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I fr... - .fimfifi :fiszmum. 2:22»... 4....v..4.......ri.r .2; This is to certify that the dissertation entitled Type III Protein Secretion and Host Cell Death in Plant-Pseudomonas syringae Interaction presented by Jing Yuan has been accepted towards fulfillment of the requirements for Ph-D. degree in Genetics W i Major professor Dr. Sheng Yang He Date 5/4/00 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LEBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/CIRC/DateDuo.p65-p 15 Type III Protein Secretion and Host Cell Death in Plant-Pseudomonas syringae Interaction By Jing Yuan A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 2000 ABSTRACT Type 111 Protein Secretion and Host Cell Death in Plant-Pseudomonas syringae Interaction By Jing Yuan Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins are components of type III secretion, which delivers bacterial virulence and avirulence (Avr) proteins into host cells. However, the molecular mechanism of type HI protein secretion is poorly understood. Similarly, although plant programmed cell death (PCD) is an integral part of a plant's defense during pathogenic plant-bacteria interactions, little is known about the underlying mechanisms. This research is focused on 1) studying the proteins secreted via the type III secretion system and their roles in interactions between plants and Pseudomonas syringae pv. tomato (Pst) DC3000, and 2) investigating possible similarities between PCD in plant defense and animal apoptosis. I showed that Pst DC3000 secreted multiple extracellular proteins under hrp- gene-inducing conditions. By creating hip gene mutations that affect type III secretion, I demonstrated that the production of at least five extracellular proteins, including HrpW, HrpZ, and HrpA, is under the control of the Pst DC3000 type III secretion system. In collaboration with Dr. Martin Romantschuk’s group at the University of Helsinki, I showed that the HrpA protein is associated with a novel filamentous appendage (named the Hrp pilus) on the bacterial surface, and that assembly of the Hrp pilus is controlled by the type III secretion system. These results provide the first evidence that the type 111 system is involved in the secretion of multiple proteins and pilus biogenesis. Furthermore, I provided evidence that HrpA is strongly associated with host cell walls. Caspases are a family of cysteine proteases that promote apoptosis in animal systems. I found that N-benzyloxycarbonyl-Val-Ala-Asp-chloromethylketone (Z-YVAD- CMK), an inhibitor of the interleukin-10 converting enzyme (ICE)-type caspases, prevented both the HR necrosis triggered by avirulent pathogens and the disease- associated necrosis caused by virulent pathogens on tobacco leaves. Caspase—like activity was detected in tobacco leaf tissues undergoing cell death. These results demonstrate that a key step in plant PCD involves caspase-like activities. Affinity labeling using biotinylated-YVAD identified three tobacco proteins that bind to the caspase inhibitor Z- YVAD-CMK. Bcl-Z is a pro-survival protein that appears to inhibit apoptosis in several animal systems. However, instead of inhibiting cell death, I found that the transgenic plants expressing the bcl-2 gene exhibited spontaneous leaf necrosis and showed increased resistance to the virulent bacterial pathogen Pst DC3000. Leaf cells undergoing cell death in these transgenic plants showed nuclear condensation, a phenomenon ofien observed in animal apoptosis. However, two functional conserved domains, BHl and BH2, of Bcl-2 were not found to be involved in the necrotic phenotype caused by Bel-2 expression in transgenic plants. These results suggest that expression of the human bcl-2 gene can trigger plant cell death and disease resistance, but that this process is independent of the anti-apoptotic activity of Bcl-2 in animals. COPyright by J ing Yuan 2000 To my parents ACKNOWLEDGEMENTS I wish to express my deepest gratitude to my supervisor, Dr. Sheng Yang He. I was fortunate to have him guiding my many research projects. His enthusiasm and drive were inspirational, contributing in many ways to the completion of my studies. I am also grateful to the members of my committee: Drs. Jonathan Walton, Ray Hammerschmidt and Frans de Bruijn, for their valuable advice, encouragement, and the time invested in the evaluation of this thesis. I would like to thank my colleagues Anne Plovanich-Jones, Wensheng Wei, Roger Thihnony, Qiaoling J in, Julie Zwiesler-Vollick, Paula Lee, Sruti Bandyopadhyay, Suresh Gopalan, and Wenqi Hu. It has been most beneficial to work in this joyful, friendly, and supportive environment. In doing my experiments and in writing this dissertation, I have consistently received their advice, support and encouragement. I would also like to thank Dr. Robin Buell, who was my advisor during my internship at the Institute for Genomic Research, for her advice, support, and encouragement throughout the writing of my thesis. I want to credit our collaborators Drs. Martin Romantschuk, Elina Roine and Alan Collmer, and wish to acknowledge the positive role of the Plant Research Laboratory in my graduate training. It is this excellent research institute that has fostered my development in a supportive environment with colleagues of high standing in the scientific community and staff who are always willing to help. Finally, I want to thank my husband, F anzhi Kong, for his consistent support and understanding. vi TABLE OF CONTENTS LIST OF TABLES ............................................................................................................... x LIST OF FIGURES ........................................................................................................... xi CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW 1.1. Pathogenesis by Plant Pathogenic Bacteria ............................................................ 1 1.1.1. Pathogenicity Islands ..................................................................................... 2 1.1.2. The Bacterial Type III Secretion System ....................................................... 3 1.1.2.1. The Type III Secretion Apparatus ......................................................... 4 1.1.2.2. Proteins Secreted Via Type III Systems in Animal Pathogenic Bacteria .............................................................................. 5 1.1.2.2.]. Sensors ......................................................................................... 5 1.1.2.2.2. Translocation Apparatus .............................................................. 6 1.1.2.2.3. Effectors ....................................................................................... 6 1.1.2.3. Secretion Signals of the Type III Pathway ........................................... 7 1.1.2.4. Regulation of Type III Secretion .......................................................... 8 1.1.3. hip Genes and Type III Secretion in Plant Pathogenic Bacteria ................. 10 1.1.3.1.hrp Genes ............................................................................................ 10 1.1.3.2. Proteins Secreted via the hip-Encoded Type III Pathway .................. 11 1.1.4. Similarities Between Type III Secretion and F lagellar Assembly ............... 14 1.2. Plant Resistance Response to Phytopathogens ..................................................... 16 1.2.1. The Gene-fonGene Hypothesis and Plant Resistance (R) Genes ................ 16 1.2.2. The Hypersensitive Response (HR) ............................................................. 18 1.2.3. Systemic Acquired Resistance (SAR) ......................................................... 19 1.3. Programmed Cell Death ........................................................................................ 20 1.3.1. Apoptosis in Animals ................................................................................... 21 1.3.2. Programmed Cell Death in Plants ................................................................ 21 1.4. Project Summary ................................................................................................... 23 1.5. References ............................................................................................................. 27 CHAPTER 2 THE PSEUDOMONAS S YRINGAE TYPE III SYSTEM CONTROLS THE SECRETION OF MULTIPLE EXTRACELLULAR PROTEINS AND ASSEMBLY OF A NOVEL PILUS 2.1 . Abstract ................................................................................................................. 40 2.2. Introduction ........................................................................................................... 41 2.3. Materials and Methods .......................................................................................... 43 2.3.1. Culture Conditions ....................................................................................... 43 2.3.2. Analysis of Bacterial EXPs and Surface Structures .................................... 43 2.3.3. Antibody Preparation ................................................................................... 44 2.3.4. N-terminal Amino Acid Sequencing ........................................................... 44 vii 2.3.5. Construction of hrcC and hrpS Mutants ...................................................... 45 2.3.6. Transmission Electron Microscopy (TEM) ................................................. 45 2.4. Results ................................................................................................................... 46 2.4.1. Production of Multiple EXPs by DC3000 in a hrp-Inducing Medium ........ 46 2.4.2. Cellular Distribution of DC3000 EXPs ....................................................... 46 2.4.3. Hrp-Dependent Production and Secretion of DC3000 EXPs ...................... 48 2.4.4. Identities of EXPs Produced by DC3000 in a hip-Inducing Medium ......... 50 2.4.5. Assembly of a hip Gene-Dependent Pilus by DC3000 ............................... 52 2.4.6. Association of the HrpA Protein with DC3000 Hrp Pili ............................. 55 2.5. Discussion ............................................................................................................. 57 2.6. References ............................................................................................................. 61 CHAPTER 3 CHARACTERIZATION OF TRANSGENIC ARABIDOPSIS T HALIANA PLANTS EXPRESSING THE HRP PILUS PROTEIN HRPA OF PSEUDOMONAS S YRINGAE PV. TOMATO DC3000 3.1. Abstract ................................................................................................................. 66 3.2. Introduction ........................................................................................................... 67 3.3. Materials and Methods .......................................................................................... 70 3.3.1. Bacterial Strains and Inoculation ................................................................. 70 3.3.2. Construction of Arabidopsis hrpA Transgenic Plants .................................. 70 3.3.3. Genomic DNA Isolation and Southern Blot Analysis ................................. 71 3.3.4. Protein Immunodetection ............................................................................. 72 3.3.5. RNA Isolation and Analysis ........................................................................ 72 3.3.6. Bacterial Population Assay .......................................................................... 73 3.3.7. Irnmunogold Labeling of the HrpA Protein ................................................. 73 3.4. Results ................................................................................................................... 74 3.4.1. Generation of Transgenic Arabidopsis Plants Expressing the HrpA Protein ................................................................................................ 74 3.4.2. Southern Blot Analysis of Arabidopsis hrpA Transgenic Plants ................. 75 3.4.3. Expression of HrpA in Transgenic Plants .................................................... 78 3.4.4. Localization of the HrpA Protein in Transgenic Plants ............................... 78 3.4.5. Plant-Expressed HrpA Does not Affect Pst DC3000 Pathogenesis ............ 80 3.4.6. Effect of the Plant-Expressed HrpA Protein on the Interaction between Arabidopsis and Pst DC3000 Mutants ......................................................... 82 3.5. Discussion ............................................................................................................. 87 3.6. References ............................................................................................................. 91 CHAPTER 4 REQUIREMENT OF CASPASE-LIKE PROTEASE ACTIVITY FOR PATHOGEN- TRIGGERED CELL DEATH IN TOBACCO 4. 1 . Abstract ................................................................................................................. 94 4.2. Introduction ........................................................................................................... 95 4.3. Materials and Methods .......................................................................................... 98 4.3.1. Plants, Pathogens, and Elicitor ..................................................................... 98 4.3.2. Qualitative Assay of Caspase-like Activity ................................................. 99 4.3.3. Assay for Inhibition of HR and Disease Necrosis ....................................... 99 viii 4.3.4. Detection of Caspase-like Proteins in Native Polyacrylamide Gels .......... 100 4.3.5. Affinity Labeling ....................................................................................... 100 4.3.6. Chromatographic Fractionation ................................................................. 101 4.4. Results ................................................................................................................. 102 4.4.1. Initial Detection of Putative Caspase Activity ........................................... 102 4.4.2. Inhibition of HR and Disease Necrosis by Caspase Inhibitors .................. 104 4.4.3. Effect of Caspase Inhibitor on Induction of Plant Defense Genes ............ 107 4.4.4. Preliminary Identification of Caspase-like Proteins .................................. 109 4.5. Discussion ........................................................................................................... 1 12 4.6. References ........................................................................................................... 1 17 CHAPTER 5 EXPRESSION OF THE HUMAN BCL-2 GENE IN ARABIDOPSIS T HALIANA RESULTS IN PLANT CELL DEATH AND DISEASE RESISTANCE 5.1. Abstract ............................................................................................................... 122 5.2. Introduction ......................................................................................................... 123 5.3. Materials and Methods ........................................................................................ 126 5.3.1. Construction of Arabidopsis bcl—2 Transgenic Plants ............................... 126 5.3.2. Protein Isolation and Immunodetection ..................................................... 127 5.3.3. RNA Isolation and Analysis ...................................................................... 128 5.3.4. Pathogen Infection ..................................................................................... 128 5.3.5. Microscopy Analysis ................................................................................. 129 5.4. Results ................................................................................................................. 129 5.4.1. Expression of the Human bcl-2 Gene in Arabidopsis Results in a Necrotic Phenotype ................................................................................. 129 5.4.2. Formation of Necrosis is Accompanied by Activation of Host Defense Responses ..................................................................................... 133 5.4.3. Detection of Nuclear Condensation in bcl-2 Transgenic Plants ................ 135 5.4.4. Expression of Mutant bcl-2 Genes in Arabidopsis Plants ......................... 136 5.5. Discussion ........................................................................................................... 138 5.6. References ........................................................................................................... 145 CHAPTER 6 CONCLUSION AND FUTURE PERSPECTIVES ........................................................ 149 References............. ..................................................................................................... 157 ix LIST OF TABLES Table 3.1. Summary of the effect of the plant-expressed HrpA protein on Arabidopsis-P. syringae interactions ................................................................ 83 Table 4.1. Effects of protease inhibitors on HR development in tobacco ....................... 106 Table 5.1. Effects of mutations in the BHI and BH2 domains of the Bcl-2 protein on Bcl-2-induced cell death and resistance in transgenic Arabidopsis plants ............................................................................................................... 140 LIST OF FIGURES Figure 1.1. The hrp gene cluster of Pseudomonas syringae pv. syringae 61 .................... 12 Figure 2.1. EXPs produced by P. syringae pv. tomato DC3000 ....................................... 47 Figure 2.2. Irnmunoblot analysis of the cellular location of P. syringae pv. tomato DC3000 EXPs .................................................................................................. 49 Figure 2.3. Amino-terminal sequences of P. syringae pv. tomato DC3000 EXPs ............ 51 Figure 2.4. Identification of P. syringae pv. tomato DC3000 Hrprst .............................. 53 Figure 2.5. Detection of Hrp-dependent Hrp pili on the surface of P. syringae pv. tomato DC3000 ............................................................................................... 54 Figure 2.6. SDS-PAGE analysis of bacterial surface proteins ........................................... 56 Figure 3.1. The leaf morphology of Arabidopsis transgenic plants expressing the Pst DC3000 hrpA gene fused to PR-I b signal peptide sequence ................... 76 Figure 3.2. Southern blot analysis of Arabidopsis hrpA transgenic plants ........................ 77 Figure 3.3. Detection of the hrpA transcript in the transgenic plants by northern blot analysis ............................................................................................................. 79 Figure 3.4. Immunogold labeling of the HrpA protein expressed in Arabidopsis transgenic plants ............................................................................................... 81 Figure 3.5. Growth of a Pst DC3000 hrpA mutant strain in leaves of Arabidopsis plants expressing hrpA ................................................................ 85 Figure 3.6. Symptom development in leaves of transgenic Arabidopsis plants inoculated with Pst DC3000 and its mutant strains hrpA and hrpS, carrying the avrB gene ..................................................................................... 86 Figure 4.1. Caspase-like protease (CLP) activity in tobacco leaf tissue .......................... 103 Figure 4.2. Inhibition of tobacco cell death by Z-YVAD-CMK ..................................... 105 Figure 4.3. Northern blot analysis of the effect of Z-YVAD-CMK on induction of an HR-associated tobacco gene, HHVI , and a pathogenesis-related gene, PR-3 ...................................................................................................... 108 xi Figure 4.4. Detection of CLPs in tobacco leaf tissue ....................................................... 110 Figure 4.5. Fractionation of tobacco proteins and CLP activity ...................................... 111 Figure 4.6. Affinity labeling of caspase-like proteins in tobacco leaf tissue ................... 113 Figure 5.1. Phenotypes of T2 bcl-Z transgenic plants ....................................................... 131 Figure 5.2. Levels of the Bel-2 protein in transgenic Arabidopsis plants ........................ 132 Figure 5.3. Activation of host defense responses in transgenic Arabidopsis plants expressing the bcl-2 gene .............................................................................. 134 Figure 5.4. Detection of nuclear condensation in leaves of transgenic Arabidopsis plants expressing the bc1-2 gene ................................................................... 137 Figure 5.5. Expression of Bel-2 mutant proteins, ml and mH, in transgenic Arabidopsis plants ......................................................................................... 139 Figure 5.6. Models of possible human Bcl-2 protein function in transgenic Arabidopsis plants ............................................................................................................. 142 xii Chapter 1 INTRODUCTION AND LITERATURE REVIEW Plants make up the majority of the earth's living environment. Directly or indirectly, plants also are the source of all the food on which humans and animals depend. Therefore, plant diseases can be disastrous to humankind. It is estimated that in the United States alone, crops worth almost $10 billion are lost to diseases each year (Agrios, 1997). The most famous example of disease-caused crop losses occurred in 1845, when an epidemic caused by the oomycete Phytophthora infestans triggered the Irish potato famine and resulted in the starvation death of a quarter million Irish people. Thus, it is easy to realize that studies on the mechanisms of plant diseases and development of protection strategies against plant diseases are important. Most plant diseases are caused by viruses, bacteria, fungi, and nematodes. Plant pathogens usually cause disease by disturbing the metabolism of plant cells via specific enzymes, toxins, growth regulators, and other substances that they secrete, and also by absorbing nutrients from host cells for their own use. Some pathogens may also cause disease by growing and multiplying in the xylem or phloem vessels of plants, thereby blocking the transportation of water and sugars. 1.1. Pathogenesis by Plant Pathogenic Bacteria Bacterial pathogens have been known since 1878 (Agrios, 1997) and are responsible for numerous diseases in plants. The most common Gram-negative plant pathogenic bacteria belong to the genera Agrobacteria, Erwinia, Pseudomonas, Xanthomonas, and Ralstonia. These pathogens cause leaf spots and blights; soft rot of fruits, roots, and storage organs; wilts; overgrowth; scabs; and cankers. Each given symptom can be caused by members of the different bacterial genera, and each genus may contain pathogens capable of causing different types of diseases. Although these pathogens have diverse host ranges that affect diverse symptoms, they have developed a number of similar mechanisms to subvert the physical barriers and the subsequent defense responses of hosts during their co-evolution with their hosts (Dangl and Holub, 1997) 1.1.1. Pathogenicity Islands Since the early 19805, genetic analysis of bacterial virulence factors has led to the discovery of large gene clusters called pathogenicity islands. Pathogenicity islands are found in both Gram-negative and Gram-positive bacteria. They are comprised of large DNA regions (up to 200 kb of DNA) that have a G+C content often differing from the rest of the bacterial genome. In most cases, pathogenicity islands are flanked by specific DNA sequences, such as direct repeats or insertion sequences (IS). In addition, these clusters have the tendency to be deleted frequently or to undergo amplification, and apparently have been acquired during evolution by horizontal gene transfer. Acquisition of pathogenicity islands enables an otherwise non-pathogenic bacterium to become virulent on a new host (Hacker et al., 1997). Pathogenicity islands have been found in many bacteria, such as Salmonella (Mills et al., 1995), Shigella (Sansonetti et al., 1982), Erwinia amylovora (Barny et al., 1990; Beer et al., 1991), Pseudomonas syringae (Lindgren et al., 1986), Xanthomonas campestris (Bonas et al., 1991), and enteropathogenic Escherichia coli (Jarvis et al., 1995). 