated seedling roots. It is possible that many of the observed localization patterns were specific to cultured cells and do not reflect the localization patterns within intact tissue. Despite the observed differences in tissue type, cell type, and differences between the antibodies used to probe these cells, all M A P K immunolabeling results revealed a general nuclear accumulation of label upon elicitation with harpin or megaspermin To effect intracellular changes M A P K s can be activated/deactivated, can activate/deactivate other proteins, and can re-locate within the cell. I have shown activated M A P K concentrates in part to the nucleus upon treatment with harpin or megaspermin. M A P K s in eukaryotic cells control gene expression through activation of downstream transcription factors, and therefore links extracellular stimuli with appropriate patterns of gene expression. These mechanisms are essential to stress perception, and reaction. 98 APPENDIX 1: A: Media Recipe for Micro-propagation of Tobacco SUM (IL) RJJvI(lL) M S salts* 4.3 g 4.3 g Vitamin mix (below) l .OmL l . O m L Glycine 0.200 g 0.200 g Sucrose 30.0 g 30.0 g Agar 3.0 g 3.0 g Phytagel 11 g 11 g N A A (1 mg/mL) 100 pL -B A ( 1 mg/mL) 2 m L -p H (w/ 1 N NaOH) 5.6 5.6 Hygromycin 0.250 g 0.250 g Carbenicil l in 0.500 g 0.500 g Cefotaxime 0.250 g 0.250 g M S salts*: Murashige & Skoog salt mixture (Murashige and Skoog, 1962) (Invitrogen) B: Vitamin Mix (1000 X) Chemical For 1 L of Stock Myo-Inositol 100.0 g Thiamine-HCl 100.0 g Nicotinic acid 500.0 g Pyr idoxine-HCl 500.0g 99 APPENDIX 2 Control Harpin Megaspermin 48 kDa 46 kDa Western Analysis of ERK1/2 from Tobacco Cultured cells treated with 40 ug/ml harpin Appendix 1.0: western analysis of 3 day old WT cultured cells at 30 min after eliciting with 40 ug/ml harpin and megaspermin-treated with 1:1000 anti-pERK overnight followed by treatment with 1:5000 anti-rabbit alkaline phosphatase for 2 h. The film was exposed for 15 min before developing. Work done by Hardy Hall. 100 A P P E N D I X 3 Appendix 3 shows the light microscope images of 3 different populations of cultured tobacco cells; Xanthi WT, overexpressing SIPK, and RNAi knock-out SIPK lines. These non-fixed cells were subcultured at 4 days, and grown in MS with hormones and shaken on a gyratory shaker in the dark. Transgenics were grown in media containing 50 mg/L kanamycin. 101 A P P E N D I X 4 CONTROL No 1°or2° Antibody CONTROL pre-immune CONTROL anti-histone Appendix 4 shows the results of immunolabelling 2 week old tobacco seedling root tips fixed with paraformaldehyde and probed with anti-pERK (1:200). Controls are the no primary or secondary application (a) shows natural autofluorescence of roots. Application of a pre-immune serum (1:200) with secondary Alexa 5 9 4 conjugated anti-rabbit IgG (1:500) (b) shows non-specific binding within root tissue. Application of anti-histone (1:500) positive control with secondary Alexa594-conjugated anti-mouse IgG (1:500) (c) shows immunolabelling of cellular histone. Anti-pERK (1:200) with secondary Alexa 5 9 4 -conjugated anti-rabbit IgG (1:500) show the subcullular location of cellular MAPKs SIPK and WIPK. Harpin (40 [xg/mL) and megaspermin elicited (40 ng/mL) samples were treated for 20 min. Samples were viewed by fluorescence microscopy and images captured at a 3 second exposure. 102 n=nucleus, c=cytoplasm, cw=cell wall, v=vacuole, d=dextran, r=resin 40X cw » 40X a-pERK Exp. 15 uM 15 uM i Appendix 5 shows toluidine blue stained (a), light and fluorescent images (b-i) of immunolabelled high pressure frozen two week old tobacco seedling roots embedded in LR white resin and sectioned (1 uM). A rabbit pre-immune serum (1:200) shows non-specific binding of the antibody(c). Anti-histone (1:500) positive control indicates cellular histone location. 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