1.1.2. The Bacterial Type III Secretion System Most pathogenic bacteria have tough, rigid cell walls and a cytoplasmic membrane. The cell wall allows the inward passage of nutrients and the outward passage of waste matter and digestive enzymes. Gram-negative bacteria also have a lipopolysaccharide layer. The architecture of the cell envelope of Gram-negative bacteria dictates that proteins destined to be delivered to the outside must traverse several barriers: the inner membrane, the periplasmic space, the peptidoglycan layer, and finally the outer membrane. Consequently, these bacteria have evolved a variety of mechanisms for the transfer of proteins from the cytoplasm to the extracellular environment (Pugsley, 1993). One of the highly specialized secretion mechanisms, termed type III, has been shown to enable the animal pathogenic bacteria to deliver proteins from the bacterial cytoplasm into the host cell cytosol. These bacterial proteins can then stimulate or interfere with host cellular processes and mediate bacterial-host cell interactions (Hueck, 1998). Type III secretion systems are present in both animal and plant pathogenic bacteria. They have three distinguishing features: 1) the absence of amino-terminal processing of the secreted proteins that is characteristic of proteins secreted via the sec-mediated general secretory pathway, 2) the requirement for specific accessory proteins (chaperones) for many of the secreted proteins, and 3) a widespread requirement for host cell contact for full activation of the secretory pathway. 1.1.2.1. The Type III Secretion Apparatus The type III secretion system has been widely studied in animal pathogenic bacteria, such as Yersim'a spp., Salmonella spp. and Shigella spp., as well as plant pathogenic bacteria, such as Pseudomonas syringae, Xanthomonas campestris, Erwim'a amylovora and Ralstonia solanacearum. The type III secretion apparatus is composed of approximately 20 proteins (Galan and Colhner, 1999; He, 1998; Hueck, 1998). Some of them, such as YscC protein from Yersinia spp., are believed to be associated with the bacterial envelope and to form a pore in the outer membrane (Koster et al., 1997). All known type HI secretion systems share a number of core structural components that are highly conserved (Hueck, 1998). Amino acid sequence comparison indicates that the most conserved components of the type III secretion system share sequence similarities with proteins involved in flagellar export systems (Bogdanove et al., 1996; He, 1997), suggesting that type III secretion machines are evolutionarily related to the flagellar apparatus. One of the conserved components of the secretion system also shares sequence similarity with a bacterial membrane-associated AT Pase, indicating the possible involvement of ATP hydrolysis in type III secretion. Type III secretion systems among different bacteria sometimes are functionally interchangeable (Anderson et al., 1999; Ham et al., 1998; Rosqvist et al., 1995; Rossier et al., 1999). For example, the Xanthomonas Hrp type 111 system was able to secrete PopA from Ralstonia solanacearum, AvrB from Pseudomonas syringae pv. glycinea, and YopE from Yersint'a pseudotuberculosis (Rossier et al., 1999). However, the actual mechanisms of type HI secretion are poorly understood. 1.1.2.2. Proteins Secreted Via Type 111 Systems in Animal Pathogenic Bacteria In animal pathogenic bacteria, such as Yersinia, Shigella, and Salmonella, most of the proteins secreted via the type HI pathway fall into one of three groups: participants in the signaling pathway leading to gene regulation, components of the translocation apparatus, or intracellular effectors (Galan and Collmer, 1999). 1.1.2.2.1. Sensors Delivery of type III secreted proteins into the host cell requires accessory proteins. In animal pathogenic bacteria, these proteins are secreted through the type III pathway to the bacterial cell surface where they function to sense contact with the host. This process is best understood in the enteropathogen Yersinia (Comelis, 1998). The sensing of host cells by Yersinia is modulated by three extracellular proteins, YopN, Lch, and TyeA, via an unknown mechanism (Boyd et al., 1998; Iriarte et al., 1998). These extracellular proteins are proposed to form a plug complex closing the bacterial secretion channel in the absence of host contact (Comelis, 1997). Contact with host cells removes them from all secretion channels, resulting in the full induction of the type HI secretion genes. 1.1.2.2.2. Translocation Apparatus Certain proteins secreted through the type HI system form the translocation apparatus that is deployed at the bacterial surface to deliver effector proteins into the host cell. In Yersinia, it has been suggested that two type III secreted hydrophobic proteins, YopB and YopD, form a translocation pore in the host cytoplasmic membrane, but their function remains controversial (Hakansson et al., 1996; Lee and Schneewind, 1999). The actual process of translocation through the eukaryotic host cell membrane is poorly understood. 1.1.2.2.3. Effectors Bacterial effector proteins secreted by the type 111 system are targeted to the host cell interior. Animal pathogenic bacteria often use effectors to disarm cells involved in the immune response. For example, YopE of Yersinia is involved in the disruption of the actin-microfilament structure of the host cell to prevent phagocytosis (Sory et al., 1995). Other animal pathogenic bacteria modulate actin cytoskeleton functions to either gain access to nonphagocytic cells or to attach to epithelial cell surfaces. For example, Salmonella typhimurium injects into the host cell a set of effector proteins that induce actin cytoskeleton rearrangements, membrane ruffling, and macropinocytosis, which ultimately result in bacterial uptake (Galan, 1999). One such effector protein is the actin- binding protein, SipA (Zhou etal., 1999). Another activity associated with effector proteins is to induce apoptosis in infected macrophages. For example, in addition to its role in translocation subsequent to bacterial invasion, Shigella IpaB protein has been shown to bind directly to the interleukin lB-converting enzyme (ICE) caspase in macrophages, thereby initiating the apoptotic program (Chen et al., 1996). In Yersinia, the induction of apoptosis is dependent on the fimction of YopJ/YopP (Mills et al., 1997; Monack et a1. , 1997). It seems that YopJ/YopP inhibits the mitogen-activated protein kinase in host cells (Orth et al., 1999). YopJ/YopP shares sequence similarity with effector proteins delivered by type III systems in other bacteria, such as the animal pathogen S. typhimurium (AvrA), the phytopathogen Xanthomonas campestris (Aerxv), and the legume-associated symbiont Rhizobium spp. (the product of the y410 gene). This is the only example of a type III-secreted effector protein family that includes members in both plant and animal pathogens (Galan, 1998). 1.1.2.3. Secretion Signals of the Type III Pathway Proteins secreted via the type III pathway show no sequence features or structural similarities that could function as common secretion signals. Experiments canied out with YopE and YopN, two type III secreted proteins of Yersinia, revealed that ~15 amino acids at amino-terminal are necessary for secretion and sufficient to direct the secretion of hybrid fiision proteins (Comelis et al., 1998; Sory et al., 1995). However, mutational analysis of the secreted proteins has shown a high tolerance for sequence changes within the amino terminus without loss of secretion. Therefore, the secretion signal probably resides in the 5' region of the mRN A that encodes the secreted proteins (Anderson and Schneewind, 1997). It has been demonstrated with Yersinia YopQ that such an RNA sequence-dependent secretion is coupled to YopQ translation (Anderson and Schneewind, 1999b). Recently, it has been reported that the type IH machinery of Erwinia chrysanthemi, when introduced into Escherichia coli, recognizes the secretion signals of yopE and yopQ. Pseudomonas syringae AvrB and AvrPto, two proteins exported by the recombinant Erwinia machine, can also be secreted by the Yersinia type III pathway (Anderson et al., 1999). Experiments designed to determine the AvrPto sequences required for the secretion of reporter fusions in Yersinia revealed the presence of an mRNA secretion signal. It is therefore proposed that the conserved components of the type HI secretion apparatus may recognize signals that couple mRNA translation to polypeptide secretion (Anderson et al., 1999). Certain type III secreted proteins can be secreted posttranslationally. The secretion signal of these proteins is located within the first 100 amino acids and specifies a discrete chaperonin-binding domain of the secreted protein. Removal of this domain alleviates the requirement for a cytoplasmic chaperonin for secretion, although it also prevents translocation of the protein into host cells (Anderson and Schneewind, 1999a; Cheng et al., 1997). Unlike other well-characterized chaperonins, type III-associated chaperones have a rather narrow binding specificity and appear to lack nucleotide-binding activity. The use of multiple targeting mechanisms may determine the timing of secretion of some effectors and contribute to the secretion of others. 1.1.2.4. Regulation of Type III Secretion Regulation of type III secretion takes place at both transcriptional and posttranslational levels. The regulatory genes involved in type III secretion are often located adjacent to genes associated with the respective type HI secretion system. Transcriptional regulation is accomplished by specific transcription factors. Most of them are members of a number of well-studied regulatory systems. One of the best- characterized systems of transcriptional regulation of type III secretion has been described for the phytopathogen Pseudomonas syringae. In Pseudomonas syringae pv. syringae 61, HrpS, HrpR, and HrpL are involved in the regulation of type III secretion. HrpS and HrpR share sequence similarities with the NtrC class of two-component response regulatory proteins (Grimm and Panopoulos, 1989). HrpL is a member of the ECF (extra cytoplasmic functions) of response regulators (Grimm et al., 1995; Grimm and Panopoulos, 1989; Xiao et al., 1994). It is proposed that HrpS and HrpR form a heterodimer that then binds to the upstream activation region of the hrpL gene. HrpL recognizes and activates promoters for all type HI secretion-associated genes identified in P. syringae (Xiao et al., 1994; Xiao and Hutcheson, 1994). These regulatory gene products enable bacteria to respond to environmental cues characteristic of the infection site, such as temperature, osmolarity, availability of nutrients, Ca2+, pH, or to the grth phase of the bacteria. Such cues can be mimicked in vitro. In P. syringae and other plant pathogenic bacteria, a nutrient—deficient minimal medium mimics the poor nutrient conditions found in plant apoplasts and thus induces expression of type III-associated hrp and avr genes (Xiao etal., 1992). The process of posttranslational regulation of the type IH secretion system involves contact with host cells. Contact-dependent type III gene activation has been found in the animal enteric pathogens Salmonella, Yersinia, and Shigella. The exact process is not well understood. However, certain components involved in this regulation, such as Yersinia Lch, have been identified. Lch is a negative regulator of the Yersinia type III system (Rimpilainen et al., 1992). It is secreted upon host-bacteria contact (Pettersson et al., 1996). The cytoplasmic concentration of Lch is thereby lowered, leading to increased expression of the genes of the type III system. This regulatory mechanism enables bacteria to fully express virulence genes only when they are in contact with host cells and deliver the virulence gene products into the host cell. 1.1.3. hrp Genes and Type III Secretion in Plant Pathogenic Bacteria Components of the type III secretion system of plant pathogenic bacteria are encoded by hrp (hypersensitive reaction and pathogenicity) genes (Lindgren et al., 1986). hrp genes are required for the bacterium to cause disease in susceptible plants and to elicit the hypersensitive reaction (HR) in resistant plants. HR is a rapid localized resistance response against invasion by an avirulent pathogen. The structure and function of hip genes has been studied in several plant pathogenic bacteria: Pseudomonas syringae, Erwinia amylovora, Ralstonia solanacearum, and Xanthomonas campestris. 1.1.3.1. hrp Genes hrp genes in plant pathogenic bacteria are normally clustered in a single 25—to 30- kb chromosomal region. A cosmid carrying the hrp gene cluster of P. syringae pv. syringae strain 61 is sufficient to enable E. coli to elicit HR in plants (Huang et al., 1988). 10 There are about 27 genes in the functional P. syringae hrp gene cluster (Fig. 1.1) (He, 1997; Huang et al., 1995). It contains the hrp genes involved in type III secretion and regulation. Some of the hip genes encode the effector proteins. Based on analysis of sequence homologies, among the 27 hrp genes of P. syringae, at least 16 encode proteins that appear to make up the type III secretion apparatus in the bacterial envelope (He 1997; Huang et al., 1992; Huang et al., 1995; Lidell and Hutcheson, 1994). Twelve genes show sequence similarities with Yersinia spp. type III secretion genes (ysc) (Kim et al., 1997) Of these twelve, nine share sequence similarities with flagellar assembly genes and appear to be highly conserved among all bacteria that contain type III secretion systems. These nine hrp genes have therefore been renamed as hrc genes (Lap conserved) (Bogdanove et al., 1996). 1.1.3.2. Proteins Secreted via the hrp-Encoded Type III Pathway Thus far, there appear to be two classes of effector proteins that traverse the Hrp secretion system. The first class of proteins, exemplified by harpins, is believed to be secreted to the extracellular space outside of plant cells. The other class of proteins, typified by avirulence (Avr) proteins, appears to be secreted directly into plant cells. HrpN of E. amylovora was the first Hrp protein shown to elicit an HR in tobacco and subsequently shown to be secreted in an hrp-dependent manner (Wei et al., 1992). The properties of HrpN define the characteristics of the class of HR elicitors known as harpins: hydrophilic, rich in glycine, heat stable, and able to elicit HR when injected into the apoplast of certain plants. Two other type III-dependent secreted proteins, PopA ll .moxon bum E 330%,: 2a 828:3 _8mEonooB sacs—:5 me 8:00 .moxon 52w 3 @2822: v.8 mfiouoa 33:08.55 @5525 8:00 53:89.2 .moxon v2 98 35 3 83063 2d 85» c2688 28 bone—$3“ deoao none 8m comatomqg no 552% 05 88%.: a8 05 so $65 6qu59. Sn mange? gosogouk mo 5520 anew RE 2:. .2 SEE ago—Eons E5 5893 55958 E 253. _. z .. . 9:803 oEOmoEoEU , ,, 33—3855 3:525 850 I m X? , 35 5:808 2935 I 3». u. . 85w bonfinwom I RE H 5on 50515 D I: I lo I I .I. x I I 1 e: I .\ \ \ I I I \ I .l \ \ Avoiomcowg on: I l anaemic « w a N mom: a6 .6 E b s wfimwma o 2 a see a u M3 I l l l 12 (Arlat et al., 1994) and HrpZ (or harpinpss) (He et al., 1993) from R. solanacearum and P. syringae, respectively, possess properties of harpins and thus are members of the harpin family. Both of them elicit HR when injected into tobacco apoplasts. However, the mechanisms by which harpins elicit the HR remain elusive. The second class of proteins is typified by the avirulence (Avr) proteins. Genetic analysis has revealed that avr genes mediate the elicitation of disease resistance in plants that contain corresponding disease resistance genes (Leach and White, 1996). More than 40 bacterial avr genes have been described in pathovars of Pseudomonas syringae and Xanthomonas spp. (Dangl, 1994). They do not have sequence homology to each other (with the exception of the avrBs3 family), and they encode hydrophilic and soluble proteins. In different P. syringae pathovars, several avr genes are located in regions flanking the hip gene cluster (Alme and Collmer, 1997; Lorang and Keen, 1995; Stevens et al., 1998). The phenotypic expression of avr genes in bacteria requires hrp genes (Huynh et al., 1989; Keen, 1990). In fact, a 25-kb hrp gene cluster of P. syringae 61 is sufficient to enable E. coli to deliver Avr proteins to the plant (Gopalan et al., 1996; Pirhonen et al., 1996). However, unlike harpins, addition of purified Avr proteins to the plant apoplast does not trigger an HR. Experiments with P. syringae pv. tomato DC3000 avrB, ath0 and aerpt2, and Xanthomonas campestris pv. vesicatoria avrBs3 have shown that expression of avr genes directly in plant cells carrying the corresponding disease resistance genes leads to HR, strongly suggesting hrp-dependent transport of Avr proteins into the interior of plant cells (Gopalan et al., 1996; Leister et al., 1996; Scofield et al., 1996). Yeast two-hybrid experiments have revealed a direct physical interaction 13 between Ath0 and tomato Pto, aputative intracellular receptor encoded by the tomato disease resistance gene Pto (Tang et al., 1996). More evidence suggesting that avr genes act inside plant cells comes from studies on the AvrBs3 family of proteins. AvrBs3 proteins contain functional plant nuclear localization signals (NLS) that can target a reporter protein to the plant nucleus (Yang and Gabriel, 1995). It has been shown that these NLSs are required for the AvrBs3 family of proteins to trigger an HR, suggesting that some members of the AvrBs3 family enter not only the plant cell, but also the plant nucleus. 1.1.4. Similarities Between Type III Secretion and Flagellar Assembly It has been demonstrated that three aspects of type III secretion show significant similarities to the protein products of the flagellar assembly machinery of Gram-negative bacteria. First, eight type III secretion genes share sequence similarities with the genes involved in flagellar assembly. For example, HrcC, which is similar to the flagellar outer- membrane protein, may be considered the functional equivalent of the flagellar P- and 0- rings of the outermembrane (Kim et al., 1997). HrcN and its flagellar counterpart, FliI, are cytoplasmic proteins that have several motifs characteristic of ATPases and are candidates for energizing both the assembly of flagella and the type III protein secretion apparatus (Lidell and Hutcheson, 1994). Second, in Yersinia, the regulation of flagellar assembly is similar to that of type III secretion. During flagellar assembly, flagellin and other subunits travel in an orderly manner through a hollow channel before being assembled at the distal end of the growing 14 flagellum (Macnab, 1992). The F lgM protein, a negative regulator of flagellar assembly, also travels through the channel. It has been shown that the flagellar assembly apparatus can sense abnormal assembly or breakage of the extracellular filament by measuring the concentration of FlgM in the cytoplasm (Hughes et al., 1993). Breakage of the sealed filament leads to the release of F lgM into the medium. The reduced cytoplasmic concentration of F lgM results in activation of expression of flagellar assembly genes. Finally, supermolecular structures similar to flagella have been found in Salmonella typhimurium (Kubori et al., 1998) and Shigellaflexneri (Blocker et al., 1999). Complex structures resembling needles (needle complex) that span the inner and outer membranes were visualized on the cell surface of non-flagellated strains of Salmonella. The basal structure contains two outer and two inner rings, similar to these of the flagellar basal body. InvG, PrgH and PrgK, all of which are components of the type 111 system, are the most abundant proteins in the purified needle complex (Kaniga et al., 1994; Pegues et al., 1995). InvG is also required for the assembly of appendage-like structures called invasomes (Ginocchio et al., 1994). The needle complex in Salmonella may function as the equivalent of the flagellar basal body, serving as a channel through which the proteins of the secretion apparatus and effector molecules are transported across the two bacterial membranes. The similarities between the flagella export machinery and the type III secretion system as well as the architecture of the needle complex in Salmonella and Shigella provide strong support for the postulated common ancestry of these structures (Bogdanove et al., 1996; Galan and Collmer, 1999). 15 1.2. Plant Resistance Response to Phytopathogens Plants are hosts to thousands of potentially infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses, and nematodes. However, only a relatively small proportion of pathogens successfully invades their plant hosts and cause disease. The interactions between plants and pathogenic bacteria can be classified as compatible and incompatible, mediated by bacterial virulence and avirulence (avr) genes, respectively. In a compatible interaction, a virulent bacterium infects a susceptible host plant, multiplies, and causes disease symptoms. In an incompatible interaction, the infection and multiplication of bacteria are severely restricted (Flor, 1971; Keen, 1990). Plants recognize and resist many invading pathogens by inducing a rapid cell-death response at the site of infection, termed the hypersensitive response (HR). The HR is dependent on interactions between resistance gene products in the plant and corresponding dominant pathogen avirulence (Avr) gene products. This local response of HR often triggers nonspecific resistance throughout the plant, a phenomenon known as systemic acquired resistance (SAR). Once triggered, SAR provides resistance to a wide range of pathogens that persists for days or even weeks. 1.2.1. The Gene-for-Gene Hypothesis and Plant Resistance (R) Genes Working with the rust disease of flax, H. H. Flor first postulated the gene-for-gene hypothesis in the 1940s (Flor, 1971). In this model, Flor proposed that a single gene in the host could confer resistance if the pathogen contains a corresponding gene for 16 avirulence. The concept has been proven generally applicable to many other disease interactions involving resistance against bacteria, viruses, fungi, insects, and nematodes. Since Staskawicz et al. (1987) first isolated a pathogen avirulence gene from Pseudomonas syringae pv. glycinea race 6, over 40 avirulence genes have been isolated from bacterial pathogens (Leach and White, 1996). The first plant gene cloned that follows the gene-for-gene resistance (R gene) is tomato Pto. It confers resistance to the bacterial speck pathogen (Martin et al., 1993). More than 20 disease resistance genes have now been cloned. R gene products can be grouped into two major classes. The first class is called the nucleotide-binding-site/leucine-rich-repeat (NBS-LRR) type. It includes receptors with cytoplasmic LRRs or extracytoplasmic LRRs. Examples of the cytoplasmic receptor-like proteins include Arabidopsis RPSZ and RPM1(conferring resistance to P. syringae harboring aerpt2 and aerme , respectively) (Grant et al., 1995; Mindrinos et al., 1994; Reuber and Ausubel, 1996) and tobacco N (resistance to tobacco mosaic virus) (Whitham etal., 1996). Cf-2 (Dixon et al., 1996) and Cf-9 (Jones et al., 1994) from tomato are transmembrane receptors with extracytoplasmic LRR domains. They confer resistance to different races of Cladosporiumfulvum. Some of the NBS-LRR type resistance proteins contain conserved leucine zipper and transmembrane domains. The second class of R gene products, represented by the tomato Pto protein, possesses a serine-threonine kinase domain. It confers resistance to P. syringae pv. tomato containing avrPto (Martin et al., 1993). Pto shares sequence homology to the mammalian Raf, IRAK, and Drosophila Pelle kinases. Interestingly, genetic analyses show that another LRR-NBS-containing protein, Prf, is necessary for Pto function. The 17 rice gene Xa21, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae, encodes a putative transmembrane receptor with both an extracellular LR and intercellular serine-threonine kinase domains (Song et al., 1995). The structure of Xa21 suggests an evolutionary link between LRR proteins like Cf-2 and the Pto kinase- type of protein. Little is known about the function of the various R gene products and their domains in pathogen recognition and resistance signaling pathways. Because of the structural similarities among many R gene products, a likely candidate motif of recognition for Avr gene products is the LRR domain (Bent et al., 1994; Grant et al., 1995). However, because of the diversity of bacterial avirulence proteins and the different predicted cellular locations of the LRR domains of plant resistance gene products, the process by which bacterial Avr proteins and plant R gene products recognize each other is largely unknown. 1.2.2. The Hypersensitive Response (HR) The hypersensitive response (HR) is a defense reaction of plants to pathogens. HR has been defined as a rapid tissue collapse around the infection site that leads to necrosis of the plant tissue and immobilization of the intruding pathogen (Keen, 1992). Plant pathogens that evoke an HR are called avirulent and the plant host is said to be resistant. In contrast to the rapid HR in incompatible interactions, slow necrosis also occurs in many compatible interactions. During HR, changes in membrane potential, ion flux, and lipid peroxidation have been observed (Atkinson et al. , 1990; Keppler and Baker, 1989). 18 Other defense responses are also induced. These include production of reactive oxygen species (ROS), strengthening of cell walls through the deposition of callose, lignin and related cell-wall-bound phenolics, production of phytoalexins, and biosynthesis of pathogenesis-related (PR) proteins (Bowles, 1990; Kiedrowski et al., 1992; Lamb et al., 1989; Rogers et al., 1996). However, the biochemical basis for the HR is not clear. 1.2.3. Systemic Acquired Resistance (SAR) Activation of defenses by pathogens also often extends to uninfected tissues, leading to systemic and long-lasting resistance to a broad range of pathogens; this process is termed systemic acquired resistance (Ross, 1961). The induction of a set of SAR genes, which include pathogenesis—related genes (PR genes), is tightly correlated with the onset of SAR in uninfected tissue (Metraux et al., 1990; Uknes et al., 1992; Ward et al., 1991). A key step in the SAR signaling pathway is the accumulation of salicylic acid (SA). A large set of data has established a correlation between increased SA concentration and enhanced disease resistance in tobacco, cucumber, and other plants (Malamy et al., 1990; Metraux et al., 1990). Exogenous SA has also been found to induce SAR and SAR- related gene expression (Uknes et al., 1992; Ward et al., 1991). Reduction of endogenous SA levels in plants via introduction of a bacterial salicylate hydroxylase gene (nahG) enhances the plant susceptibility to both virulent and avirulent pathogens (V emooij et al., 1994). All these data suggest that SA accumulation is required for SAR induction. However, whether SA is the translocated signal during the SAR signaling process remains controversial (Shulaev et al., 1995). Another well-known plant gene that is 19 involved in the regulation of SAR is Arabidopsis NPR] . Mutants with a defect in NPRI fail to respond to various SAR-inducing treatments, displaying little expression of PR genes and exhibiting increased susceptibility to infections (Cao et al., 1994). NPR] encodes a novel protein containing ankyrin repeats (Cao et al., 1997). It has been suggested that NPR] may regulate PR gene expression by interacting with a subclass of basic leucine zipper protein transcription factors (Zhang et al., 1999) 1.3. Programmed Cell Death In multicellular organisms, programmed cell death (PCD) is a physiological process that removes cells that are no longer needed (Ellis et al., 1991). It is a highly organized form of cell death in which cells are sacrified to maintain the normal firnctions of the rest of the organism. These unwanted cells include those that have served temporary functions; cells that are overproduced; cells that are present in inappropriate positions; and cells that die during the process of cell specialization. PCD can also serve as a mechanism to remove cells that have been damaged, and this may be important for protection against pathogens (Williams and Smith, 1993). Some pathogens have also evolved methods to take advantage of PCD and can effectively trick the host into this process, thereby avoiding the activation of host defenses (Khelef et al., 1993). Thus, PCD plays an important role in cell and tissue homeostasis and specialization, and disease. In recent years, extensive studies on animal programmed cell death have lead to the elucidation of the conserved mechanisms in programmed cell death pathways. 20 1.3.1. Apoptosis in Animals The best-characterized form of PCD in animal systems is apoptosis. Animal cells undergoing apoptosis exhibit certain morphological characteristics such as DNA fragmentation at the nucleosome linker sites, condensation and fragmentation of the nucleus, membrane blebbing, and cytoplasmic condensation (Cohen, 1993). However, not all forms of PCD involve these changes (Schwartz et al., 1993). Many of the components involved in animal apoptosis have now been identified. Growth factor deprivation (Duke and Cohen, 1986; Raff, 1992) and reactive oxygen species (ROS) including 02’ and H202 (Jacobson, 1996; Pierce et al., 1991) can trigger signal pathways leading to PCD. Oncoproteins in the Bel-2 family regulate the onset of apoptosis signaling pathways in many animal cells (Farrow and Brown, 1996). Caspases, a family of cysteine proteases, trigger proteolytic cascades that cleave and activate the execution proteins of cell death (Salvesen and Dixit, 1997), and endonucleases cause the fragmentation of nuclear DNA (Eastman et al., 1994). Ca2+ and changes in protein phosphorylation status also participate in apoptosis signaling pathways (McConkey and Orrenius, 1995; Stewart, 1994). 1.3.2. Programmed Cell Death in Plants In plants, PCD is essential for development and survival, including tracheary element (TE) formation, somatic embryogenesis, senescence, and the phytohormone- regulated degeneration of the aleurone cell layer of monocots. Characteristics of animal 21 apoptosis, such as DNA fragmentation and cell condensation, are sometimes present in these processes. Arabidopsis senescence-associated genes (SA G2 and SA GI 2) (Lohman et al., 1994) and one of the genes induced during TE formation in Zinnia (Demura and F ukuda, 1994; Minami and F ukuda, 1995) encode cysteine proteases, and presumably could function in the plant PCD pathway somewhat like caspases. A single-stranded DNA nuclease is also induced during xylogenesis in Zinnia. This nuclease is activated by Ca2+ and is likely to be responsible for the observed fragmentation of DNA (Mittler and Lam, 1995). PCD is also involved in pathogen resistance and in the development of disease- related symptoms. One hallmark of the resistance responses of plants, HR, appears to be a form of PCD. HR cell death occurs at the site of attempted attack by an avirulent pathogen, and is likely to be important for limiting a pathogen's nutrient supply (Dangl and Holub, 1997). Staining for dead cells shows that many of the cells that die during the HR lie close to the veins of the host plant (Harnmond-Kosack and Jones, 1996). These observations suggest that the bundle sheath cells surrounding veins may be more susceptible to death-inducing signals than mesophyll cells. The localized death of bundle sheath cells may prevent pathogens from gaining entry to the vascular system and spreading systemically (Carrington and Whitharn, 1998). HR-mediated cell death is an active process. It can be triggered by the purified bacterial elicitor, harpin, and it requires the host transcriptional and translational machineries (He et al., 1993). Mutants of Arabidopsis called acd2 (accelerated cell death 2) and lsdl (lesions simulating disease 1) activate the HR and multiple defenses in the absence of any pathogen (Dietrich et al., 1994; Greenberg et al. , 1994), suggesting that 22 HR cell death is under genetic control. Further study of these “lesion mimic” mutants may therefore lead to a better understanding of the general mechanism of PCD in plants. Several groups have observed rapid production of reactive oxygen species during HR. The enhanced production of H202 during HR leads to a dramatic increase in the amount of cell death observed in a soybean cell culture system (Levine et al., 1994). However, the isolation of bacterial mutants that fail to induce cell death in tobacco suspension cells, yet still induce the oxidative burst, has led to the suggestion that H202 is not sufficient to trigger the HR but may act in conjunction with other factors to activate cell death (Glazener et al., 1991). Cells that die during HR in Arabidopsis condense and shrink and ultimately look much like apoptotic cell corpses (Levine et al., 1996). A tobacco nuclease, NUC3, which is activated during the HR and is likely to participate in the fi'agmentation of DNA, has been isolated and characterized (Mittler and Lam, 1995). During HR in soybean and cowpea (Levine et al., 1996; Ryerson and Heath, 1996), as well as in tomato plants treated with host-selective Alternaria alternata phytotoxins (Wang et al., 1996), ~50 kb and oligonucleosome-sized pieces of DNA have been observed. Despite some similarities between PCD in plants and animals, it remains to be demonstrated conclusively that the mechanisms underlying PCD in plants and animals are conserved. 1.4. Project Summary When I started my Ph.D. thesis, it was known that phytopathogenic bacteria, such 23 as Pseudomonas syringae and Erwinia amylovora, secrete harpins to trigger the plant hypersensitive response. It was not clear whether other proteins were secreted by the type III secretion systems of plant pathogenic bacteria or what their functions are in plant- bacteria interactions. Therefore, I was interested in the study of proteins secreted by type III secretion systems of plant pathogenic bacteria. 1 was also interested in understanding the molecular mechanisms of HR cell death triggered by plant pathogenic bacteria, and specifically, I wanted to know whether highly conserved animal apoptosis genes, such as caspases and Bcl-2 family proteins, affect HR cell death. My Ph.D. thesis research addresses some of these questions. Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is used as a model pathogenic bacterium for my thesis project. It is a pathogen of both tomato and Arabidopsis, and it causes a hypersensitive response in tobacco leaves. Pst DC3000 contains functional hip gene clusters as well as several avirulence genes. Similarly, model plants, such as Arabidopsis and tobacco, were used to study the molecular mechanism of plant cell death triggered by pathogens. Chapter 2 summarizes my study of extracellular proteins secreted via the type 111 system of Pst DC3000. I found that DC3000 secretes multiple proteins under hrp gene- inducing conditions. By analyzing DC3000 hrcC and hrpS mutants, I demonstrated that at least three of the extracellular proteins are secreted via the type IH pathway. The secreted proteins were analyzed by amino-terminal sequencing analysis. Furthermore, in collaboration with Dr. Martin Romantschuk's group at the University of Helsinki, Finland, I demonstrated that one extracellular proteins, HrpA, is associated with a pilus- 24 like structure and that the production of this pilus-like structure (named Hrp pilus) is under the control of the type III secretion system. Chapter 3 describes my efforts to determine the firnction of HrpA in planta. Since the HrpA protein appears to be associated with the Hrp pilus, which may interact with a receptor located on the host cell surface, I tested the hypothesis that expression of HrpA in planta may interfere with the assembly of the Hrp pilus or may pre-occupy a plant receptor to prevent plant-Pst DC3000 interactions. I generated Arabidopsis plants expressing the Pst DC3000 hrpA gene. I found that the plant-expressed secreted HrpA protein is primarily located in the plant cell wall and the transgenic plants show unique leaf morphology. These results implicate a possible interaction between the HrpA protein and the plant cell wall. However, pathogenesis assays did not reveal an effect of the plant- expressed HrpA protein on the interaction between plant and Pst DC3000. Chapter 4 reports my research on the involvement of caspase-like proteases in the development of hypersensitive cell death. Caspases are a well-conserved family of cysteine proteases that promote apoptosis in animal systems. I am interested in determining whether caspase activity is required for HR cell death. By using synthetic tetrapeptides as caspase substrate, I detected an ICE-type caspase-like activity during HR. I also found that an inhibitor of the ICE-type family of caspases, Z-YVAD-CMK, inhibited HR cell death. Furthermore, by using a biotinylated Z-YVAD-CMK, I was able to detect three tobacco proteins that bind to YVAD. Chapter 5 describes my experiments to study the effect of animal Bel-2 protein on plant cell death. Bcl-2 is an oncogenic protein that acts by inhibiting programmed cell death in human cells (Garcia et al. , 1992; Vaux et al., 1988). Homologs have been found 25 in mouse, chicken, C. elegans, and Drosophila. Expression of the human bcI-2 gene in C. elegans reduced the number of cells undergoing apoptosis (V aux et al., 1992). I was interested in determining whether HR in plants can also be modulated by Bcl-2 and whether the Bcl-2 type regulatory mechanism exists in plants. For this purpose, I expressed the human bcl-2 gene in Arabidopsis. I found that transgenic plants expressing the bcl-2 gene exhibited spontaneous leaf necrosis. The transgenic plants also showed a high level of resistance to bacterial infection and a high level of PR1 expression. The nuclei in the necrotic region of the transgenic plants were condensed. 26 1.5. References Agrios, G. N. (1997) Plant Pathologi, 4th ed. (San Diego: Academic Press), pp. 25-37. Alfano, J. R., and Collmer, A. (1997). The type HI (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. J Bacteriol 179, 5655- 62. Anderson, D. M., Fouts, D. E., Collmer, A., and Schneewind, O. (1999). 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Raine, E., Wei, W., Y n J , Nurmiaho—Lassila, E. L., Kalkkinen, N ., Romantschuk, M., and He, S. Y. (1997). Hrp pilus: An hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci U S A 94, 3459-64. (as co-first author) Charkowski, A. O., Alfano, J. R., Preston, G., Yu n J., He, S.Y., and Collmer, A. (1998). The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate. J Bacteriol 180, 521 1-17. 39 2.1. ABSTRACT Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Pseudomonas syringae pv. tomato DC3000 was found to produce seven major extracellular proteins (EXPs) in a minimal medium that induces hrp genes. Secretion of five EXPs (EXP-60, EXP-45, EXP-43, EXP-22, and EXP-10) was under the control of the type 1H system. Sequence analysis revealed that three of the EXPs, EXP-60, EXP-45 and EXP-10, are HrpW, HrpZ, and HrpA, respectively. HrpZ and HrpW are HR elicitors and trigger the HR in tobacco leaves. In collaboration with Dr. Martin Romantschuk's group, we observed a filamentous surface appendage, called the Hip pilus, on the surface of DC3000 grown on solid minimal media. I showed that assembly of the Hrp pilus was dependent on at least two hrp genes, hrpS and hrcC, which are involved in type H1 gene regulation and protein secretion, respectively. I also showed that the production of the HrpA protein is correlated with the assembly of the Hrp pilus. The production of the Hrp pilus was later demonstrated to be required for plant-Pseudomonas syringae interactions. In conclusion, results described in this chapter show that the type IH system of Pst DC3000 controls the production and secretion of multiple extracellular proteins, some of which play important roles in plant-Pseudomonas syringae interactions. 40 2.2. INTRODUCTION Plant pathogenic bacteria, including the members of the genera Erwinia, Pseudomonas, Ralstonia, and Xanthomonas, are intercellular pathogens that do not invade host cells. All signal exchanges between plant cells and bacteria occur in (or through) the plant apoplast. The ability of these plant-pathogenic bacteria to elicit the hypersensitive reaction (HR) in resistant plants and to cause disease in susceptible plants is controlled by hrp genes (He, 1998; Lindgren, 1997). hrp genes are also required for the ability of all known bacterial avirulence (avr) genes to elicit the plant genotype-specific HR and resistance (Gopalan et al., 1996a; Huynh et al., 1989; Knoop et al., 1991; Pirhonen et al., 1996). The 25-kb hrp gene cluster of Pseudomonas syringae pv. syringae strain 61 is sufficient to enable nonpathogenic strains of Pseudomonasfluorescens and Escherichia coli to elicit the HR in nonhost plants (Huang et al., 1988). Sixteen of the 25 genes in this completely sequenced hrp gene cluster encode proteins that are components of the type III secretion pathway (He, 1997; Huang et al., 1992, 1995; Lidell and Hutcheson, 1994). Nine of these hrp genes, recently renamed hrc genes (Bogdanove et al., 1996a), are broadly conserved among P. syringae, Erwinia, Xanthomonas, and Ralstonia pathovars (Bogdanove et al., 1996b; F enselau and Bonas, 1995; Huang et al., 1995; Van Gijsegem et al., 1995). hrc genes, including hrcC (formerly hrpH ), share sequence similarities with ysc/lcr genes of Yersinia (Bergman et al., 1994; Fields et al., 1994; Michiels et al., 1991), mxi-spa genes of Shigella (Sasakawa et al., 1993), and inv genes of Salmonella (Galan et al., 1992; Ginoechio et al., 1992), all of which are predicted to be components of the type 41 III secretion pathway in Gram-negative bacteria and firnction in secretion of proteins required for pathogenesis. Some components of this secretion pathway are also used for flagellar assembly (Kihara et al., 1989). In plant pathogenic bacteria, the type HI secretion system encoded by hrp genes is called the Hrp secretion system (He at al., 1993) Three hrp genes of Pseudomonas syringae encode proteins involved in gene regulation: HrpS and HrpR, two homologous positive transcriptional regulators of the NtrC family, and HrpL, an alternative sigma factor (Grimm et al., 1995; Grimm and Panopoulos, 1989; Xiao et al., 1994). HrpL recognizes a consensus sequence motif ("harp box") that has been identified in the upstream regions of all hip and avr genes of P. syringae (Xiao and Hutcheson, 1994). In P. syringae, hip genes have been shown to be induced in "hrp-inducing minimal medium"; the nutrient-limiting nature presumably mimics the plant apoplast environment (He et al., 1993; Huynh et al., 1989). These hrp- inducing minimal media have been used quite extensively for studies on the regulation of hip genes and avr genes. Using a cloning approach based on HR-eliciting activity, He et al. (1993) discovered an extracellular protein from P. syringae pv. syringae 61 that depends on the P. syringae Hrp secretion apparatus. This protein was identified as HrpZ (formerly harpin”). When infiltrated into the apoplast, HrpZ is able to elicit the HR (He at al., 1993), induce systemic acquired resistance (Strobel et al., 1996), and induce plant defense-related genes in certain plants (Gopalan et al. , 1996b). However, nonpolar hrpZ deletion mutants of P. s. pv. syringae 61 still elicit HR, albeit to a lesser extent (Alfano and Collmer, 1997), suggesting the involvement of other factors in eliciting the HR. To 42 search for additional Hrp-dependent extracellular proteins in P. syringe, I examined and characterized the secreted proteins of P. s. pv. tomato DC3000, a pathogen of tomato and Arabidopsis. 2.3. MATERIALS AND METHODS 2.3.1. Culture Conditions For detection of bacterial extracellular proteins (EXPs) in liquid cultures, bacteria were first grown at 30°C to an OD600 of 0.8-1.0 in 50 ml King's medium B broth (King et al., 1954), supplemented with 100 ug/ml rifampicin. Bacteria were then pelleted and resuspended in 50 ml hrp-inducing broth (He et al., 1993) or King’s medium B broth and incubated with shaking (250 rpm) at room temperature (21-23°C) for 24 hours. For preparation of bacterial surface-associated proteins, bacteria were grown on solid hrp- inducing medium at room temperature (21-23°C) for 2 days. 2.3.2. Analysis of Bacterial EXPs and Surface Structures For preparation of EXPs, bacteria were pelleted by centrifugation at 10,000 g for 10 min. The supernatant was concentrated 50-fold using centricon concentrators with a molecular weight cutoff of 3,000 daltons (Amicon), and 10 ul was analyzed on sodium dodecyl sulfate-15% PAGE followed by staining with 0.025% Coomassie brilliant blue R-250 in 40% methanol and 70% acetic acid. For preparation of bacterial surface- associated proteins, bacteria from a 76-mm agar plate containing solid hrp-inducing 43 medium were resuspended in 1 ml of 10-mM sodium phosphate (pH 5.5), pelleted by centrifugation at 13,000 g for 10 minutes to partially remove proteins not associated with cell surface structures, and then resuspended in 0.2 ml of 10-mM sodium phosphate (pH 5.5). The bacterial suspension was pushed through a 25 G needle 4 to 5 times to shear surface structures and proteins (e. g., flagella and pili) fiom the bacteria, and was then centrifuged at 13,000 g for 10 minutes at 4°C. Twenty microliters of the supernatant was used for SDS-15% PAGE analysis. 2.3.3. Antibody Preparation Concentrated DC3000 EXPs were desalted by dialyzing against 500 volumes of phosphate-buffered saline (pH 7.0) overnight at 4 °C. Each of two New Zealand White rabbits was injected twice, two weeks apart, with 0.5 ml concentrated EXP preparation (1 mg/ml of protein) mixed with 0.5 ml Freund’s incomplete adjuvant. The third injection was performed in the same way one week after the second injection. One week after the third injection, approximately 50 ml antiserum was collected, passed through a 0.22 pm pore filter, and stored at 70°C. The initial crude EXP antiserum reacted with numerous nonspecific DC3000 cellular proteins. Therefore, we performed preabsorption of the antiserum with lysates of DC3000 and E. coli DH50L grown in King's medium B broth. 2.3.4. N-terminal Amino Acid Sequencing For analysis of the N-tenninal amino acid sequences of the EXPs, proteins were 44 separated on a large preparative SDS-15% PAGE gel and transferred to a polyvinylidene difluoride membrane. The membrane was stained with 0.025% Coomassie brilliant blue R-250 in 40% methanol and 70% acetic acid and individual protein bands were excised. The arrrino-terrninal sequence of each protein was analyzed with an Applied Biosystems protein sequencer at the University of Kentucky Macromolecular Structural Analysis Facility. 2.3.5. Construction of hrcC and hrpS Mutants For the construction of hrcC and hrpS mutants, a 10.5-kb KpnI/XbaI fragment of pSHY25 (He, S. Y. unpublished results) containing hrpRS and hrcC of P. syringae pv. tomato strain DC3000 was subcloned into pRK415 (Keen et al., 1988) to produce pJY 1 , which was used as the template for transposon mutagenesis (Taylor et al., 1989). pJY 1 was first mutagenized with mini-Tn5 Cm. Tn5 Cm insertions into hrcC and hrpS genes were screened by restriction enzyme digestion. The mutagenized hrcC and hrpS genes were then marker-exchanged into the DC3000 chromosome. Marker exchange events were selected following a standard procedure (Huang et al., 1992). Tn5 Cm insertions in the DC3000 hrcC and hrpS genes were confirmed by Southern blot analysis. 2.3.6. Transmission Electron Microscopy (TEM) For TEM observation of bacterial surface structures, a drop of bacteria or flagellum plus pilus suspension was applied to a copper grid coated with pioloforrn and carbon, followed by staining with 1% potassium phosphotungstic acid adjusted to pH 6.5 45 with potassium hydroxide. The grids were then examined with a transmission electron microscope at the Center for Optical Electronics at Michigan State University. 2.4. RESULTS 2.4.1. Production of Multiple EXPs by DC3000 in a hip-Inducing Medium In a "hrp-inducing minimal broth" that induces the hip genes of P. syringae, strain DC3000 produced at least eight major EXPs, two of which (EXP-50 and E)G’-21) were also produced in nutrient-rich, hrp-repressing King's B meditun (Fig. 2.1). EXP-36, which was not present in the experiment shown in Fig. 2.1 (lane 2), appeared in many King's B cultures, but the amount varied greatly. EXP-60, EXP-45, EXP-43, EXP-22 and EXP-10 were never observed in the supernatant of DC3000 grown in King's B medium. Thus, expression of hip genes was correlated with the production of at least five EXPs. 2.4.2. Cellular Distribution of DC3000 EXPs To determine the cellular distribution of the DC3000 EXPs and to facilitate future cloning of the EXP-encoding genes, we developed a polyclonal antibody against the DC3000 EXPs. The antibody reacted well with EXP-60, EXP-50, EXP-45, EXP-36 and EXP-21, but relatively poorly with EXP-43, EXP-22 and EXP-10, presumably because of their low antigenicity or low concentration (Fig. 2.2, lane 1). The antibody still reacted with several non-specific cell-associated proteins (Fig. 2.2, lanes 2, 4 and 6), but these the 46 EXP-60 — W . EXP-50 — . W I .\ EXP-45 — ‘ ‘ EXP-43 - ”y -. EXP-36 — ~‘ W Ni» ,1" W ,ufi' EXP-22 — W EXP-21 ' .li‘rl‘rtli EXP-10 — Figure 2.1. EXPs produced by P. syringae pv. tomato DC3000. Lane 1, Ems from a hrp- induced culture; lane 2, EXPs from 3 King's B culture. The SDS-15% PAGE gel was stained with Coomassie blue R-250. Proteins are named according to their molecular masses (in kDa). 47 proteins did not interfere with the detection of EXPs. Lane 1 in Fig. 2.2 was loaded with an EXP sample concentrated 50-fold to visualize all major EXPs. Other lanes in Fig. 2.2 were loaded with EXP samples concentrated only lO-fold to avoid detecting too many nonspecific cellular proteins. EXP-60 and EXP-45 were found to be located mainly in medium, whereas EXP-50, EXP-36, and EXP-10 were more or less equally distributed in the medium and the cell (Fig. 2.2, lanes 2 and 3). The concentrations of EXP-43 and EXP-22 in the medium were found to be low and were not detectable in EXP samples concentrated only 10-fold (Fig. 2.2, lane 3). 2.4.3. Hrp-Dependent Production and Secretion of DC3000 EXPs To examine whether the production and/or secretion of those DC3000 EXPs produced only in hrp-inducing medium were under the control of hrp genes, I constructed DC3000 hrcC and hrpS mutants via transposon mutagenesis. The DC3000 hrpS gene is required for the expression of all known hrp and avr genes in this bacterium (Grimm and Panopoulos, 1989). The hrcC gene product is a bacterial outer membrane protein that is involved in the assembly of the type III secretion apparatus (Huang et al., 1992). A mutation in the hrpS or hrcC gene would be expected to block the production or secretion of the extracellular proteins controlled by the Hrp system. The hrcC and hrp S mutants and the wild-type DC3000 bacteria were grown in hip-inducing medium. The distributions of EXPs in the three bacterial cultures were determined. As shown in Fig. 2.2 (lanes 1 and 3), strain DC3000 produced and secreted all detectable EXPs, although EXP-43 and EXP-22 were less abundant and could be visualized only when the medium 48 DC3000 hrpS hrcC EXP-60 — EXP-SO — EXP-45 — EXP-43 '— EXP-36 — ' EXP-10 — mum-I ,lr““fl\«\‘l\\u‘v“| r , WW‘ 1w!“ Hall,» .L Figure 2.2. Irnmunoblot analysis of the cellular location of P. syringae pv. tomato DC3000 EXPs. DC3000 and hrpS and hrcC mutants were grown in a hrp-inducing medium. Lane 1 was loaded with a DC3000 EXP sample concentrated 50-fold to permit visualization of all major EXPs. Lanes 2, 4, and 6 were loaded with cell suspensions. Lanes 3, 5, and 7 were loaded with 10—fold concentrated culture medium fractions. Dots to the right of bands indicate major EXPs. 49 fraction was concentrated 50-fold (Fig. 2.2, lane 1). In the hrpS mutant, EXP-60, EXP-45, EXP-43, EXP-22 and EXP-10 were not produced, whereas the production and secretion of EXP-50, EXP-36 and EXP-21 were not affected (Fig. 2.2, lanes 4 and 5). EXP-43 and EXP-22 were not detected even when the medium fraction was concentrated 50-fold (data not shown). The hrcC mutant produced EXP-60, EXP-45, EXP-43, EXP-22, and EXP- 10, but did not secrete them into the medium (Fig. 2.2, lanes 6 and 7); instead, these EXPs accumulated in the cell in the hrcC mutant (Fig. 2.2, lane 6). Interestingly, at least one additional protein of 56 kDa in size was detected in the cell fraction of the hrcC mutant. This protein could be the truncated product of EXP-60. Alternatively, it could be an additional hrp-controlled extracellular protein that normally would escape detection by Coomassie staining or by antibody binding because of its low concentration in the medium. In the hrcC mutant, EXP-56 may have accumulated to higher levels and therefore became detectable on an irnmunoblot. 2.4.4. Identities of EXPs Produced by DC3000 in a hrp-Inducing Medium To identify the DC3000 EXPs, their amino terminal sequences were determined. Figure 2.3 shows the arnino-terminal sequences of several DC3000 EXPs. The N- terrninal sequence of EXP-60 (MRIGITPRPQQ) matched the predicted product of the hipW gene, which is located near the DC3000 aer gene (Lorang and Keen, 1995). The N-terminal sequence of EXP-45 (MQALNSISSLQTSASLFPVVLNAD) matched the predicted N-tenninal sequence of HrpZ ,5, (Preston et al., 1995). The first 35 amino acids (VAFAGLTSKLTNLGNSAVGGVGGALQGVNTVASNA) of EXP-10 matched that of 50 fl p-dgpendent EXP-45 MQALNSISSLQTSASLFPVVLNAD EXP-10 MVAFAGLTSKLTNLGNSAVGGVGGALQ GVNTVASNA EXP-60 MRIGITPRPQQ Hrp-independent EXP-36 ALTVNTNVASLNVQKNLGRASDALST EXP-50 ASPITS'I'I‘GLGSGLAI EXP-21 AQPNTMTLNGYA HrpZ HrpA HrpW Flagellin Unknown Unknown Figure 2.3. Amino-terminal sequences of P. syringae pv. tomato DC3000 EXPs. A highly concentrated DC3000 EXP preparation was loaded onto a preparative SDS-15% PAGE. Separated proteins were electrophoretically transferred to a polyvinylidene fluoride membrane. Individual protein bands visualized alter Coomassie staining were cut out, and the amino-terminal sequences were analyzed at the University of Kentucky Macromolecule Structure Analysis Facility. 51 the predicted product of the hrpA gene (Preston et al., 1995). Among the Hrp- independent EXPs, the N-terrninal sequence (ALTVNTNVASLNVQKNLGRASDALST) of EXP-36 was almost identical to that of flagellins of Pseudomonas aeruginosa and Pseudomonas putida (Totten and Lory, 1990; Winstanley et al., 1994), suggesting that EXP-36 is the DC3000 flagellin. The N—termini of EXP-50 and EXP-21 were also determined, but they showed no similarity to any protein sequences deposited in the sequence databases. To determine whether EXP-45 is a homolog of HrpZ from Pseudomonas syringae pv. syringae 61 , a polyclonal antibody against the Pss 61 HrpZ protein was used to identify DC3000 HrpZm EXPs of Pss 61 and Pst DC3000 grown in hip-inducing broth were concentrated 10-fold and separated on a 15% SDS-PAGE gel. Figure 2.4 A shows the EXP profiles of Pss 61 and Pst DC3000 in a hrp-inducing medium. Major EXPs were shown in the supernatant with different gel profiles in two strains. A western blot of the gel followed by antibody detection identified EXP-45 as the DC3000 HrpZ (HrpZPsJ protein (Fig. 2.4 B). The identification of HrpZPst in the DC3000 EXP mixture confirmed that the method of using hrp-inducing medium is appropriate for identification of Hrp- controlled EXPs of DC3000 and that EXP-45 is DC3000 HrpZ. 2.4.5. Assembly of a hrp Gene-Dependent Pilus by DC3000 Examination of bacteria grown on solid hrp-inducing medium by transmission electron microscopy revealed that DC3000 produced two to three polar flagella (15-18 nm in diameter) on both King's B medium (Fig. 2.5 A) and hrp-inducing agar plates (Fig. 52 l 2 1 2 1' «b—-—-- M“ -EXP-60 —EXP-6O : B -EXP-50 -EXP-50 -EXP-45 *m b ‘ -EXP-45 HrpZPss - ”- .—.—. ~EXP-43 -EXP-43 all. ~Exp-35 -EXP-36 an. -EXP-21 -EXP-21 W .......'.-.- &+EXP-10 -EXP-10 Figure 2.4. Identification of P. syringae pv. tomato DC3000 HrpZm The culture medium of bacteria grown in a hrp-inducing medium was concentrated 10-fold. Ten-nricroliter protein samples were electrophoresed in two identical SDS-15% PAGE gels. (A) Samples were stained with Coomassie blue to visualize the EXPs of P. syringae pv. syringae 61 (lane 1) and P. syringae pv. tomato DC3000 (lane 2). (B) Samples were irnmunoblotted with a rabbit polyclonal antibody against P. syringae pv. syringae 61 HrpZPss to identify DC3000 HrpZP,,. HrpZPst has a larger molecular weight than Hrprss. 53 Y \ . lw :;..t In“ t ‘. ‘ IMMIMt' Figure 2.5. Detection of Hrp-dependent Hrp pili on the surface of P. syringae pv. tomato DC3000. (A) DC3000 grown on hrp gene-repressing King's medium B agar plates, and (B) DC3000, (C) hrcC and (D) hrpS mutants grown on solid hrp-inducing medium were examined with a transmission electron microscope after staining with potassium phosphottmgstic acid. One to three polar flagella of 15-18 nm in diameter are present on most rod-shaped bacteria (surrounded by dark shadows) in samples (A-D); in panel B, many Hrp pili of 6-8 nm in diameter are also present (indicated by arrows). Scale bars = 200 nm. 54 2.5 B). In addition, DC3000 also produced many pilus-like appendages (6-8 nm in diameter) on solid hrp-inducing medium (Fig. 2.5 B), but not on King's B medium plates (Fig. 2. 5A). The pilus-like appendages were found both on the bacterial surface, with no consistent distribution pattern, and as detached pilus clusters. The pilus-like appendages were easily fragmented during sample preparation, as evidenced by the presence of many short pieces of pili (Fig. 2.5 B); therefore, the length of these pilus-like appendages could not be determined. To determine whether the formation of pilus-like appendages is under the control of the Hrp system, I examined the surface of the DC3000, hrcC, and hrpS mutants grown on solid hrp-inducing medium. Polar flagella, but not the pilus-like appendages, were seen on the surfaces of the hrcC and hrpS mutant bacteria (Fig. 2.5 C and D). These results demonstrate that the formation of the pilus-like appendages, but not flagella, is under the control of hrp genes. Based on its hip-dependent property, we propose the name "Hrp pilus" for this pilus-like structure. 2.4.6. Association of the HrpA Protein with DC3000 Hrp Pili To analyze subunits of the Hrp pilus, cell surface structures (flagella and Hrp pili) were prepared from DC3000 and its hrcC and hrpS mutants, and subjected to SDS-PAGE analysis (Fig. 2.6). Only DC3000 grown on solid hrp-inducing medium produced the HrpA protein (Fig. 2.6, lane 1). Neither DC3000 grown on King's B agar medium (Fig. 2.6, lane 4), nor the hrcC and hrpS mutant bacteria grown on solid hrp-inducing medium, produced the protein (Fig. 2.6, lanes 2 and 3). Besides the HrpA protein, EXP-50 and 36- 55 M l 2 3 4 “yawn—‘11,?“ 1‘“ ‘.\‘1 1 w" 11‘ 1‘ 1 (11“ 11 1 " “,1 1‘“11\\\§\ i W .1111 111‘ 1 (11:;‘1111 ‘“\“\\\11\1“\\1\“‘ ‘11“\ l\ “1“ 111“" 1‘“, 1" ‘ , . “'1‘ 11‘1‘“\:“: \\\““1\l'“\\11 “I \ “‘“| 31““; 1 ““31" W“ ‘ 1‘ I 1““ 11 “‘.111\‘“\.“\““ "‘ I‘“i“1\"““ \ 1‘1““11“, “1"““111"“1“““\1“ \ 111“:\:11“\\ 1\‘ Figure 2.6. SDS-PAGE analysis of bacterial surface proteins. An SDS-15% PAGE gel loaded with protein samples prepared from the surface of DC3000 (lane 1), hrcC mutant (lane 2), and hrpS mutant (lane 3) grown on solid hrp-inducing medium, or DC3000 grown on King's medium B agar plates (lane 4). The gel was stained with Coomassie blue. Lane M, molecular mass markers (Bio-Rad) in kDa. Arrowhead indicates the 10- kDa protein. 56 kDa flagellin proteins were also found on the cell surface. However, hrcC and hrpS mutations did not affect the production or secretion of these two proteins in hrp-inducing solid medium. Thus, the presence of the Hrp pilus is specifically correlated with the appearance of the HrpA protein on the bacterial surface. 2.5. DISCUSSION I identified five DC3000 Hrp-controlled EXPs in this study: HrpW, HrpZ, EXP- 43, EXP-22, and HrpA. Among these five Hrp-controlled EXPs, HrpZ is a homolog of harpin”, of DC3000; HrpW was subsequently shown to be a harpin-like protein that triggers HR in tobacco (Charkowski et al., 1998); HrpA was subsequently demonstrated by mutagenesis analysis to be required for Hrp pilus assembly and plant-Pst DC3000 interactions (Roine et al., 1997). The identities of EXP-43 and EXP-22 remain unclear. These Hrp-controlled EXPs may serve several functions during plant-P. syringae interactions. First, EXPs such as HrpZ and HrpW are harpin-like proteins and both have elicitor activity (Charkowski etal., 1998; He et al., 1993). Avr proteins now appear to be both essential and sufficient for elicitation of the genotype-specific HR once they are delivered to the plant cytoplasm (Gopalan et al., 1996a). Thus, the ability of isolated P. syringae HrpZ and HrpW proteins to elicit the HR in plant apoplast may not reflect their primary biological fimctions as bacterial elicitors. Instead, HrpW and HrpZ may be possible virulence factors that cause plant cells to release nutrients required for bacterial grth in the apoplast. Second, some EXPs may be involved in sensing the apoplast environment, inducing the expression of hrp and avr genes through activation of hrpS, 57 hrpR, and hrpL. In Yersinia spp., an extracellular protein (YopN) secreted via the Ysc type III secretion apparatus was shown to be involved in the detection of lower calcium levels in vitro and host-bacterial contact in vivo. These are both required for expression of yop genes (F orsberg et al., 1991; Rosqvist et al., 1994). It is not known whether a similar mechanism exists in P. syringae. Finally, some of the EXPs, such as HrpA, may be involved in the assembly of a translocation apparatus that delivers bacterial virulence proteins into the plant cell. This idea is consistent with the observations that the AvrB protein of P. syringae can elicit a genotype-specific HR when produced inside Arabidopsis cells containing the corresponding RPM! resistance protein (Gopalan et al., 1996a) and that P. syringae produces a hrp-dependent pilus-like structure (the Hrp pilus). During Avr protein delivery, some hrp-dependent EXPs may interact with and thus modify the plant cell wall and facilitate the translocation of the bacterial proteins into the host cell. Harpins appear to act on the plant cell walls. HrpZ associates with the walls rather than the membranes of plant cells, and the protein elicits no response from protoplasts lacking walls (Hoyos etal., 1996). C-terminal amino acids of HrpW share similarities to several fungal pectate lyases from Nectria haematococca and several bacterial Pel homologs (Charkowski et al., 1998). The Pel domain in HrpW binds to pectate, which is the component of the cell wall matrix that controls porosity (Guo et al., 1995; Shevchik et al., 1997). The association of bacterial harpins with the plant cell wall suggests possible modification of the plant cell wall by the infecting bacteria. The nucleic acid sequence of the P. syringae pv. tomato DC3000 hrpA gene, the first gene of the hrpZ operon, was previously determined (Preston et al., 1995). hrpA codes for a hydrophilic protein with a predicted molecular weight of 10 kDa. The primary 58 amino acid sequence of HrpA does not show any significant homology to those of characterized pilin proteins. However, computer analysis using the PROPSEARCH program (Hobohm and Sander, 1995), which identifies structural similarities between proteins without looking for primary amino acid sequence homology, indicates that HrpA is structurally similar to several pilin proteins, especially to the AF /R1 pilus chain A precursor of E. coli (with a 41% reliability). A nonpolar hrpA mutant strain does not produce HrpA or form the Hrp-pilus, and loses the ability to initiate pathogenesis or to elicit the HR in plants, a typical phenotype of all hrp mutant strains (Roine et al., 1997). These results strongly suggest that HrpA is essential for the Hrp pilus assembly and Pst DC3000 to initiate interactions with plants. However, the exact function of the Hrp pilus remains to be determined. Our finding that the Hrp pathway is involved in the formation of a pilus structure is consistent with the observation that many Hrp proteins are structurally related to those that participate in the construction of bacterial flagella (Kihara et al., 1989). This suggests an involvement of Hrp proteins in the assembly of an extracellular supermolecular structure. Salmonella, Shigella, and Yersinia strains, all of which contain type III secretion systems, secrete proteins required for pathogenesis (Hueck et al., 1995; Parsot et al., 1995; Persson et al., 1995; Rosqvist et al., 1994). Both Salmonella typhimurium and Shigellaflexneri are enteroinvasive pathogens. S. typhimurium transiently produces filamentous surface appendages of 60 nm in diameter upon contact with epithelial cells during its invasion of host cells (Ginocchio et al., 1994). The structural components of these appendages have not been identified. Yersinia spp. are not intracellular pathogens, but during infection they secrete virulence proteins through contact zones between 59 bacteria and host cells (Rosqvist et al., 1994). The secreted proteins of S. flexneri and Yersinia spp. form various aggregates and protein complexes in liquid, stationary-phase cultures (Michiels et al., 1990; Parsot et al., 1995). However, the relationship between these protein aggregates and possible formation of surface appendages in these bacteria remains to be determined. Agrobacterium tumefaciens was shown to produce pili involved in T-DNA transfer (F ullner et al., 1996). Pili of 3.5 nm in diameter were formed under vir gene-inducing conditions. These pili were proposed to function as conjugation pili in T-DNA transfer between bacteria and plant cells. The protein components of the T- DNA secretion pathway encoded by virB genes share sequence similarities with proteins involved in the assembly of conjugative pili, but not with protein components of the type III secretion system. The structural proteins of the A. tumefaciens pilus have yet to be identified. Recent results suggest that the action sites of P. syringae AvrB and possible X. campestris pv. malvacearum Avr/Pth proteins are inside the plant cell. A previous study showed that close bacterial contact is required for bacterial elicitation of the HR (Stall and Cook, 1979). The Hrp pilus identified in this study may be involved in the delivery of AvrB and possibly other virulence and avirulence proteins to the plant cell. Alternatively, it may be involved in mediating contact between bacteria and plant cells in the plant intercellular space. The exact function of the Hrp pilus in protein transfer or cell-cell contact remains to be determined. 60 2.6. REFERENCES Alfano, J. 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J Bacteriol 176, 3089-91. 65 Chapter 3 CHARACTERIZATION OF TRANSGENIC ARABIDOPSIS T HALIANA PLANTS EXPRESSING THE HRP PILUS PROTEIN HRPA OF PSEUDOMONAS S YRINGAE PV. TOMA T 0 DC3000 3.1. ABSTRACT Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. The hrp genes of Pseudomonas syringae pv. tomato (Pst) DC3000 are involved in the assembly of a surface appendage called the Hrp pilus. The HrpA protein is identified as a major component of the Hrp pilus. A Pst DC3000 hrpA deletion mutant is unable to elicit HR or cause disease in plants, suggesting an essential function of the HrpA protein in plant-Pst DC3 000 interactions (Roine etal., 1997). In this study, I explored the possibility that the HrpA protein expressed in planta would interfere with the function of the Hrp pilus and alter the interactions between the plant and Pst DC3000. I expressed the hrpA gene fused to the tobacco PR-I b signal peptide sequence in A. thaliana plants. Leaves of hrpA transgenic plants exhibited a unique pocked surface. Immunogold localization studies showed that the expressed HrpA protein accumulated in the Arabidopsis cell wall but not in the intercellular space. The HrpA protein in the Arabidopsis cell wall could not be extracted by boiling in a 0.1% SDS solution, indicating a strong interaction between the HrpA 66 protein and the Arabidopsis cell wall. Pathogenesis assays showed that the HrpA protein expressed in the transgenic plants had no effect on disease symptoms caused by Pst DC3000. However, hrpA transgenic plants showed I-IR-like cell death in response to infiltration of DC3000 hrpA and hrpS mutants carrying the avrB gene, indicating that the hrpA transgenic plants may be more sensitive to bacteria-induced tissue damage. 3.2. INTRODUCTION Bacteria belonging to the genera Pseudomonas, Xanthomonas, Ralstonia and Erwinia are responsible for many diseases in plants. However, not all pathogenic bacteria cause disease on all plants. The interactions between plants and pathogenic bacteria can be classified as compatible and incompatible, mediated by bacterial virulence and avirulence (avr) genes, respectively. In a compatible interaction, a virulent bacterium infects a susceptible host plant, multiplies, and causes disease symptoms. In an incompatible interaction, the infection and multiplication of bacteria are severely restricted (Flor, 1971; Keen, 1990). Study of host-pathogen interactions in recent years has led to the discovery that many Gram-negative bacterial pathogens, including Pseudomonas syringae pv. tomato DC3000, possess a unique protein secretion system referred to as type IH. The type IH secretion system has been shown to play a critical role in bacterial infection of plants and animals (Galan and Collmer, 1999; He, 1998; Hueck, 1998). Rosqvist et al. (1994) provided the first evidence for the transport of a bacterial (Yersinia Pseudotuberculosis) virulence protein into animal cells through the ysc-encoded type III secretion system. 67 Subsequently, research has demonstrated that such a protein transfer mechanism is common among many pathogenic bacteria of plant and animal hosts. Several general features of type III protein secretion have been revealed. First, type III protein secretion appears to be activated fully upon contact with the host cells in viva (Pettersson et al., 1996). Second, extracellular filamentous appendages are often associated with type III protein secretion (Ginocchio et al. , 1994; Knutton et al., 1998; Roine et al., 1997). Third, the secretion apparatus is genetically and morphologically similar to the bacterial flagellum (Kubori et al., 1998). In plant pathogenic bacteria, the type IH protein secretion system is encoded by hrp (for hypersensitive reaction and pathogenicity genes) (Lindgren et al., 1986). The Hrp secretion system of Pseudomonas syringae has been shown to secrete two families of proteins that elicit a host response: harpins, such as HrpZ and HrpW (Charkowski et al., 1998; He et al., 1993; Yuan and He, 1996), and Avr proteins (Mudgett and Staskawicz, 1999; van Dijk et al., 1999). Unlike harpins, which trigger the HR when infiltrated into the apoplast of certain plants, Avr proteins are thought to be delivered to the plant cell interior to elicit a host response (Gopalan et al., 1996; Leister et al., 1996; Scofield eta1., 1996; Tang et al., 1996). How Avr proteins are delivered through bacterial and plant envelopes is unclear. In a previous study, we showed that Pseudomonas syringae pv. tomato DC3000 produces a hrp-dependent pilus (the Hrp pilus). One of the Hrp-dependent extracellular proteins, HrpA, was shown to be the main component associated with the Hrp pilus (Raine et al., 1997a, 1997b; Taira et al., 1999). Furthermore, a hrpA mutant no longer produced the Hrp pilus and lost the ability to initiate both compatible and incompatible interactions with its plant host, suggesting an essential role of the Hrp pilus in Pst 68 DC3000 pathogenesis. Formation of hrp-dependent pili appears to be a common function of hrp genes in plant pathogenic bacteria. So far, Hrp pili have been found in E. amylovora, X. campestris and R. solanacearum (S. Y. He, personal communication). However, the exact role of the Hrp pilus in the transport of virulence and Avr proteins during bacterial infection is not known. The Hrp pilus may serve as a channel to deliver Avr proteins into the host cells or as an attachment apparatus to bring bacteria close to host cells during infection. Either way, a physical interaction between the Hrp pilus and the host cell surface would seem to be important for Hrp pilus function. It has been shown that bacterial binding to plant cells in the apoplast is essential for the elicitation of the HR (Hoyos et al., 1996). This raises the possibility that a plant receptor for the Hrp pilus may exist on the surface of the plant cell and that contact between such a plant receptor and the Hrp pilus may be important for initiating interactions between plant and Pst DC3000. Although the exact composition of the Hrp pilus has yet to be resolved, the HrpA protein appears to be the major component of the Hrp pilus (Roine et al., 1997a, 1997b). We hypothesized that overexpression of the HrpA protein in the plant apoplast may alter the ratio of the Hrp pilus subunits and thus disturb the Hrp pilus assembly in planta. Alternatively, overexpression of the HrpA protein in planta may preoccupy the putative plant cell surface receptor(s) for the Hrp pilus. In either case, Arabidopsis plants overexpressing HrpA protein may have increased resistance to Pst DC3000. In this study, I explored these possibilities with the aim of creating a new disease control strategy. 69 3.3. MATERIALS AND METHODS 3.3.1. Bacterial Strains and Inoculation The virulent bacterial strains used in this study were Pseudomonas syringae pv. tomato DC3000 and its hrpA and hrpS mutant derivatives (Roine et al., 1997b; Yuan and He, 1996). The avirulent strains used were Pst DC3000 and its hrpA and hrpS mutant derivatives carrying the avrB gene (Gopalan et al., 1996). Bacterial strains were grown at 30°C to OD600 =1.0 in 50 ml Luria-Bertani (LB) broth (Sambrook et al., 1989), supplemented with appropriate antibotics. Bacteria were then pelleted and resuspended in water to 2x108 cells/ml. For the HR assay, bacterial suspensions were hand-infiltrated into Arabidopsis leaves using a needleless syringe. For pathogenicity assay, bacterial suspensions were diluted to 1x106 cells/m1, unless otherwise indicated, and then hand- infiltrated into leaves. 3.3.2. Construction of Arabidopsis hrpA Transgenic Plants The hrpA coding region devoid of the start codon was amplified from Pseudomonas syringae pv. tomato DC3000 by PCR. Primer A (5’— TCGCCGCGGGTCGCATTTGCAGGATTAAC—3’) and primer B (5’— GGGTCTAGATCAGTAACTGATACCTTTAGCGTT—3’) were used. The amplified hrpA fragment was purified from an agarose gel using the Prep-A-Gene Kit (Bio-Rad). The hrpA PCR product was cloned into the SacII/XbaI site of pKYLX71 ::35S2 carrying a 70 tobacco PR-I b signal peptide coding sequence (Comelissen et al., 1987; Gopalan et al., 1996). The nucleotide sequence surrounding the start codons of the PR-I b signal peptide sequence was modified to A'3CCATGG” to conform to the consensus sequence for high- level translation in eukaryotic cells. The hrpA expression plasmid was introduced into Agrobacterium strain GV3 850 (Zambryski et al., 1983) by tri-parental mating (F igurski and Helinski, 1979). The plant transformation protocol followed that of Bechtold et al. (1993). Seeds were plated on Murashige-Skoog (MS) media containing 0.8% agar and 1x vitamins, supplemented with 50 ug/ml kanamycin. Individual transfonnants were analyzed for the expression of hrpA by northern gel blot analysis. 3.3.3. Genomic DNA Isolation and Southern Blot Analysis Genomic DNA was isolated from 6-week-old Arabidopsis plants of the T3 generation. One leaf disc of Arabidopsis was ground in 400 pl 100 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1% SDS. Proteins were removed by extraction with 400 pl of phenol:chloroform:isoamyl alcohol (25:24: 1). The tubes were vortexed and then spun for 5 minutes and the upper phase was transferred to a new tube. One-tenth volume of 3 M sodium acetate and 2 volumes of 100% ethanol were added. After incubation for 15 minutes at room temperature, the DNA was pelleted at 15000 g for 10 minutes. The pellet was washed with 70% ethanol, dried, and redissolved in 20 ul of water. A lO-ul DNA sample was then digested with XbaI at 37°C ovemight. DNase-free RNase (0.25 ug) was added into the reaction buffer to remove the contaminating RNA. Southern blot analysis was performed according to Sarnbrook et al. (1989). 71 3.3.4. Protein Immunodetection For analysis of expression of the HrpA protein in transgenic plants, equal amounts of leaf discs were homogenized in SDS-PAGE loading buffer, and boiled. Total leaf protein was separated by SDS-15% PAGE. Proteins were stained with Coomassie Brilliant Blue R-250. For immunodetection of the HrpA protein, proteins were electroblotted from an SDS-PAGE gel to an Immobilon-P membrane (Millipore), which was incubated with a rabbit polyclonal antibody raised against the HrpA protein (Roine et al., 1997b). After reaction with an anti-rabbit IgG antibody conjugated with alkaline phosphatase, the HrpA protein band was visualized by incubating the membrane with 5- bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (Sigma). 3.3.5. RNA Isolation and Analysis Arabidopsis leaves were collected and frozen in liquid nitrogen. RNA was extracted by using the RNAgents total RNA isolation system (Promega). Three pg RNA samples were fractionated on a 1.2% formaldehyde gel and transferred to an Irnmobilon- N transfer membrane (Millipore). The membranes were hybridized with DNA probes of the hrpA gene. The DNA probes were labeled by random priming using the Prime-It H kit (Stratagene). Hybridization and washing of blots were conducted as recommended by the manufacturer. The blots were exposed to X-ray films to visualize the corresponding transcripts. 72 3.3.6. Bacterial Population Assay Bacterial inoculations were performed on 5-week-old Arabidopsis thaliana Col glI plants. Whole young leaves of wild-type and hrpA transgenic plants were infiltrated with a Pst DC3000 suspension of 2x106 cells/ml for compatible interaction and 2x108 cells/ml for incompatible interaction by using a needleless syringe. Leaves were removed from intact plants 5 days after inoculation for photography. For bacterial growth assay, bacterial numbers were monitored over a 3-day time course. At each time point, four leaf disks were removed using a No. 5 cork borer from four different plants, surface sterilized, and ground in a 1.5-ml microcentrifuge tube containing 0.5 ml of dHZO. Dilutions were made in dH,O and plated on selective medium. Colonies were counted 48 hours later. 3.3.7. Immunogold Labeling of the HrpA Protein For detecting the plant-expressed HrpA protein in the transgenic plants, Arabidopsis leaf tissue was collected and fixed for 2 hours in 2% glutaraldehyde dissolved in 0.1 M sodium cacodylate, pH 7.2, at room temperature. The samples were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate (pH 7.2) for one hour at room temperature, followed by dehydration in a series of acetone dilutions, and embedded in the resin "Spurtol". Ultra-thin sections (70-90 nm) were cut using a diamond knife and placed on uncoated nickel grids. The grids were then treated with a saturated aqueous solution of sodium metaperiodate (Sigma) (Bendayan and Zollinger, 1983). After incubation in 20 uM Tris, pH 7.0, 0.5% BSA and 0.05% Tween 20 (TBS buffer) for 73 30 minutes at room temperature, the grids were washed in TBS buffer four times, for 5 minutes each, and incubated with goat anti-rabbit IgG conjugated with 10-nm gold particles (Sigma) for 2 hours. The grids were washed in TBS four times, each for 5 minutes, and stained with 1% potassium phosphotungstic acid (pH 7.0). Before examination by electron microscopy, grids were post-stained first with 1% uranyl acetate for 30 minutes, rinsed in distilled water, and then stained with Hanaichi’s lead solution (I-Ianaichi et al., 1986) for 5 minutes, followed by rinsing in 20 mM NaOH and deO. After staining, grids were observed with a CM10 transmission electron microscope (Philips). Micrographs were taken at an accelerating voltage of 80 kV. The experiment was performed at the Center for Optical Electronics at Michigan State University, with the help from Dr. Wenqi Hu. 3.4. RESULTS 3.4.1. Generation of Transgenic Arabidopsis Plants Expressing the HrpA Protein To examine whether the HrpA protein expressed in plants would interfere with Hrp pilus function by either pro-occupying the hypothetical plant cell wall receptor or disrupting the assembly of a functional Hrp pilus in planta, I expressed the P. syringae pv. tomato DC3000 hrpA gene in Arabidopsis plants. The coding region of the hrpA gene from the Pst DC3000 was cloned into a plant transformation vector with a signal peptide sequence. Therefore, the HrpA protein was expected to be expressed and secreted into the plant apoplast. The construct was introduced into Arabidopsis thaliana plants via 74 Agrobacterium-mediated transformation. About 25 independent lines of T1 transgenic plants with the hrpA gene were obtained. The leaf surface of all transgenic Arabidopsis plants showed an irregular pockmark appearance (Fig. 3.1). The number of pockmarks varied among different transgenic lines and the pockrnarks were more obvious in older leaves. The pockmark appearance was a stable phenomenon and it was passed to the T2 and T, transgenic plants. Eighty percent of the lines of the hrpA transgenic plants exhibited a stunted morphology. Control plants transformed with pKYLX::35S2 vector alone showed normal leaf morphology and growth. 3.4.2. Southern Blot Analysis ofArabisapsis hrpA Transgenic Plants To confirm that the kanamycin-resistant Arabidopsis plants contained the hrpA gene, Southern blot analysis was performed. Genomic DNA was extracted from seven independent T, transgenic lines as well as a control Arabidopsis plant. The genomic DNA was digested with XbaI, which cuts once within the T-DNA; therefore, the number of hybridization bands detected in the Southern blot analysis could be used to estimate the copy number of the hrpA transgene in the genome. As shown in Fig. 3.2, the hrpA transgene was detected in all of the seven hrpA transgenic plants, but not in the control plant transformed with vector alone. Lines 3 and 6 contained a single hybridization band, suggesting that they contained a single copy of the hrpA transgene. Transgenic lines 1, 2, 5, 8, and 9 contained multiple hybridization bands, suggesting multiple insertions of the hrpA transgene in the genome. The exact copy number of the hrpA transgene could not 75 Figure 3.1. The leaf morphology ofArabidopsis transgenic plants expressing the Pst DC3000 hrpA gene fused to PR-I b signal peptide sequence. (A) An Arabidopsis plant transformed with vector. (B) A hrpA transgenic plant. (C) Leaves from the hrpA transgenic (right) and control (lefi) plants. 76 .\ 1‘ -‘ (hi |V\ it w. “ . , ‘ .‘u . ‘.‘ at)?“ u“. 1““ Figure 3.2. Southern blot analysis of Arabidopsis hrpA transgenic plants. Individual transgenic lines are indicated by the numbers. C represents the control plant transformed with the vector. The last lane, “hrpA”, was loaded with the hrpA gene as a positive control. 77 be determined because the multiple bands could be generated from incomplete enzyme digestion. 3.4.3. Expression of HrpA in Transgenic Plants To test whether the hrpA transgene was expressed and whether the pockmark appearance was related to the expression level of the hrpA gene, northern blot analysis was performed. The hrpA transcript was detected in ten randomly selected T, transgenic lines (Fig. 3.3). Most importantly, the hrpA expression level was correlated with the degree of the pockmark appearance of the leaves. Transgenic lines 3, 7, and 9, which exhibited higher hrpA expression levels, showed more pockmarks. These three lines also exhibited an overall stunted appearance, presumably because of the over-production of HrpA protein. This result correlates the pockmark appearance of the leaves of hrpA transgenic plants with the hrpA gene expression level. I then attempted to detect the HrpA protein in the transgenic plants. No HrpA protein was detected using western blot analysis in any of the transgenic lines (data not shown). 3.4.4. Localization of the HrpA Protein in Transgenic Plants Because HrpA is a major component of the Hrp pilus, we speculated that it may have a strong affinity for plant cell walls, and this may prevent the release of the HrpA protein into the soluble fraction during sample preparation for immunoblot analysis. Therefore, I conducted qualitative immunogold-labeling experiments to examine 78 hrpA rRNA Figure 3.3. Detection of the hrpA transcript in the transgenic plants by northern blot analysis. Ten independent hrpA T, transgenic lines are indicated by the numbers on the top. C indicates the control Arabidopsis line transformed with the expression vector pKYLX::35S2. Upper panel was hybridized with the DC3000 hrpA gene. Lower panel shows ethidium bromide-stained rRNA. 79 whether HrpA was produced in transgenic Arabidopsis plants, and if so, where the expressed HrpA protein was localized. Transgenic lines 3 and 9, which had a more pronounced pockmark appearance on the leaves, were chosen for this experiment. The HrpA protein expressed in the plants had the PR-Ib signal peptide fused to its N- terminus, and was expected to be secreted out of the plant cell. However, the preliminary result showed that the majority of the HrpA protein, as indicated by gold particles, was detected in the plant cell wall. The HrpA protein was not observed in the extracellular space, and a few gold particles were detected inside the plant cells (Fig. 3.4). This result suggests that the HrpA protein is retained in the plant cell wall. The fact that boiling in the SDS buffer could not release HrpA from the transgenic plant cell wall indicates a strong association between the Arabidopsis cell wall and HrpA. Failure to detect the HrpA protein by western blot analysis could therefore be attributed to a strong association with the host cell wall. 3.4.5. Plant-Expressed HrpA does not Affect Pst DC3000 Pathogenesis Since HrpA is a major component of the Hrp pilus and has a strong association with the plant cell wall in the transgenic Arabidopsis plants, it may occupy sites in the cell wall with which the Hrp pilus normally interacts. Alternatively, HrpA expressed in plants may interfere with the assembly of Hrp pili. In either case, Pst DC3000 may exhibit altered pathogenicity in the hrpA transgenic plant. To test this hypothesis, hrpA transgenic plants were challenged with Pst DC3000 at a concentration of 1x106 cells/ml. The hrpA plants showed disease necrosis and chlorosis four days after inoculation, the 80 Intercellular Space Figure 3.4. Immunogold labeling of the HrpA protein expressed in Arabidopsis transgenic plants. Arrow indicates 10-nm gold particles. Gold particles were exclusively located in the plant cell wall in the examined grids. Bar indicates 100 nm. 81 same as the wild-type plants (Table 3.1). The bacterial population of Pst DC300 in the transgenic plants was monitored over four days. The results showed that the population increase of Pst DC3000 in the hrpA transgenic plants and in the control plants was not significantly different (data not shown). Because high inoculum levels may mask subtle differences in disease symptom or bacterial grth in the hrpA plants vs. the control plants, I also infiltrated the hrpA plants with Pst DC3000 at a lower concentration (105 or 10‘ cells/ml). However, again no difference in symptoms and bacterial populations was observed in the control plants vs. the hrpA transgenic plants. I also examined the effect of plant-expressed HrpA protein on incompatible interactions. Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene avrB triggers the hypersensitive response in Arabidopsis plants containing the cognate disease resistance gene Rpm] (Grant et al., 1995). A Pst DC3000 strain carrying the avrB gene on a plasmid was infiltrated into the hrpA plants as well as control plants at 1x108 cells/ml. The HR symptom was observed 8 hours after infiltration in both transgenic plants and control plants. This suggests that the HrpA protein expressed in the transgenic plants had no detectable effect on the incompatible interaction between Pst DC3000/avrB and Arabidopsis plants (Table 3.1). 3.4.6. Effect of the Plant-Expressed HrpA Protein on the Interaction between Arabidopsis and Pst DC3000 Mutants The Pst DC3000 hrpA mutant strain cannot form the Hrp pilus and thus does not cause disease in a host plant or trigger the hypersensitive response in a resistant plant 82 Table 3.1. Summary of the effect of the plant-expressed HrpA protein on Arabidopsis-P. syringae interactions. Interactions Bacterial Strains Control Plants hrpA Transgenic Plants Compatible DC3000 Disease Disease hrpA“ No symptom No symptom Incompatible DC3000/avrB HR HR hrpA'/avrB No symptom Necrosis hrpS/avrB No symptom Necrosis For pathogenesis assay, an aqueous solution of 1x106 cells/ml Pst DC3000 and its mutant strains was infiltrated into the apoplast of Arabidopsis plants. The disease symptoms were recorded 12 days after infiltration. For I-IR assay, an aqueous solution of 1x108 cells/ml of Pst DC3000 and its mutant strains carrying the avrB gene were infiltrated into the apoplast of Arabidopsis plants. The HR appeared 12 hours after infiltration, while necrosis appeared in the hrpA transgenic plants from 20 hours to 3 days after infiltration. HR: hypersensitive response. 83 (Roine et al., 1997a , 1997b). The compatible interaction between Arabidopsis and the hrpA mutant strain can be restored by introduction of the hrpA gene canied on a plasmid into the Pst hrpA mutant. To determine whether the HrpA protein expressed in the transgenic plants could recover disease symptoms caused by the DC3000 hrpA mutant, control and hrpA transgenic plants were inoculated with the hrpA mutant strain at concentrations of 10°, 105, and 10“ cells/ml. Symptom development and bacterial titres were monitored over 12 days, instead of 4 days, after inoculation. However, I did not observe a difference in the number of the hrpA mutant in the transgenic plants compared to that in the control plants at the three tested inocula (Fig. 3.5). There was no difference in the disease symptoms in these plants. These results indicated that the plant-expressed HrpA protein cannot complement the DC3000 hrpA mutation. I also tested whether the hrpA gene expressed in the transgenic plants could complement the DC3000 hrpA mutation in triggering the HR. For this purpose, the Pst DC3000 hrpA mutant carrying the avrB gene was infiltrated at a high concentration (2x108 cells/ml) into the leaves of hrpA transgenic and control plants. The Pst DC3000 hrpA mutant carrying the avrB gene did not cause any symptom in the control plants. However, it caused necrotic symptoms in the hrpA transgenic plants after 24 hours, and the necrotic symptom progressed over the next two days (Fig. 3.6). The necrosis observed in the hrpA plants could be due to the complementation of the DC3000 hrpA mutation by the hrpA gene expressed in the transgenic plants, or to the nonspecific reaction of the transgenic plants to bacterial inoculation. To test these possibilities, the transgenic and control plants were inoculated with the DC3000 hrpS mutant carrying the avrB gene. The hrpS mutation affects the expression of the Hrp 84 ’6‘ 4 ‘ =3 h a 3 . i -—<= 3 “*5 g «”6 a +8 9 I I I +9 '5- '; 0 4 8 12 Days after infiltration Figure 3.5. Growth of a Pst DC3000 hrpA mutant strain in leaves of Arabidopsis plants expressing hrpA. Pst DC3000 hrpA mutant was infiltrated into leaves of the hrpA and control plants at a concentration of 106 cells/ml. Bacterial grth was monitored over a 12-day time course. Each data point represents the mean titer of four leaf disks from four individual leaves. C represents the control plants. 5, 6, 8, and 9 represent independent T3 hrpA transgenic lines. 85 l 2 3 5 C DC3000/avr £3 a o o hrpA'lavrB 0 £3 @- 0 A hrpS/avrB Q Q @- & Figure 3.6. Symptom development in leaves of transgenic Arabidopsis plants inoculated with Pst DC3000 and its mutant strains hrpA and hrpS, carrying the avrB gene. T3 transgenic lines 1, 2, 3, and 5, and the control plant (C) were inoculated by hand with DC3000/avrB at the concentration of 2x108 cells/ml. Plants inoculated with DC3000/avrB showed HR symptoms within 12 hours, while the transgenic plants inoculated with DC3000 hrpA and hrpS mutants carrying the avrB gene showed necrosis 24 hours to 2 days after inoculation. Leaves were removed from plants 2 days after inoculation for photography. 86 system in Pst DC3000. Therefore, the hrpS mutant carrying avrB does not trigger the HR in the wild-type Arabidopsis plants (Fig. 3.6). However, the hrpS mutant carrying avrB caused a necrotic symptom on the hrpA transgenic plants. This result suggested that the observed necrotic symptoms caused by the hrpA mutant carrying the avrB gene were not due to complementation of the hrpA mutation by the HrpA protein expressed in the transgenic plants. My explanation for the necrotic symptom is that the hrpA transgenic plants are generally more sensitive to bacterial inoculation at high inocula. Consistent with this explanation, infiltration of hrpA transgenic plants with water or buffer did not result in any leaf necrosis. 3.5. DISCUSSION In this study, I examined the effect of the expression of HrpA protein in Arabidopsis on the interaction between Arabidopsis and Pst DC3 000. Although northern blot analysis indicated that at least 10 transgenic lines expressed hrpA gene, an antibody against HrpA failed to detect the HrpA protein on western blots of any of the transgenic lines. However, I did detect the HrpA proteins in the cell wall of the transgenic plant leaves using TEM-coupled immunogold labeling. This result indicated that the HrpA protein in the transgenic plants may have been trapped in or bound to the cell wall. I also observed a unique pockmark appearance on the transgenic leaf surface. The unique leaf morphology and the cell wall localization of the HrpA protein in the transgenic plants indicate a possible interaction between the HrpA protein and components of the Arabidopsis cell walls. 87 The initial goal of this study was to test the hypothesis that expression of HrpA in plants may preoccupy the putative pilus receptor site, thus affecting bacterial pathogenesis. Alternatively, because the HrpA protein is mainly associated with the Hrp pilus, overexpression of HrpA may interfere with the assembly of the Hrp pilus in planta. However, I found that hrpA expression in the transgenic plants had no effect on either compatible or incompatible interactions. These results suggested the following: First, if the Hrp pilus receptor hypothesis is correct, the receptor may not interact with the Hrp pilus through the HrpA protein. Another unidentified protein on the Hrp pilus may mediate cell wall interaction. Second, if the assembly hypothesis is correct, the Hrp pilus assembly apparently was not affected by cell wall-localized HrpA protein in the transgenic plants. I also examined whether expression of the HrpA protein in Arabidopsis can complement the DC3000 hrpA mutation. As shown in Figure 3.5, the HrpA transgenic plant did not complement the bacterial hrpA mutant for causing disease symptoms on Arabidopsis plants. However, the hrpA mutant carrying the avrB gene did cause HR-like necrosis in hrpA transgenic plants, but not in control Arabidopsis plants. The symptoms appeared more than 20 hours after bacterial infiltration. The DC3000 hrpS mutant carrying the avrB gene also elicited the same HR-like necrosis in the hrpA transgenic plants. The hrpS mutant is defective in the expression of the type 111 protein secretion system; HR-like necrosis caused by the DC3000 hrpA mutant carrying avrB is therefore not Hrp-dependent. I suggest that expression of HrpA in the transgenic plants and its localization to the plant cell wall result in increased sensitivity of the transgenic plants to high levels of bacterial inocula during the HR assay. Indeed, the low levels of hrpS or 88 hrpA mutants carrying the avrB gene did not cause necrotic symptoms in the hrpA plants (data not shown). Evidence for interactions between bacterial secreted proteins and plant cell walls has been reported for several Hrp-dependent extracellular proteins. For example, HrpZ, which belongs to the harpin family of proteins, has been found to be associated with plant cell walls, and the protein elicited no response from wall-less protoplasts (Hoyos et al., 1996). Another Hrp-controlled extracellular protein, HrpW of Pst DC3000, also appears to interact with the plant cell wall. HrpW is also a harpin-like protein that elicits the HR when infiltrated into tobacco leaves. Moreover, HrpW has a Pel domain that is similar to that in pectate lyases from Nectria haematocacca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis. The Pel fragment of HrpW binds to calcium pectate, a major constituent of the plant cell wall. However, neither the fiill-length HrpW nor the Pel fragment showed Pel activity (Charkowski et al., 1998). A possible role for the interaction between Hrp-controlled bacterial extracellular proteins and the host cell wall could be to change the texture of the host cell wall, which is a physical barrier for type III protein secretion in plant-bacteria interaction. Modification of plant cells by these Hrp proteins may help the delivery of the bacterial proteins into the host cell. Alternatively, the interaction between hrp-controlled EXPs, including HrpA, and the plant cell wall may provide a means of attachment of the Hrp pilus to the plant cell wall. Screening for mutations that disrupt the cell wall-HrpA interaction in the hrpA transgenic plant may lead to identification of the plant cell wall component and HrpA amino acid residues involved in the interaction between the Hrp pilus and Arabidopsis cell walls. 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EMBO J 2, 2143-50. 93 Chapter 4 REQUIREMENT OF CASPASE-LIKE PROTEASE ACTIVITY FOR PATHOGEN-TRIGGERED CELL DEATH IN TOBACCO 4.1. ABSTRACT Plant cell death is an integral part of plant reproduction, xylogenesis, senescence, and pathogen defense. Little is known about its mechanism. In animals, a family of aspartic acid (Asp)-directed cysteine proteases (named caspases) plays a critical role in the execution of programmed cell death. We found that N-benzyloxycarbonyl-Val-Ala- Asp-chloromethylketone (Z-YVAD-CMK), an interleukin-10 converting enzyme (ICE)- type caspase inhibitor, prevented hypersensitive necrosis in tobacco leaves triggered by two avirulent pathogens, Pseudomonas syringae pv. tomato and tobacco mosaic virus, as well as by a protein elicitor, harpin. Z-YVAD-CMK also effectively prevented disease- associated necrosis in tobacco leaves caused by a virulent pathogen, P. s. pv. tabaci. Affinity labeling identified three tobacco proteins that bind to Z-YVAD-CMK. Our results are consistent with the idea that a key step in the execution of both HR- and disease-associated cell death is sensitive to caspase inhibitors. The identification of the three Z-YVAD-CMK-binding proteins provides a foundation for future analysis of the plant cell death process during plant-pathogen interactions. 94 4.2. INTRODUCTION Cell death is an integral part of plant reproduction, xylogenesis, senescence, and defense against pathogen infection. Plant cell death triggered by infection of an avirulent pathogen is known as the hypersensitive response (HR) (Goodman and Novacky, 1994). HR cell death is accompanied by induction of multifaceted defense responses, including the production of reactive oxygen species (ROS) (Lamb and Dixon, 1997) and phytoalexins (Hammerschmidt, 1999), rapid cross-linking of plant cell wall proteins (Bradley et al., 1992; Brisson et al., 1994), and resistance to invading pathogens (Dangl et al., 1996; Greenberg, 1996). It is believed that HR-mediated cell death results from the execution of an endogenous programmed cell death pathway, based on activation of HR by plant mutations and inhibition of elicitor-triggered HR cell death by inhibitors interfering with host metabolism (He at al., 1993; Dietrich et al., 1994; Greenberg et al., 1994; Mittler and Lam, 1995; Mittler et al., 1996; Dang] et al., 1996; Greenberg, 1996). In some cases, plant cells appear to undergo morphological changes (e. g., DNA fi'agmentation, formation of apoptotic bodies, condensation of nuclei) similar to those characteristics of animal apoptosis (Levine et al., 1996; Ryerson and Heath, 1996; Wang et al., 1996). However, these changes are not always observed (Bestwick et al., 1995). Several plant genes, mutations of which trigger cell death in plants, have recently been cloned and they encode a putative membrane protein (Buschges et al., 1997), a transcription factor (Dietrich et al., 1997), a dioxygenase (Gray et al., 1997), and uroporphyrinogen decarboxylase (Hu et al., 1998). In addition, ROS are involved in the control of death of 95 Arabidopsis leaf cells caused by the 13d] (lesions simulating disease resistance 1) mutation (Jabs et al., 1996) and of cultured soybean cells triggered by P. syringae pv. glycinea (Levine et al., 1994). Despite these pioneering studies on HR cell death, the key biochemical processes underlying the execution of HR cell death remain elusive. Also, the question as to whether plants and animals share common molecular mechanisms in the execution of cell death awaits answers. In plant-pathogen interactions, in addition to the rapid and highly localized HR cell death associated with incompatible interactions between resistant (non-host) plants and avirulent pathogens, there is also a slow plant cell death that spreads beyond the site of pathogen infection during some compatible interactions between susceptible (host) plants and virulent pathogens. Such host cell death, eventually manifested as tissue necrosis, is the primary symptom of many diseases caused by necrotizing pathogens. An example is the extensive tissue necrosis caused by the tobacco “wild fire” bacterium P. s. pv. tabaci (Psta) strain WF4 (Gopalan et al., 1996). It is an important, unresolved question whether HR and disease cell death processes share similar mechanisms. In animals, a growing family of cysteine proteases (named caspases), including CED—3, ICE, ICHl/NEDDZ, ICH2/TX, CPP32/YAMA/apopain, and MCH2, play a critical role in apoptosis (Yuan et al., 1993; F emandes-Alnemri et al., 1994; Kumar et al., 1994; Faucheu et al., 1995; Karnens et al., 1995; Alnemri et al., 1996; Wang et al., 1994; Villa at al., 1997; Solary et al., 1998). Activation of these caspases, usually caused by proteolytic cleavage of the respective inactive zymogens, appears to be a universal mechanism during animal apoptosis. The necessity of these caspases for animal apoptosis has been established by use of highly specific inhibitors such as the cowpox virus CrmA 96 protein, baculoviral protein p35, and peptide methyl ketones and peptide aldehydes that specifically and irreversibly bind to the active site cysteine of the caspases (Ray et al., 1992; Miura et al., 1993; Thornberry et al., 1994; Boudreau et al., 1995; Enari et al., 1995; Los et al., 1995; Nicholson et al., 1995; Schlegel et al., 1995; Tewari and Dixit, 1995; Tewari et al., 1995; Dubrez et al., 1996). For example, the tetrapeptide Tyr-Val- Ala-Asp* (YVAD*) corresponds to the consensus recognition/cleavage site of ICE-type caspases, whereas the Asp-Glu-Val-Asp* (DEVD*) corresponds to the consensus recognition/cleavage site of CED-3/CPP32—type caspases (Villa et al., 1997; Solary et al., 1998). Cleavage occurs immediately after the Asp residue indicated by asterisks. In plants, no caspase has been directly shown to be involved in programmed cell death. However, several proteases have been implicated in programmed cell death (PCD) in several plant systems. For example, inhibitors of serine proteases inhibit T richoderma viride-induced hypersensitive cell death in cultured tobacco cells (Y ano et al., 1999). In barley, cell degeneration of nucella afler ovule fertilization is characteristic of PCD. Nucellin, an aspartic protease, is expressed synchronally with nucellar cell degeneration (Chen and F oolad, 1997). In cultured soybean cells, the synthetic protease inhibitors such as leupeptin, PMSF and AEBSF effectively suppress PCD triggered by oxidative stress or by infection with avirulent pathogens (Levine et al., 1996). All these inhibitors are active against cysteine proteases. In Zinnia elegans, tracheary element differentiation that ends in cell death is blocked by the addition of E-64, a specific cysteine protease inhibitor (Minami and Fukuda, 1995). Ectopic expression of cystatin, an endogenous cysteine protease inhibitor, represses the PCD activated by oxidative stress and pathogen attack in soybean cells (Solomon et al., 1999). Furthermore, an extract from fungus-infected 97 cowpea plants undergoing HR cleaves exogenous poly (ADP-ribose) polymerase, a substrate for CPP32-type caspase in apoptotic animal cells (D'Silva et al., 1998), although the proteolytic cleavage pattern is somewhat different fi'om that in animals. In this Chapter, I show that a tetrapeptide inhibitor (Z-YVAD-CMK) of caspase inhibits cell death in tobacco leaves caused by a purified HR elicitor, harpin, and virulent and avirulent pathogens. I use affinity labeling to identify three specific tobacco proteins that may function in Z-YVAD-CMK-mediated inhibition of cell death. An independent study has recently shown inhibition of HR cell death triggered by avirulent P. s. pv. phaseolicola and tobacco mosaic virus (TMV) by Ac—YVAD-ACO (del P020 and Lam, 1998) 4.3. MATERIALS AND METHODS 4.3.1. Plants, Pathogens, and Elicitor Nicotiana tabacum cv. Samsun plants were grown in the greenhouse. Fully expanded leaves of 6- to 8-week-old plants were used for HR or disease assay. Pseudomonas syringae pv. tomato (Pst) DC3000 and P. s. pv. tabaci (Psta) WF4 cause HR and disease necrosis, respectively, in tobacco leaves. Bacteria were infiltrated into the leaf apoplast at 2x108 bacteria/ml. Erwinia amylovora harpin was prepared as described (He at al., 1994) and was infiltrated into the tobacco leaf apoplast at 2 uM. Under the conditions used in this study, HR necrosis caused by Pst DC3000 and harpin and disease necrosis caused by Psta WF4 occurred within 10 hours post-infiltration. 98 4.3.2. Qualitative Assay of Caspase-like Activity Two synthetic fluorescent substrates were used: Z-YVAD-7-amino—4- trifluoromethyl coumarin (Z-YVAD-AF C), a substrate for ICE-type caspases; and Z- DEVD-AF C, a substrate for CED-3/CPP32—type caspases. Two leaf disks of 10 mm in diameter taken from normal tobacco leaves or leaves that showed confluent HR necrosis (triggered by 2 pM harpin) were homogenized in 0.5 ml of 0.1 M HEPES buffer (pH 7.4) containing 2 mM DTT, 0.1% CHAPS, and 1% sucrose. The homogenates were ‘ centrifuged at 14,000 g for 10 minutes at 4°C. Supernatant (200 pl) was placed in each microtitre plate well and incubated with the substrates. The caspase reaction was carried out at room temperature for 3 hours, unless indicated otherwise. For in planta caspase- like activity assay, when the leaf panel infiltrated with substrate plus harpin or DC3000 showed HR necrosis, two leaf disks of 10 mm in diameter were taken from each panel and homogenized. The homogenate supematants were pipetted into microtitre plate wells and, without incubation, photographs were taken under a UV lamp (UVL-56, UVP) with a peak wavelength of 366 nm. For detection of caspase-like activity in chromatographic fractions, 200 pl of each fiaction was used. 4.3.3. Assay for Inhibition of HR and Disease Necrosis Pst DC3000, Psta WF4, or harpin was mixed with DMSO or inhibitors dissolved in DMSO immediately before infiltration into the apoplast of fully expanded leaves of 4- to 6-week-old, greenhouse-grown tobacco plants using a needleless syringe. For TMV 99 challenge, the tobacco leaf surface was first rubbed with a highly purified TMV suspension (a gift from Dr. Gus de Zoeten, Michigan State University) containing carborundum. The rub-inoculated leaf panels were infiltrated with 2% DMSO or inhibitors in 2% DMSO 24 hours later. Inoculated plants were kept on the laboratory bench for 3 days for symptom observation. 4.3.4. Detection of Caspase-like Proteins in Native Polyacrylamide Gel Two tobacco leaf disks (10 mm in diameter) were taken from normal or HR leaf panels and homogenized in 0.5 ml caspase assay buffer (0.1 M HEPES, pH 7.0, 2 mM DTT, 0. 1% CHAPS, 1% sucrose) at room temperature. The tissue suspension was clarified at 4°C for 10 minutes at 14,000 g. Equal volumes of the supernatant and 20% glycerol solution were mixed. Proteins in 20 pl of each mix were separated on a native 15% polyacrylarnide gel at 4°C. The gel was then incubated in the caspase assay buffer for 15 minutes. Active caspases were detected by overlaying the gel with Enzyme Overlay Membrane AF C-120 (Enzyme Systems Products, Dublin, CA) for 3 hours at room temperature. Photographs were taken under a long wavelength UV lamp (UVL-S 6, UVP). 4.3.5. Affinity Labeling Tobacco leaves were infiltrated with 4x108 bacteria/m1 of Pst DC3000. Leaves showing visible HR necrosis were collected by immersion in liquid N,. Caspase labeling 100 followed the protocol of F aleiro et al. (1997). Biotinyl-YVAD-CMK was used at a final concentration of 5 pM. The initial incubation time was 2 hours at room temperature. The labeled proteins were separated by 15% SDS-PAGE electrophoresis and transferred to immobilon-P membrane (Millipore) for 45 minutes at 100mA using the standard transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). Membranes were pre-blocked in PTB buffer (20 mM Tris, 150 mM NaCl, 0.02% Tween-20) supplemented with 5% nonfat milk. Membranes were then incubated in avidin horseradish peroxidase conjugate at 1 pg/ml (Molecular Probes) in PTB-milk. The labeled proteins were visualized by ECL (Amersham). 4.3.6. Chromatographic Fractionation Tobacco leaf tissue undergoing hypersensitive cell death was collected and frozen in liquid nitrogen 4 hours after infiltration with harpin. The frozen tissue was homogenized in caspase activity buffer, and the homogenate was centrifuged at 4°C at 15,000 rpm for 30 minutes. Cleared extracts were precipitated with ammonium sulfate (80% saturation, 0.516 g/ml). The protein pellet was collected after centrifugation and redissolved in caspase assay buffer. After passing through an Econo-Pac 10 DG desalting column (Bio-Rad), the protein extract was loaded onto a DEAE-SPW anion exchange column (TosoHaas) and proteins were eluted with a linear NaCl gradient (0 — 600 mM for 30 minutes at 1 ml/minute) in caspase activation buffer. The HPLC used was equipped with a Beckrnan model 114M pump and detection was at 280 nm. Fractions containing 101 caspase-like activity were pooled and further fractionated using a TSK 3000 SW column (7.5 mm x 7.5 cm) (Beckman) in caspase activity buffer at a flow rate of 4 ml/minutes. 4.4. RESULTS 4.4.1. Initial Detection of Putative Caspase Activity To test whether a caspase is required for the HR in plants, we examined caspase activity in plant leaf tissues. For this purpose, two synthetic fluorescent substrates were used: Z-YVAD-AF C, a substrate for ICE-type caspases; and Z-DEVD-AF C, a substrate for CED-3/CPP32—type caspases. Cleavage of these two blue fluorescent substrates by caspases results in the release of the AFC group, which emits green fluorescence. We found that homogenized HR tobacco (Nicotiana tabacum L. cv. Samsun NN) leaf tissues exhibited ICE-type caspase activity, but not CED-3/CPP32—type caspase activity (Fig. 4.1 A). In this experiment, Pseudomonas syringae pv. tomato strain DC3000 (Psta DC3000) and Erwinia amylovora harpin (Wei et al., 1992), both of which trigger HR in tobacco leaves, were used to induce HR. As will be discussed further in this chapter, at present we cannot absolutely ascertain that the ICE-type caspase activity detected in tobacco in fact results from a genuine caspase; therefore, we will hereinafter call this enzyme caspase-like protease (CLP). The CLP activity in HR tobacco leaf tissue was sensitive to heat (100°C, 10 minutes) and the cleavage reaction was competitively inhibited by addition of a 10-fold higher amount of Z-YVAD-CMK, a specific inhibitor of animal ICE-type caspases, but 102 Figure 4.1. Caspase-like protease (CLP) activity in tobacco leaf tissue. (A) Detection of CLP activity in vitro. Well 1, 100 pM substrate Z-YVAD-AF C alone; well 8, 100 pM substrate Z-DEVD-AF C alone; wells 2 and 9, HR leaf tissue homogenates only; well 3, HR tissue homogenate plus 100 pM Z-YVAD-AF C, no incubation; well 10, HR tissue homogenate plus 100 pM Z-DEVD-AF C, no incubation; well 4, HR tissue homogenate plus 100 pM Z-YVAD-AF C, 3-hours incubation; well 11, HR tissue homogenates plus 100 pM Z-DEVD-AF C, 3-hours incubation; well 5, normal tissue homogenate plus 100 pM Z-YVAD-AF C, 3-hours incubation; well 12, normal tissue homogenate plus 100 pM Z-DEVD-AF C, 3-hours incubation; well 6, boiled HR tissue homogenate plus 100 pM Z- YVAD-AF C, 3-hours incubation; well 13, boiled HR tissue homogenate plus 100 M Z- DEVD-AF C, 3-hours incubation; well 7, HR tissue homogenate plus 100 pM Z-YVAD- AFC plus 1 mM ICE inhibitor Z-YVAD-CMK, 3-hours incubation; well 14, HR tissue homogenate plus 100 pM Z-YVAD-AF C plus lmM CED3/CPP32 inhibitor Z-DEVD- CMK, 3-hours incubation. The substrates Z-YVAD-AF C and Z-DEVD-AF C emit blue fluorescence. The AFC group released after protease cleavage emits green fluorescence (wells 4, 5 and 14). Tobacco leaf tissue homogenate alone emits dark purple autofluorescence. Caspase reaction was carried out at room temperature for 3 hours, except for wells 3 and 10, for which there was no incubation time. (B) Detection of CLP activity in planta. Z-YVAD-AF C (200 pM) was infiltrated into tobacco leaf panels together with 2% DMSO (well 1), 2 pM harpin (well 2), or 2 pM harpin plus 1 mM Z- YVAD-CMK in 2% DMSO (well 3). Leaf panels infiltrated with the solvent, 2% DMSO, were used as control. 103 not acetyl (Ac)-DEVD aldehyde (CHO), a competitive inhibitor of animal CED- 3/CPP32—type caspases (Fig. 4.1 A). Unexpectedly, homogenized normal leaf tissue also exhibited CLP activity after a 3-hours incubation in vitra (Fig. 4.1 A). We suspected that the CLP activity in homogenized normal leaf tissue resulted from mechanically killed leaf cells. Because the activation of the caspases in animal systems involves proteolytic cleavage, it is likely that mechanical killing of the normal leaf cells in vitro may release certain proteases that activate the CLP during the 3-hours incubation period. We therefore performed an in planta caspase activity assay. Tobacco leaf panels were infiltrated with Z-YVAD-AF C with or without harpin. When leaf panels infiltrated with both harpin and Z-YVAD-AF C showed HR necrosis, leaf disks were taken from infiltrated normal and HR leaf tissues and quickly homogenized. CLP activity was determined without the 3- hours incubation. No CLP activity was detected in normal leaf tissue, whereas CLP activity was present in HR leaf tissue (Fig. 4.1 B). This result suggests that CLP activity in homogenized normal leaf tissue resulted from cell death following tissue homogenization and subsequent incubation. 4.4.2. Inhibition of HR and Disease Necrosis by Caspase Inhibitors Is the CLP activity detected in HR leaf tissue required for the HR? I found that the I C E-type caspase inhibitor Z-YVAD-CMK, but not the CED-3/CPP32-type caspase inhibitor AC-DEVD-CHO, consistently and completely inhibited the HR in tobacco triggered by Pst DC3000, E. amylovora harpin protein, or tobacco mosaic virus (TMV) (Fi g. 4.2, A and B, and Table 4.1). This result is in accordance with the detection of only 104 Fat-WT. .. inf-Wm Figure 4.2. Inhibition of tobacco cell death by Z-YVAD-CMK. (A) Tobacco leaf panels were infiltrated with 2x108 bacteria/ml of Pseudomonas syringae pv. tomato strain DC3000 (panels 1 and 4), 2x108 bacteria/ml P. s. pv. tabaci strain WF4 (panels 2 and 5), or 2 pM Erwinia amylovora harpin (He et al., 1994) (panels 3 and 6). Leaf panels 1 to 3 were co-infiltrated with 1 mM Z-YVAD-CMK in 2% DMSO, whereas panels 4 to 6 were co—infiltrated with 2% DMSO only. Photographs were taken 12 hours post-infiltration. Gray necroses indicate HR. (B) Inhibition of HR necrosis in tobacco leaf elicited by tobacco mosaic virus (TMV). Tobacco leaf panels 1 and 2 were first rub-inoculated with purified TMV, followed by infiltration with 2% DMSO (panel 1) or 1 mM Z-YVAD- CMK in 2% DMSO (panel 2) 24 hours later. Some 200-300 small HR lesions started to appear 48 h afier viral inoculation in panel 1 only. Expanding HR lesions coalesced in panel 1 on day 3, when this photograph was taken. 105 Table 4.1. Effects of protease inhibitors on HR development in tobacco. Inhibitor Concentration Known targets Inhibition of HR (pM ) development Z-YVAD-CMK >500 ICE-type caspases + Ac-DEVD-CHO 1000 CED-3/CPP32-type caspases -* Iodoacetamide >50 Caspases and other cysteine + proteases E-64 1000 Papain and several other - cysteine proteases Chyrnostatin 1000 Chymotrypsin - Leupeptin 1000 Serine and thiol proteases - Aprotinin 1000 Serine proteases - Antipain 1000 Papain, etc. - Pepstatin A 500 Aspartic proteases - PMSF 1000 Serine and some cysteine - proteases Aqueous solution of 2x108 bacteria/ml Pst DC3000 or 2 pM harpin was mixed with various inhibitors before infiltration into the apoplast of tobacco leaves. Pst DC3000 and harpin induced HR necrosis within 10 hours after infiltration. Complete inhibition of HR triggered by both Pst DC3000 and harpin was scored as “+”. The cell permeability and degradation of various inhibitors in planta were not checked. Stock solutions of protease inhibitors were made in DMSO. "' In some experiments, this compound at this concentration partially inhibited HR. 106 ....,, 1.1:.- . . the ICE-type caspase activity in HR tobacco leaf tissue. HR caused by Pst DC3000 or harpin was also efficiently inhibited by iodoacetarnide, a general inhibitor of caspases and many other proteins. However, inhibitors of other classes of proteases did not significantly affect HR (Table 4.1). Thus, the HR inhibition results showed that the CLP activity is specifically required for HR cell death in plants. Z-YVAD-CMK also inhibited tissue necrosis caused by Psta WF4 (Fig. 4.2 A), suggesting that not only HR but also disease cell death triggered by Psta WF4 requires activation of ICE-type caspases. Z- YVAD-CMK does not exert a toxic effect on Pst DC3000 or Psta WF4 because the growth curves of both bacteria in LB broth (Sambrook et al., 1989) supplemented with 2% DMSO or 1 mM Z-YVAD-CMK-2% DMSO were indistinguishable (data not shown). 4.4.3. Effect of Caspase Inhibitor on Induction of Plant Defense Genes In contrast to its ability to completely inhibit HR cell death, Z-YVAD-CMK did not eliminate local activation of an HR-associated tobacco gene, HINI , and a pathogenesis-related gene, PR-3, in response to harpin or Pst DC3000 (Fig. 4.3). The HINI and PR-3 transcripts were accumulated in the presence of Z-YVAD-CMK, although the levels of the HINI and PR-3 transcripts were lower at each time point in Pst IDC3000-treated tissue (Fig. 4.3 A). This suggests that the CLP inhibited by Z-YVAD- CNIK is involved primarily in the execution of HR cell death, but not in the induction of Plant defense genes. A logical explanation is that CLP fimction downstream of a point Where signaling pathways leading to cell death and induction of HR-associated genes has 107 DC3000 Harpin DMSO YVAD DMSO YVAD 048048(h) 0610061001) rRNA mm m DMSO WAD DMSO WAD 0481004810(h) 0481004810(h) PR-3 7‘3; 2. “*3 ill . i, rRNA mm m Figure 4.3. Northern blot analysis of the effect of Z-YVAD-CMK on induction of an HR- associated tobacco gene, HINI, and a pathogenesis-related gene, PR-3. Tobacco leaves were infiltrated with 2x108 cells/ml Pst DC3000 (A) or 2 pM harpin (B) suspended in 2% DMSO or 2% DMSO containing 1 mM Z-YVAD-CMK. Leaf tissues were collected at the indicated time points for RNA isolation. A total of 3 mg RNA isolated from each sample was analyzed by northern blot using the HINI gene (Gopalan et al., 1996) and the PR-3 gene (Ward et al., 1991) as probes. The largest rRNA species visualized after staining with ethidium bromide was used as a reference for loading in each lane. HR necrosis appeared at 8 and 10 hours in leaves infiltrated with DC3000 plus DMSO and harpin plus DMSO, respectively. No HR developed in leaves infiltrated with DC3000 or harpin plus Z-YVAD-CMK. 108 diverged. Uncoupling of HR cell death and resistance or activation of I-IR-associated genes was also observed by others under certain conditions (Jakobek and Lindgren, 1993; Century et al., 1995; Gopalan et al., 1996; Yu et al., 1998). 4.4.4. Preliminary Identification of Caspase-like Proteins I employed three methods in an attempt to identify the CLP. First, I used Enzyme Overlay Membrane AF C-120 (EOM AF C-120, Enzyme System Products) coupled with native polyacrylamide gel electrophoresis (PAGE) to identify CLP activity bands. EOM AF C—120 is impregnated with Z-YVAD-AF C and is therefore specific for detection of ICE-type caspases. An apparently identical caspase activity band was detected in both mechanically killed and HR tissues near the top of the native PAGE gel (Fig. 4.4). Because of poor resolution of proteins at the top of the native 15% PAGE gel, I could not determine whether the CLP activity band was due to a single or multiple CLPs. This CLP activity band was not detected when HR tissue homogenate had been boiled for 10 minutes prior to loading samples to the PAGE gel (Fig. 4.4). I then used anion exchange and gel filtration columns to begin the purification of the CLP. Specifically, we extracted the tobacco proteins 4 hours after infiltration with harpin. The protein extract was first fractionated using an anion exchange column (Fig. 4.5 A). All fractions were collected and tested for CLP activity with Z-YVAD-AF C. Fractions 17 and 18 (indicated with the arrowheads in Fig. 4.5 A) showed CLP activity. These two fractions were pooled and fractionated again on a gel filtration column. Assay using the fluorescent substrate Z- 109 Top _ Bottom — Figure 4.4. Detection of CLPs in tobacco leaf tissue. A profile of active CLPs in tobacco leaf homogenates revealed by an activity gel overlay method. Lanes: 1, Pst DC3000- induced HR leaf tissue; 2, harpin-induced HR leaf tissue; 3, harpin-induced HR leaf tissue boiled for 10 minutes before loading; 4, homogenized (mechanically killed) normal leaf tissue. To trigger HR, 2x108 bacteria/ml of Pst DC3000 and 2 pM harpin were used. Gel top and bottom are indicated. Arrow indicates caspase activity bands. 110 1.0“? 0.6 E s :s: 8 / Z 0 E 0.5-1*- ‘25 i l l l | l 0 Fraction No. 10 ‘20 30 40 CLP activity H B A 8 f 0.5-- 8 S .e 0.25-- o m .0 < ‘ j l 1 Fraction No. 10 ‘ 20 30 CLP activity ++ Figure 4.5. Fractionation of tobacco proteins and CLP activity. Tobacco leaves were infiltrated with 2 pM harpin. After 4 hours, proteins were extracted as described in Materials and Methods. (A) A total protein extract was chromatographed using a DEAE- SPW anion exchange column, with a continuous gradient of NaCl. Each fraction was assayed for CLP activity. Active fractions 17 and 18 are indicated with arrowheads. (B) Active fractions 17 and 18 were then combined and chromatographed through a gel filtration column. Each fraction was assayed for CLP activity. Active fractions 14 and 15 are indicated with arrowheads. "+" indicates CLP activity. 111 YVAD-AF C revealed CLP activity in fractions 14 and 15 (indicated with the arrowheads in Fig. 4.5 B). Finally, I performed biotin-YVAD-CMK—based affinity labeling coupled with SDS- PAGE (F aleiro et al., 1997) to estimate the molecular weights of the CLP in tobacco. Three proteins of ca. 82 to 85 kDa were detected in homogenized normal and harpin- induced HR leaf tissues (Fig. 4.6 A). In addition to these three proteins, a ca. 65-kDa protein was labeled in the Pst DC3000-induced HR (Fig. 4.6 A). An excess amount of the ICE-type caspase inhibitor Z-YVAD-CMK, which inhibits cell death in planta (Fig. 4.2), efficiently competed with biotin-YVAD-CMK in the labeling of only the 82- to 85-kDa proteins, whereas the labeling of the 65-kDa protein was not affected by Z-YVAD-CMK (Fig. 4.6, B and C). The effective competition by Z-YVAD-CMK suggests that the 82- to 85-kDa proteins are candidate CLPs. Because our CLP activity assay (Fig. 4.1) showed that mechanically killed leaf tissue contains active caspases, labeling of the 82- to 85-kDa proteins in homogenized normal tobacco leaf tissue was expected. The absence of the 65- kDa protein in harpin-induced HR tissue and the inability of Z-YVAD-CMK to compete in labeling of this protein argue against its being a CLP. Furthermore, this protein was also labeled in protein extracts from tobacco leaf tissues infiltrated with hrpS and hrcC mutants of DC3000 (Yuan and He, 1996), which are defective in the expression of bacterial HR elicitors. We conclude that the 65-kDa protein is a Pst DC3000-specific protein nonspecifically labeled by biotin-YVAD-CMK. 4.5. DISCUSSION Data presented in this paper show that the ICE-type caspase inhibitors (e. g., Z- 112 104- 82— IN .,:\ M M ‘11-. rtl“»’t.l\l W 33— 28- WW“! Figure 4.6. Affinity labeling of caspase-like proteins in tobacco leaf tissue. (A) Lanes: 1, homogenized leaf tissue; 2, Pst DC3000-induced HR tissue; 3, harpin-induced leaf tissue. Putative caspases were affinity labeled with biotinyl-YVAD-CMK and detected by biotin-avidin-based immunoblot following the protocol described in Faleiro et al. (1997). (B) Competition of protein labeling by Z-YVAD-CMK. Proteins in Pst DC3000-induced (lanes 1 and 2) or harpin-induced (lanes 3 and 4) leaf tissue were labeled in the presence of 1 mM Z-YVAD-CMK—2% DMSO (lanes 1 and 3) or 2% DMSO (lanes 2 and 4). (C) A portion of lanes 3 and 4 was enlarged to visualize the three specifically labeled proteins. 113 YVAD-CMK) effectively prevent HR triggered by Pst DC3000, TMV, and a purified protein elicitor, harpin. Z-YVAD-CMK also inhibit the disease cell death triggered by Psta WF4. I provide evidence that tobacco leaf tissue contains caspase-like protease (CLP) activity that can be activated by HR cell death in viva and mechanical killing in vitro (Figs. 4.1 and 4.4). I further characterize caspase-like proteases by in-gel activity staining, chromatographic fractionation, and affinity labeling. Collectively, my results independently confirm and extend the observation of del P020 and Lam (1998), which showed inhibition of HR cell death triggered by P. s. pv. phaseolicola and TMV by A0- YVAD-CMK. Together, these two studies suggest a role of a Z-YVAD-CMK-inhibitable caspase-like protease in the execution of cell death caused by virulent and avirulent pathogens as well as harpin. It is important to point out that although our experimental results are consistent with the idea that pathogenesis-associated cell death requires genuine caspase activity, other interpretations of our results must be considered. For example, 1) the accumulation of the fluorescent AFC moiety (Fig. 4.1) could result from the combined action of a non- caspase protease cleaving elsewhere in the tetrapeptide and an aminopeptidase activity, but not by endoproteolytic cleavage of the substrate after aspartic acid, as predicted in the caspase cleavage reaction. However, if the former is true, the reaction should not have been inhibited by an ICE-type caspase inhibitor, Z-YVAD-CMK (Fig. 4.1). In fact, the specific inhibition of the accumulation of the fluorescent AFC moiety by Z-YVAD-CMK strongly argues for the authenticity of a true ICE-type caspase-like enzyme in the tobacco tissue. F urtherrnore, detection of a specific CLP activity band by enzyme overlay membrane (Fig. 4.4) after native polyacrylamide gel eletrophoresis and of the CLP 114 activity in specific fractions after chromatography are also not compatible with possibly combined action of multiple proteases, unless the multiple proteases co-migrate to the exact same position in the gel or column. 2) The ICE-type caspase inhibitor Z-YVAD- CMK may affect processes other than caspase-like proteins in plants. We have shown that the Z-YVAD-CMK inhibited the ICE-type caspase activity in tobacco HR leaf tissue in vitro, therefore we can conclude that at least one of the targets of Z-YVAD-CMK in viva appears to be the caspase-like protease. Also, the concentration of Z-YVAD-CMK required to inhibit plant cell death as demonstrated in this study was much higher than that used to inhibit apoptosis in animals (Solary et al., 1998; Villa et al., 1997). Although plant caspase-like proteins can recognize the YVAD tetrapeptide derivatives as substrates (or competitive inhibitors), their optimal substrate may be different from the animal caspases. Different substrate preferences for different families of animal caspases have been well documented (Villa et al., 1997; Talanian et al., 1997 ; Solary et al., 1998). 3) Affinity labeling of tobacco proteins revealed three binding proteins of 82 to 85 kDa in size (Fig. 4.6), instead of the expected ca. 20-kDa large subunit of active animal caspases (F aleiro et al., 1997). However, the labeling pattern is reminiscent of the animal caspases (i. e., labeling of multiple caspases with similar molecular weights; Faleiro et al., 1997). Further purification and enzymatic characterization of the three Z-YVAD-CMK binding proteins should help us to determine whether they are tobacco caspases. A homology search using animal caspase sequences did not reveal any caspase like genes in the current plant gene databases. With over 70% of expressed Arabidopsis genes partially sequenced (T. Newman, personal communication) and the existence of multiple caspases and isoforms in animals (Alnemri et al., 1996), one would hope to 115 .... arr-ra- f- an -55. detect such sequence homologies. It may be that plant and animal caspase genes share such low levels of sequence homology that they are beyond detection with available search programs. Another possibility is that such sequence homologies do exist, but only in regions beyond the current plant EST and genomic sequences. Detection of such homologies therefore waits genomic sequencing and/or sequencing of internal regions of expressed genes, both of which are underway. In this study I have also demonstrated that Z-YVAD-CMK not only prevents the formation of HR necrosis, it also effectively prevents execution of the disease-associated necrosis elicited by a virulent bacterium, Psta WF4 (Fig. 4.2). Previously, we showed that HR necrosis elicited by P. s. pv. tabaci WF4 in tobacco leaves is prevented by inhibitors of calcium channels and ATPase (Gopalan et al., 1996). Taken together, these two studies argue that the execution of HR and disease necrosis involves several common steps. In addition, I have shown that although Z-YVAD-CMK effectively prevents both HR- and disease-associated cell deaths, it does not have a significant effect on the harpin- induced activation of an HR-associated gene, HINI , and a pathogenesis-related gene, PR- 3 (Fig. 4.3 B). 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Cell 75, 641-52. 121 Chapter 5 EXPRESSION OF THE HUMAN BCL-Z GENE IN ARABIDOPSIS THALIANA RESULTS IN PLANT CELL DEATH AND DISEASE RESISTANCE 5.1. ABSTRACT ‘7”! f“ *‘ynuo- .«3‘: Programmed cell death (PCD) fulfills the same roles in both plants and animals: elimination of unwanted cells during development and sacrifice of diseased cells. However, although mammalian and plant PCD share some similarities with respect to ultrastructural and physiological hallmarks, evidence for common pathways leading to cell death in animals and plants is limited. The human Bel-2 protein is a pro-survival member of the Bel-2 family of proteins, which includes key regulators of both cell survival and cell death. Structural and functional Bel-2 homologs have been found in C. elegans, mammals, and some viruses (Hengartner and Horvitz, 1994b; Hsu et al., 1997). To determine whether an animal PCD gene, such as bcl-2, can affect plant cell death, we expressed the human bcl-2 gene in Arabidopsis thaliana. Transgenic plants expressing bcl-Z exhibited tissue necrosis/chlorosis and showed increased resistance to a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Necrosis and chlorosis phenotypes became prominent in 3- to 4-week-old plants and co-segregated with production of the Bcl-2 proteins. The PR-I gene, a molecular marker for systemic acquired resistance in higher plants, was induced in bcl-2 plants. Leaf cells undergoing cell death in the transgenic plants showed nuclear condensation, a phenomenon often 122 observed in animal apoptosis. However, the conserved BHl and BH2 domains of Bel-2 were found not to be involved in the necrotic phenotype caused by Bel-2 expression in the transgenic plants. These results suggest that expression of human Bel-2 protein can trigger plant cell death and disease resistance independent of the BHl and BH2 firnctional domains of Bel-2. 5.2. INTRODUCTION Programmed cell death (PCD) is involved in the removal of superfluous and damaged cells (V aux and Korsmeyer, 1999). In most multicellular eukaryotes, PCD plays a major role during development, homeostasis, and in many diseases. Signals that trigger PCD occur as part of normal development and adult homeostasis or as stimuli indicating that a cell is potentially harmful or abnormal. The concept of a functionally conserved, gene-directed process for PCD has been established in animal systems (Gerschenson and Rotello, 1992; Hengartner and Horvitz, 1994b). This orderly process of programmed cell death, known as apoptosis, is regulated by a number of conserved signaling pathways and well-characterized genes (Schwartzman and Cidlowski, 1993; Williams and Smith, 1993). Well-characterized key regulators of animal apoptosis include the Bel-2 family of proteins. Members of this family of proteins can promote or inhibit apoptosis. At least 15 Bel-2 family members have been identified in mammalian cells and several others in viruses (Adams and Cory, 1998; Chao and Korsmeyer, 1998). Pro- and anti-apoptotic family members can heterodimerize and titrate one another’s functions. Their relative 123 concentration acts as a control for the cell suicide program. All members of the Bel-2 family possess at least one of four conserved motifs known as Bel-2 homology domains (BHl to BH4). Mutagenesis analyses have established that BH domains strongly influence homo- and hetero-dimerization between the Bel-2 family of proteins and are thus essential for their fimction in apoptotic pathways (Chittenden et al., 1995; Yin et al., 1994). The human Bel-2 protein was first identified as an oncogenic protein involved in human tumor formation (Tsujirnoto et al., 1984), and later was shown to have the fimction of inhibiting apoptosis. Structural homologs of Bel-2 have been identified in C. elegans as Ced-9 and in chicken as Bcl-XL (Boise et al., 1993; Xue and Horvitz, 1997). The ability of the human bcl-2 gene to prevent cell death in C. elegans strongly suggests that bcl-2 and ced-9 are orthologous genes and functionally interchangeable (Hengartner and Horvitz, 1994b; Vaux et al., 1992). Knowledge about the detailed mechanism by which the Bel-2 protein inhibits cell death is limited. Bel-2 and related death-antagonist proteins may promote cell survival by blocking the activation of a family of proteases termed caspases, required for the final execution phase of the cell death program (Kroemer, 1997). In contrast, the pro-apoptosis members of the Bel-2 family, such as Bax, appear to disrupt the protective effect of Bel-2 by the formation of Bcl-2/Bax heterodimers (Kroemer, 1997; Oltvai et al., 1993). The pro-survival proteins also seem to maintain organelle integrity. It has been suggested that Bel-2 resides on the cytoplasmic face of the mitochondrial outer membrane, endoplasmic reticulum (ER), and nuclear envelope, and modifies the flux of small molecules or proteins to prevent apoptosis (Green, 1998; Kroemer et a]. , 1997). Although the cell 124 death process controlled by bcl—2 can occur in many cell types, it is still not clear whether there are multiple programmed cell death processes in eukaryotes. An unsolved question in plant biology is whether the plant PCD shares a mechanistic similarity with animal apoptosis. In recent years, there have been suggestions that resistance-associated hypersensitive cell death (HR) in incompatible plant-microbe interactions follows a cell death pattern similar to apoptosis in animals (Mittler and Lam, 1995). This killing of host cells during HR may block further pathogen multiplication and spread. With the onset of the HR, other defense responses to pathogens are induced. These include strengthening of cell walls through callose deposition, lignin, and related wall-bound phenolics, production of antimicrobial phytoalexins, and biosynthesis of pathogenesis-related (PR) and other defense-related proteins (Bowles, 1990; Kiedrowski et al., 1992; Lamb et al., 1989; Rogers et al., 1996). HR can be triggered by different elicitors. It requires active metabolism and depends on transcription and translation (He at al., 1993). Furthermore, plant mutants spontaneously displaying cell death lesions have been reported in maize, barley, and Arabidopsis (Buschges et al., 1997; Dietrich et al., 1994; Morris et al., 1998). These disease lesion mimics develop lesions that resemble necrotic disease or HR in the absence of a pathogen. The occurrence of these mutants supports the hypothesis that HR cell death is under genetic control. Plants may contain a pathway for cell death that can be spontaneously activated in the absence of a pathogen; whether it shares mechanistic similarity to the PCD pathway in other multicellular organisms remains an open question. Nevertheless, the HR provides a model for study of PCD in plants. 125 Recently, overexpression of the hrnnan bax gene was found to trigger HR-like cell death in tobacco, suggesting a possible mechanistic similarity between plant and animal PCD (Lacomme and Santa Cruz, 1999). I investigated whether expression of bcl-2 can inhibit HR cell death by expressing the human bcl-2 gene in Arabidopsis thaliana. I found that expression of the bcl-2 gene in transgenic Arabidopsis unexpectedly resulted in a necrotic/chlorotic phenotype. Cells adjacent to lesions of bcl-2 plants showed nuclear condensation. Transgenic plants expressing the bcl-2 gene also exhibited heightened disease resistance against a bacterial pathogen, Pst DC3000, and expressed an increased level of PR-l, a marker gene for systemic acquired resistance (SAR). However, I found that the BHl and BH2 domains of Bel-2 were not involved in the necrotic phenotype caused by Bel-2 expression in the transgenic plants. These results suggest that Bel-2 induced cell death and disease resistance are independent of the anti-apoptotic activity of Bel-2 in animal cells. 5.3. MATERIALS AND METHODS 5.3.1. Construction of Arabidopsis bcl-2 Transgenic Plants Human bcl-2 and its BHl and BH2 mutant genes were amplified from the cDNA clones (Yin et al., 1994) by polymerase chain reaction, using primer A (5’— CCCAAGCTTACCATGGCGCACGC —3’ ) and primer B (5’- GGGAAGCTTTCACTTGTGGCTCAG—3’). The amplified DNA was purified from an agarose gel using the Prep-A-Gene Kit (Bio-Rad). The nucleotide sequences surrounding 126 the start codon of bcl-2 were modified to A‘3 CCATGG+4 to conform to the consensus sequence for hi gh-level translation in eukaryotic cells. The bcl-2 PCR products were cloned directly into the HindIII site of the plant transformation vector pKYLX7l ::3SS2 (Maiti et al., 1993). The bcl-2 expression plasmids were introduced into Agrobacterium strain GV3 850 (Zambryski et al., 1983) by triparental mating (F igurski and Helinski, 1979). The plant transformation protocol used followed that of Bechtold et a]. (1993). T1 seeds were plated on Murashige-Skoog (MS) media containing 0.8% agar, 1 x vitamins, and 50 pg/ml kanamycin to select for transformants. Individual transformants were confirmed for expression of bcl-2 transgenes by protein gel blot analysis. 5.3.2. Protein Isolation and Immunodetection For analysis of expression of Bel-2 and the mutant proteins in transgenic plants, equal numbers of leaf discs of 0.5 cm in diameter were homogenized in SDS-PAGE sample buffer, and boiled. Total leaf protein extracts were separated on a 15% SDS- PAGE gel. Proteins were stained with Coomassie Brilliant Blue R-250. For immunodetection of the Bel-2 protein, separated proteins were electroblotted from the SDS-PAGE gel to an Irnmobilon-P membrane (Millipore). A hamster monoclonal antibody raised against human Bel-2 protein was used as the primary antibody. The blot was incubated further with a second rabbit anti—hamster antibody and a third goat anti- rabbit conjugated alkaline phosphatase. The Bel-2 proteins were visualized by incubating the membrane with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCTP/NBT) (Sigma). 127 5.3.3. RNA Isolation and Analysis Arabidopsis leaves were collected and frozen in liquid nitrogen and ground to a frne powder. Total RNA was isolated using the RN Agents total RNA isolation system (Promega). RNA (3 pg) fiom each sample was added to a loading buffer (17.5% forrnarnide, 15% formaldehyde and 1 x MOPS), denatured at 65°C for 15 minutes, run on an agarose-formaldehyde gel, and transferred to an Irnmobilon-N transfer membrane (Millipore). The membranes were hybridized with the PR-I gene probe, which was amplified from Arabidopsis genomic DNA by PCR. The DNA probes were labeled by random priming using the Prime-It II kit (Stratagene). Hybridization and washing of blots were conducted as recommended by the manufacturer. The blots were exposed to X-ray film to visualize the corresponding transcripts. 5.3.4. Pathogen Infection The bacteria strain used in this experiment was Pseudomonas syringae pv. tomato (Pst) DC3000, a virulent pathogen of Arabidopsis. Bacterial injection and grth curves were done on 5-week-old plants. For disease symptom observation, Pst DC3000 was grown with shaking in Luria-Bertani (LB) broth at 30°C overnight. Bacteria were harvested by centrifugation and resuspended in 5 mM MgCl,. Arabidopsis plants were inoculated by dipping whole rosettes in a suspension of Pst DC3000 at a concentration of 8x108 cells/ml. Plants were kept at 22°C covered with a humidity dome. Leaves were removed from intact plants 5 days after inoculation for photography. For determining 128 bacterial growth curves, young leaves were infiltrated with a suspension of Pst DC3000 at 2x10° cells/ml using a needleless syringe. Bacterial growth was monitored over a 3-day time course. In each time point, five leaf disks were removed using a No. 2 cork borer (2 leaf disks per sample) from five infiltrated leaves of five different plants, surface sterilized, and ground in a 1.5-ml microcentrifuge tube containing 1 ml deO. Dilutions were made in dH,O and bacteria were plated on selective medium (LB containing 100 pg/ml rifamycin). Colonies were counted 48 hours later. 5.3.5. Microscopy Analysis Arabidopsis leaves were fixed in 75% ethanol and 25% glacial acetic acid for 24 hours, and then incubated overnight in 70% ethanol. The fixed leaf tissue was rinsed in distilled water, and stained for DNA with 0.001% ethidium bromide for 30 minutes. Following several washings, stained leaf tissue was mounted on a glass slide in 100% glycerol, and viewed microscopically, using blue light epifluorescence irradiation. 5.4. RESULTS 5.4.1. Expression of the Human bcl-2 Gene in Arabidopsis Results in a Necrotic Phenotype The human Bel-2 protein has been shown to suppress apoptotic cell death when expressed in C. elegans (V aux et al., 1992). This suggests that the bcl-2 cell death 129 pathway is conserved in human and C. elegans (Hengartner and Horvitz, 1994a, 1994b). To determine whether such a pathway also firnctions in plant HR cell death, I transformed Arabidopsis plants with the human bcl-2 gene. The PCR-amplified bcl-2 gene was cloned into pKYLX71::3SS2 (Maiti et al., 1993) in both sense and antisense orientations. The antisense transgenic plants, as well as the transgenic plants with the transformation vector alone, were used as controls. We selected a total of 25 independent lines of sense transgenic plants that varied in the level of expression of the bcl-2 gene, presumably due to insertion of the transgene into different regions of the plant genome and/or differences in the copy number of the transgene. All transgenic plants exhibited a necrotic/chlorosis phenotype. Two representative T, lines are shown in Fig. 5.1. The spontaneous necrosis observed progressed with the aging of the transgenic plants. Necrosis usually appeared on fully developed leaves on 3- to 4-week-old plants. The necrotic leaves eventually died. The severity of the spontaneous necrosis was correlated with the level of Bel-2 expression in the transgenic plants. The S-6 line, which had more extensive necrosis than the S-21 line, had a higher level of Bel-2 expression (Fig. 5.2). The necrosis was stable and co-segregated with the bcl-2 transgene in subsequent generations. In the T, generation, the plants inheriting the bcl-2 construct developed the same degree of necrosis as their respective Tl parental plants. T, plants that did not inherit the bcl-2 construct had no necrosis. Disease diagnosis revealed no significant bacterial or fungal growth on the leaves of transgenic plants, suggesting that the formation of spontaneous necrosis did not result from pathogen infection (data not shown). Symptoms were found on the leaves, but not on the roots or the flowers. 130 Col Col (bcI-2) S-6 Col (bcI-2) Col (bcl-2) S-21 antisense Figure 5.1. Phenotypes of T2 bcl—2 transgenic plants. Spontaneous necrosis and chlorosis (indicated by arrows) is observed on older leaves of transgenic Arabidopsis lines, S-6 and S-21. The plants were stunted compared with wild-type plants or plants transformed with the bcl—2 antisense construct. The large T2 plant in the S-6 pot does not contain the bcl-2 gene. 131 9ta NA? \J’o'wé‘ first? {- Bel-2 protcln Figure 5.2. Levels of the Bcl-2 protein in transgenic Arabidopsis plants. Protein extracts from leaves of wild-type (Columbi gll ) plants and plants transformed with the vector pKYLX71, antisense bcl-2 construct, and sense bcl-2 construct (S-6 and S-21) were analyzed by immunoblot using a monoclonal hamster anti-Bcl-2 antibody. 132 Furthermore, Arabidopsis plants transformed with the vector or the antisense bcl-2 construct did not develop leaf necrosis. Finally, the bcl-2 transgenic plants exhibited overall stunting. The severity of stunting was also correlated with the expression of the bcl-2 gene. Stunting in this case likely resulted fi'om the continuous death of the leaf tissue. 5.4.2. Formation of Necrosis is Accompanied by Activation of Host Defense Responses To determine whether the cell death induced by Bel-2 activates plant defense responses, I examined the expression of the PR-I gene, a molecular marker for SAR in higher plants (Comelissen et al., 1987; Uknes et al., 1993). RNA was isolated from young lesion-negative or partial necrotic leaves of 4- to 5- week-old S-6 and S-21 plants, as well as from healthy leaves of antisense bcl-2 or vector transgenic plants. Transgenic plants expressing the bcl-2 gene accumulated high levels of the PR-I transcript in both healthy and necrotic tissues (Fig. 5.3 A), but PR-l expression was enhanced in leaves that contained lesions. To determine whether the constitutive activation of the PR-I gene in bcl-2 transgenic plants is accompanied by enhanced disease resistance against pathogens, as was determined for Arabidopsis mutants expressing constitutive SAR (Bowling et al., 1994; Dietrich et al., 1994; Greenberg et al., 1994; Hunt et al., 1997), I inoculated the transgenic plants with the virulent bacterium Pseudomonas syringae pv. tomato DC3000. Transgenic S-6 and S-21 plants expressing the bcl-2 gene developed no or very few 133 A cs-21se -+-+-+ a. :4 PR-1 - i r - we :mwaq‘ B c s-21 s-e O +Co| ‘ +S-6 Bacterial Population (109 cfulcnr‘) O A 0 1 2 3 Days Altar Inoculation Figure 5.3. Activation of host defense responses in transgenic Arabidopsis plants expressing the bcl-2 gene. (A). Expression of PR-I in leaves of transgenic plants expressing bcl-2. Young (—) or senescent (+) leaf tissues of plants transformed with the cloning vector (C), and lesion-negative (—) or lesion-positive (+) leaf tissues of S-21 and S-6 plants were subjected to RNA gel blot analysis. Hybridization was conducted using the PR-I cDNA as a probe. (B). Symptom development in the leaves of transgenic Arabidopsis plants inoculated with Pst DC3000. Transgenic plants S-6 and S-21 and the control plant were inoculated with Pst DC3000. Leaves were removed from plants 5 days after inoculation for photographing. Yellow chlorosis on the leaf of control plants indicates the disease symptom caused by Pst DC3000. (C). Growth of Pst DC3000 in leaves of Arabidopsis plants expressing bcl-2. Pst DC3000 were infiltrated into leaves of wild-type and bcl-2 (S-6) plants. Bacterial growth was monitored over 3 days. Each data point represents the mean titer of five leaf disks from five individual leaves of different plants. C: the control plant transformed with the cloning vector pKYLX. 134 disease symptoms after Pst DC3000 infection, compared with wild-type Columbia plants or transgenic plants expressing the antisense bcl-2 gene (Fig. 5.3 B). I next tested whether the reduced disease symptoms in the bcl-2 plants was due to inhibition of bacterial multiplication. The population increase of Pst DC3000 in the young lesion-negative leaves of bcl-2 plants that bore lesions on the older leaves was compared to that in leaves of wild-type plants at a similar development stage. As shown in Figure 5.3 C, Pst DC3000 multiplied less efficiently in the bcl-2 plants compared to wild-type plants. The bacterial titer was 10-30 times lower in the S-6 line than that in the wild-type Arabidopsis plants. A similar decrease in bacterial multiplication was observed in S-21 plants, but the level of bacterial titer in S-21 plants was slightly higher compared with that of S-6 plants (data not shown). This difference in resistance to bacterial multiplication was correlated with less necrosis development in S-21 plants compared with that in S-6 plants. This result suggests that the resistance of bcl-2 plants to Pst DC3000 is associated with the necrotic cell death in the bcl-Z transgenic plants. 5.4.3. Detection of Nuclear Condensation in bcl-2 Transgenic Plants Nuclear fragmentation and chromosome condensation, which lead to formation of apoptotic bodies, are often observed in animal apoptosis and are considered as halhnarks of apoptosis in human cells (Cohen, 1993). We have therefore examined whether nuclei of bcl-2 plants undergo any morphological changes similar to the changes observed in nuclei of animal cells during apoptosis. Partial necrotic leaf tissue of bcl-2 plants contained many cells at different stages of cell death, thus providing a gradient of cells at 135 various stages of cell death in a single section. Using ethidium bromide staining for DNA, nuclei from the healthy parts of the leaf tissue of bcl-2 plants were compared with that of the necrotic region. As shown in Figure 5.4 A and B, nuclei in the wild-type leaf showed a normal nuclear morphology with characteristic chromatin-dense spots. However, cells adjacent to lesions in the bcl-2 transgenic plants showed a high degree of nuclear condensation with no individual chromatin-dense patches (Fig. 5.4 D and E). Leaf tissue in this region still remained green and normal. As a control, nuclei in leaves undergoing extensive senescence were examined and did not condense (Fig. 5.4 C). Although some of the nuclei were misshapen, individual chromatin patches were still evident in cells undergoing senescence. At the very late stages of bcl-2-induced cell death and senescence, no green cells could be seen under light microscopy. Cytoplasmic structures were disorganized. The nuclear material was eventually diffused out in both cases (Fig. 5.4 D and F). In contrast to apoptosis in animal systems, only one ethidium bromide-positive body was seen in each plant cell. This indicates that no apoptotic bodies occurred in either bcl-2 induced cell death or leaf senescence of the control plant. 5.4.4. Expression of Mutant bcl-2 Genes in Arabidopsis Plants In the Bel-2 family, the Bel-2 homology domains BHl to BH4 are essential for their functions (Adams and Cory, 1998; Chao and Korsmeyer, 1998). These domains are thought to be involved in dimerization of members of the Bcl-2 family of proteins.Point mutaions, m1 and mII, in BHl and BH2, respectively, of bcl—2, which did not affect Bcl-2 protein expression, abolishes the ability of Bel-2 to inhibit apoptosis in animal cells 136 Figure 5.4. Detection of nuclear condensation in leaves of transgenic Arabidopsis plants expressing the bcl—2 gene. (A) to (C) show nuclear changes from cells distant (A), adjacent to (B), and in (C) necrotic/chlorotic tissue due to natural senescence in wild-type plants. (D) to (F) show cells distant (D), adjacent to (E), and in (F) necrotic tissue due to Bel-2 expression. Plant leaf tissue was cleaned, fixed, and stained with a DNA counterstain, ethidium bromide. Nuclei of cells adjacent to necrosis induced by Bel-2 are condensed and stained more heavily (E). In the cells of completely senesced (C) and necrotic (F) leaf tissues, nuclei break down and nuclear material has diffused. 137 (Hengartner and Horvitz, 1994a; Yin et al., 1994). To investigate whether the resistance response and cell death in the bcl-Z trangenic plants were dependent on the BH domains, we generated Arabidopsis transgenic lines expressing mutant bcl-2 genes carrying point mutations in BHl (m1) and BH2 (mII), respectively. I selected 20 independent transgenic lines for each mutant transgene. Protein gel blot analysis confirmed that the Bel-2 ml and mII proteins were expressed in transgenic lines (Fig. 5.5). However, the m1 or m]! transgenic plants all showed necrosis in the leaves, similar to the transgenic plants expressing the wild-type bcl-2 gene (Table 5.1). As in bcl-2 transgenic plants, the necrosis in the leaves of the ml and mII plants was correlated with the levels of the ad and mII proteins. These results suggest that cell death in the bcl-2 transgenic plants were not dependent on the BHl and BH2 domains that mediate dimerization of the Bel-2 family of proteins during apoptosis in animals. 5.5. DISCUSSION In this study, I investigated the effect of the human Bel-2 protein on the plant HR. I unexpectedly found that expression of bcl-2 in Arabidopsis resulted in a necrotic and chlorotic phenotypes in leaves (Fig. 5.1). The Arabidopsis defense response was activated in these plants, as indicated by increased expression of PR-I and enhanced resistance to Pst DC3000 (Fig. 5.3). These results suggest that Bcl-2 activated an endogenous resistance-associated cell death program in Arabidopsis. Although our data clearly indicate that the Bel-2 protein triggers cell death and disease resistance in Arabidopsis, expression of the pro-survival protein Bel-XL, a 138 4- Bel-2 Figure 5.5. Expression of Bel-2 mutant proteins, ml and Hill, in transgenic Arabidopsis plants. Proteins fi'om leaves of Arabidopsis plants transformed with the vector (pKYLX) (lane 1) and the bcl-Z (lanes 2 and 3), m1 (lanes 4 and 5) and mII (lanes 6 and 7) constructs were analyzed by immunoblot using monoclonal anti-Bcl-2 antibody. The degree of tissue necrosis/chlorosis is correlated with the Bel-2 level in transgenic plants. Transgenic lines represented by lanes 2, 5, and 6 have more severe necrosis than those represented by lanes 3, 4, and 7. 139 Table 5.1. Effects of mutations in the BHl and BH2 domains of the Bel-2 protein on Bcl- 2-induced cell death in transgenic Arabidopsis plants. Phenotype Bel-2 ml (BHl) mII (BH2) Transgene expression + + + Necrosis + + + Stunting +/- +/- +/- Transgene expression was determined in transgenic bcl-2, m1 and mII plants by western blot analysis. "+" : phenotype occurred; "-": phenotype did not occur. Stunting was observed in some lines of transgenic Arabidopsis plants. 140 chicken homolog of human Bel-2 protein, did not activate the cell death program in tobacco or inhibit the N-gene-mediated HR cell death response to tobacco mosaic virus (Mittler and Lam, 1996). This discrepancy may be due to different expression levels or structures of Bel-2 and Bcl-XL in different plants. Recently, Bax, a pro-apoptosis member of the Bel-2 family of proteins, has been shown to trigger HR-like cell death in tobacco (Lacomme and Santa Cruz, 1999). In that case, BHl and BH3 domains are required for promoting rapid cell death, and the carboxyl-terminal transmembrane (TM) domain is essential for Bax-induced cell death in tobacco. Similar to the Bel—2 protein, the cell death-promoting function of Bax in tobacco correlated with accumulation of PR-I . The ability of human Bax and Bel-2 to trigger cell death and disease resistance in tobacco and Arabidopsis, respectively, suggest their possible interactions with components or processes of the plant cell death pathway. As human Bel-2 protein functions as a pro-survival regulator for animal apoptotic pathways, the results showing cell death induction in Arabidopsis plants by expression of Bel-2 are somewhat surprising. We propose two models to explain Bel-2 -triggered cell death and disease resistance in Arabidopsis (Fig. 5.6). In the first model, Bel-2 functions as a membrane-associated protein and affects the permeability of important cytoplasmic organelles and thus perturbs the natural homeostasis, which indirectly activates the PCD and resistance response in Arabidopsis (Fig. 5.6 A). In animal cells, Bcl-2 is located on the cytoplasmic face of the mitochondrial outer membrane, ER, and nuclear envelope, and appears to maintain organelle integrity by suppressing Bax-mediated cell death activity. A key event in animal PCD is the release of cytochrome c from mitochondria into the cytosol, which then triggers caspase activation and initiation of the final 141 MODEL 2 MODEL 1 i 6% “ilr -. ._, h, r r PCD sensor? i i PCD PiD * "5 t i i SAR Figure 5.6. Models of possible human Bcl-2 protein function in transgenic Arabidopsis plants. SAR: systemic acquired resistance; SA: salicylic acid; L: living factor; D: death factor. 142 degradation phase of the cell death program (Green, 1998). Recently, altered mitochondrial functions in tobacco cells have been found to be associated with harpin- induced cell death in tobacco (Xie and Chen, 2000). The necrotic phenotype in the bcl-Z transgenic plants therefore may be caused by perturbation of mitochondrial and/or membrane function due to imprOper insertion of Bcl-2 into the plant mitochondria. In the second model, we propose that there might be an apoptotic machinery containing a Bcl-2/Bax-like complex in plant cells. The plant-expressed Bel-2 may compete with a plant death factor (D) in binding to a plant “living” factor (L) through the BH domains (Fig. 5.6 B). The resulting unbalanced ratio of D factor to L factor leads to formation of a cell death homodimer (DD), which accelerates programmed cell death in plant cells via the same mechanism as in human cells (Fig. 5.6 B). However, I have shown that point mutations in the BHl and BH2 domains of Bcl-2 do not affect the B01- 2-induced cell death. Thus, this model does not appear to be likely. Although we have eliminated the involvement of BHl and BH2 domains in possible dimerization of Bcl-2 with a plant cell death factor, we could not rule out a possible interaction of other Bel-2 sequence with a plant protein. Although the cell death-promoting activity of full-length Bcl-2 in Arabidopsis was somewhat unexpected, a truncated Bel-2 protein has been shown to trigger cell death in the animal system (Cheng et al., 1997), whereas cleavage-resistant mutants of Bel-2 have increased protection against apoptosis. However, Bcl-2 produced by the transgenic Arabidopsis plants was the expected size for the full-length Bel-2 protein. The nuclear condensation that accompanied cell death in the necrotic leaf tissue of bcl-2 transgenic plants showed similarity to that observed in animal apoptosis (Fig. 5.4). 143 However, other hallmarks of animal apoptosis were not observed in the bcl-2 transgenic plants. 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EMBO J 2, 2143-50. 148 Chapter 6 CONCLUSION AND FUTURE PERSPECTIVES The first part of my doctoral dissertation research was focused on studying the extracellular proteins regulated by the type IH secretion system in Pseudomonas syringae pv. tomato (Pst) DC3000. Type III secretion is controlled by hrp genes in Pseudomonas syringae and other phytopathogenic bacteria. It is believed to be responsible for secretion and translocation of virulence and avirulence factors into the plant cells to trigger host responses. Therefore, study of the proteins secreted through the type HI pathway is of crucial importance for our understanding of plant-bacteria interactions. I have shown that Pst DC3000 secretes several proteins under hrp-inducing conditions. By constructing hrcC and hrpS mutants of DC3000, I demonstrate that at least five extracellular proteins, including HrpW, HrpZ and HrpA, are under the control of the type III secretion system. Both HrpW and HrpZ are harpin-like proteins, whereas HrpA protein is associated with a novel pilus structure (named the Hrp pilus). In collaboration with Martin Romantschuk’s group at the University of Helsinki, we have found that the Hrp pilus is about 6-8 nm in diameter. Its assembly is dependent on the type 1H secretion system in Pseudomonas syringae. I believe that HrpZ, HrpW, and the Hrp pilus play important roles in transporting bacterial virulence and avirulence proteins from the bacterial cytoplasm to the plant cell interior. The supermolecular structure that constitutes type III secretion has been found in animal pathogenic bacteria. In Salmonella typhimurium, a structure called the needle 149 complex spans both the inner and outer membranes of the bacterial envelope. It was suggested that S. typhimurium uses this needle complex to send bacterial effector proteins into host cells (Kubori et al., 1998). The needle structure is a stiff, straight tube 80 nm long and 13 nm wide; it is much shorter than the Hrp pilus, which can grow up to 2 pm long (personal observation). This may reflect bacterial adaptation to the different surface barriers of the host. Unlike animal pathogenic bacteria, plant pathogenic bacteria have to overcome a thick cell wall (~100 nrn). Cell surface appendages similar to the Hrp pilus have been found in all major Gram-negative plant pathogenic bacteria, including Erwinia amylovora (S.Y. He, unpublished results), Xanthomonas campestris (U. Bonas, personal communication), and Ralstonia salanacearum (C. Boucher, personal communication). Thus, formation of the Hrp pilus appears to be a common feature of bacteria containing hrp genes. In Agrobacterium tumefaciens, another pilus-like structure has also been identified. Agrobacterium pili, 3.8 nm in diameter, are required for transfer of DNA to plant cells in a process similar to that of bacterial conjugation (F ullner et al., 1996). Morphologically, the Hrp pilus of P. syringae pv. tomato strain DC3000 characterized in this study resembles most closely the pilus produced by A. tumefaciens. Both pili are much thinner than the surface appendages of S. typhimurium. This similarity may reflect an adaptation of the two bacteria in the infection of wall-bound plant cells. Conditions for pilus production by the two bacteria are also very similar. As for the Hrp pilus, far fewer A. tumefaciens pili are produced at higher temperatures (e.g., 28°C) than at lower temperatures (e.g., 19°C). Furthermore, formation of the Hrp pilus requires solid growth medium, the condition also used for growing A. tumefaciens to promote pilus production. 150 This may reflect the requirement for physical contact between bacteria and plant cells for pilus formation in planta. The exact function of the Hrp pilus during bacterial infection of the plant is not yet known. It may serve as a conduit for bacterial protein delivery, or just as an attachment to keep the bacteria closer to the plant cell. Our lab has recently found that the hrpA gene is required for the secretion of Hrp and Avr proteins in culture and maximal expression of hrp and avr genes, suggesting a dual function of the Hrp pilus in protein secretion and coordinate regulation of the Hrp system (Wei et al., 2000). This evidence argues against attachment being the sole function of the Hrp pilus. In addition to HrpZ, HrpW, and HrpA, my work also revealed at least two additional Hrp-dependent extracellular proteins, EXP-43 and EXP-22 (Fig. 2.2). However, the concentrations of the two EXPs were low. This observation indicates that they are minor secreted proteins controlled by the type HI secretion pathway. The function of these proteins during bacterial infection remains to be determined. One possibility is that these proteins are also components of the Hrp pilus and/or the type III secretion apparatus. In bacterial flagella, besides the major flagellin protein, other minor proteins, such as junction proteins and capping proteins, are also secreted (Macnab, 1992). In DC3000, EXP-43 and EXP-22 might play a similar role in Hrp pilus assembly. Alternatively, they could be unidentified bacterial virulence or avirulence factors that are targeted to the host cell in vivo. The utility of the method I used to identify the Hrp-dependent secreted proteins is limited by the amount of secreted proteins and their antigenicity to the antibody against total EXPs. Thus, some Hrp-dependent EXPs that are important for bacterial infection 151 and type III secretion may have escaped detection. Nevertheless, my efforts resulted in the first identification of multiple Hrp-dependent EXPs in any plant pathogenic bacteria. Alternative approaches need to be developed to identify possibly additional Hrp- controlled EXPs in the future. One such approach would be to exploit the HrpS regulatory mechanism to clone potentially all P. syringae genes that are under the control of the Hrp-regulatory/secretion system. As shown in this study, the production of all P. syringae Hrp-controlled EXPs detected in my experiments was coordinately regulated by the hrpS gene. None of them appeared to be produced in the hrpS mutant (Fig. 2.2). This suggests that the Hrp secretion pathway is likely to be very specific in the proteins that pass through it. Perhaps only proteins that are essential in establishing the initial stages of plant-P. syringae interactions are secreted via this pathway. In the Pst DC3000 hrp gene cluster, the functions of several hrp gene products, such as Her, are still unknown. Whether they are secreted remains to be determined. An antibody against these hrp gene products could be developed and then used to determine either their location in the Hrp secretion apparatus or whether they are secreted in hrp-inducing medium. These hrp genes could also be individually mutated to see if the mutations affect secretion and bacterial pathogenesis. To test possible effects on bacterial pathogenesis of the HrpA protein expressed in plant, I constructed transgenic Arabidopsis plants expressing hrpA. The leaves of the transgenic plants showed a unique pockmark appearance. To my knowledge, this phenotype has not been observed in any Arabidopsis plant expressing any foreign gene. HrpA firsed with the PR-I b signal peptide was found to accumulate in the cell wall of transgenic plants and could not be released by boiling in SDS-PAGE extraction buffer. 152 This is an indication that the interaction between the HrpA protein and plant cell wall components is very strong, and may be the cause of the pockrnarks on the transgenic leaves. The Hrp pilus is suspected to connect the plant cell to the infecting bacteria. As a major component of the Hrp pilus, the affinity of the HrpA protein to the plant cell wall was not surprising. This interaction could change the plant cell wall structure to facilitate the delivery of bacterial proteins into the host cell. Alternatively, the HrpA protein could also frmction as a ligand to specifically bind to a plant cell wall receptor and to trigger a signaling process leading to the host cell response. The specificity of the interaction and the component of the plant cell wall that binds to HrpA needs to be further characterized. Screening for mutations that disrupt the cell wall-HrpA interaction in the hrpA transgenic plant would identify the plant cell wall component and HrpA amino acid residues involved in the interaction between the Hrp pilus and Arabidopsis cell walls. Further analysis of the effect of such hrpA and Arabidopsis mutations on bacterial pathogenesis and type III protein secretion would elucidate whether the cell wall-association of the HrpA protein is biologically significant to bacterial pathogenesis. Although the HrpA protein is believed to be a major structural protein of the Hrp pilus and the formation of the Hrp pilus is essential for plant-P. syringae interactions, HrpA expressed in Arabidopsis did not show any effect on either compatible interaction or incompatible interaction. This result suggested that the Hrp pilus assembly was not disturbed by the expression of HrpA in plants, or that the HrpA protein expressed in plants did not affect the interaction between the Hrp pilus and the host cell (Table 3.1). I hypothesized that the HrpA protein expressed in plants might occupy a receptor site in the 153 plant cell wall and affect the interaction of plant cell walls with the Hrp pilus. The results do not support this hypothesis, in spite of the plant cell wall localization of the HrpA protein. My current hypothesis is that the Hrp pilus assembly is a highly regulated and integrated process and that the interaction between the plant cell and HrpA is very intricate. Simple expression of the HrpA protein alone in plants may not be sufficient to manipulate the interactions between the plant cell and infecting bacteria. As we learn more about the mechanisms of type III delivery and components of the type HI secretion structure, we may be able to construct additional transgenic plants that express selected components of the type III secretion apparatus and to examine the effects of such transgenic expression on plant-Pseudomonas syringae interactions. The second part of my thesis project was to investigate the mechanisms underlying plant programmed cell death and any possible similarities between plant PCD and animal apoptosis. I examined whether animal apoptosis gene products or inhibitors would modulate the hypersensitive response or disease cell death in plants. I found that expression of the human anti-apoptosis bcl-2 gene in Arabidopsis transgenic plants unexpectedly induced spontaneous leaf necrosis and disease resistance (Fig. 5.1). Using the synthetic peptide inhibitors of animal caspases, I showed the existence of ICE-type caspase-like activity in cell death in tobacco plants. These results provide preliminary evidence that plant cell death and animal apoptosis may share certain components, but differ mechanistically. Programmed cell death in plants is an important process in normal plant development and during infection by pathogens (Greenberg, 1996; Mittler and Lam, 1996). It appears to share several features with apoptosis in mammalian cells. However, 154 current evidence linking PCD in plants and animals is still circumstantial, and not all of the classical halhnarks of programmed cell death in animal systems are observed in plants. For example, no apoptotic bodies have been found in plants. This may reflect the differences in cell structure between plants and animals. The formation of the apoptotic bodies is thought to facilitate phagocytosis of their contents by neighboring cells. But in plants, such a process would not be possible because of the cell wall. In recent years, more evidence has suggested that plant PCD shares mechanistic similarities with animal apoptosis. Several components involved in animal apoptosis are found to function in plant PCD. However, no plant genes encoding Bel-2 family members or caspases have been identified in the public plant databases so far. This indicates either a divergence of genes involved in programmed cell death in plants and animals, or that sequence similarity has decreased to undetectable levels during evolution. Consequently, animal apoptosis gene products may or may not function properly in plants. Therefore, the approach involving solely transgenic plants expressing animal apoptosis genes may not be sufficient for understanding the mechanisms of plant PCD. Additional approaches have to be employed to uncover the genes involved in plant PCD. One of the approaches we employed was to use a peptide inhibitor of animal caspases to identify plant caspases. In my study and a recent report by another group (del P020 and Lam, 1998), YVAD, a peptide inhibitor, was shown to inhibit disease-associated cell death, indicating that caspase activity is required for plant PCD. We have also detected three tobacco proteins that specifically bind to the inhibitor YVAD. Further characterization and mutant analysis of the genes encoding these tobacco proteins promise to provide useful information about the plant PCD pathway. 155 Finally, analysis of plants with mis-regulated cell death would be another approach to use to identify the components involved in plant PCD pathways. Several PCD-related disease resistance genes have been cloned. One is the Arabidopsis LSDI gene, which encodes a zinc finger protein and may function as a negative transcriptional regulator of cell death in response to a superoxide-dependent signal (Dietrich et al., 1997). Another example is the barley MLO gene. The MLO gene encodes a novel transmembrane protein (Buschges et al., 1997). However, a potential problem with this approach is that plant cell death might be activated by many mutations that are not normally in the PCD pathway. Therefore, interpretation of the results of cell death mutants can be complicated. In the future, with the tremendous progress on plant genome projects and the rapid development of computation-aided gene analysis, the search for plant genes that are either frmctionally or physically homologous to animal apoptosis genes may result in more fruitful discoveries. 156 References Buschges, R., Hollricher, K., Panstrnga, R., Simons, G., Walter, M., Frijters, A., van Daelen, R., van der Lee, T., Diergaarde, P., Groenendijk, J., Topsch, S., Vos, P., Salamini, F., and Schulze-Lefert, P. (1997). The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88, 695—705. del Pozo, O., and Lam, E. (1998). 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