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CHARACTERIZATION OF THE ROLE OF PSEUDOMONAS SYRINGAE TYPE III EFFECTOR HopAF1 IN VIRULENCE Erica Jania Washington A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology. Chapel Hill 2013 Approved by: Jeff Dangl Miriam Braunstein Joseph Kieber Jason Reed Matt Wolfgang © 2013 Erica Jania Washington ALL RIGHTS RESERVED ii ABSTRACT Erica Jania Washington: Characterization of the role of Pseudomonas syringae type III effector HopAF1 in virulence (Under the direction of Jeff Dangl) Many plant pathogens, including Pseudomonas syringae, encode the type III secretion system for translocating effector proteins into the host during infection. Strains of P. syringae which are not capable of delivering the type III effectors are nonpathogenic. Therefore, the functions of type III effectors are essential for disease. Although several type III effectors have been demonstrated to block components of the plant defense response, the functions of most type III effectors are unknown. Our lab is interested in the type III effector HopAF1, a type III effector that is present in eleven of the nineteen sequenced strains of P. syringae and other plant pathogens. Although the presence of HopAF1 in multiple strains of P. syringae suggests that it plays an important role in virulence, no function has yet been associated with HopAF1. We generated a tertiary structural prediction for HopAF1, which suggests that HopAF1 is structurally related to bacterial deamidases. Deamidation, the irreversible substitution of an amide group with a carboxylate group, is the mechanism by which several bacterial virulence factors manipulate the activity of a specific substrate. To identify a potential target of HopAF1 activity, we employed a yeast two-hybrid screen and identified Arabidopsis methylthioadenosine nucleosidase (MTN1) as a putative target of HopAF1. This interaction, which we extended to include Arabidopsis MTN2, was confirmed in planta using coimmunoprecipitations and bimolecular fluorescence complementation assays. MTNs are enzymes in the Yang cycle, a cycle essential for high iii levels of ethylene biosynthesis. Ethylene is a key plant hormone for plant developmental processes, such as senescence and flowering. Ethylene is also induced during PTI. Therefore, we hypothesized that HopAF1 inhibits PTI by manipulating MTNs and levels of ethylene in plants. To this end, we used gas chromatography to measure ethylene biosynthesis in plants treated with bacterially-delivered HopAF1. We determined that HopAF1 inhibits ethylene biosynthesis in a manner dependent on putative catalytic residues. Additionally, Yang cycle mutants mtn1 mtn2 and mtk are more susceptible to weak pathogens. This data is consistent with the idea that HopAF1 targets a novel component of the plant immune system, the Yang cycle. iv This work is dedicated to my parents. v ACKNOWLEDGEMENTS I am grateful to many colleagues, friends, and family members for the support and encouragement that have made this dissertation possible. It would be impossible to adequately thank each individual; therefore I would like to specifically thank a few individuals for their contributions to my graduate school career. First, I would like to thank my advisor, Jeff Dangl, for his ongoing support and enthusiasm. Jeff taught me how to fearlessly follow science where it takes you and in doing so, always provided me with the resources necessary to help my project progress. In addition, Jeff took the time to teach me how to think scientifically and critically. I also strongly believe that without Jeff’s support during a critical time, I would not be able to write this dissertation. Finally, I would like to thank Jeff for providing me a home in the Dangl-Grant lab. During my time in the Dangl-Grant lab I have worked with so many wonderful individuals that taught me about science and life. I would like to thank Sarah Grant for the time she invested in supporting me and my research. I would also like to thank Petra Epple for her guidance, Terry Law for her friendship, Marc Nishimura for mentoring me when I first joined the lab and Nuria Sanchez Coll for being a role model and a friend. I would also like to thank Vera Bonardi, Tatiana Mucyn, and Derek Lundberg for many good times over the years. Additionally, I would like to thank the scientific community at UNC. I have received great support from across the campus from professors and members of core facilities. I specifically would like to thank Tony Perdue of the UNC Biology Microscopy Facility. In vi addition to spending many hours at the microscope with me, Tony has always been generous with his encouragement and support. Thank you to Ashutosh Tripathy of the UNC Macromolecular Interactions Facility, Brenda Temple of the UNC Center for Structural Biology, Lee Graves and John Sondek. Thank you to my committee for your patience as I developed as a scientist. Thanks to Miriam Braunstein and Matt Wolfgang for teaching me bacteriology in my first year at UNC and for continuing to support me while on my committee. I would also like to Thank you to Jason Reed for being thoughtful and constructive during committee meetings and journal clubs. I would also like to thank Joe Kieber. I rotated in the Kieber lab and always felt like an honorary member of the Kieber lab. The Kieber lab doors remained open to me when I needed to use the gas chromatography machine to do some of my most important experiments. Thank you to Gyeong Mee Yoon and Smadar Harpaz-Saad for being so patient and kind while teaching me how to perform these experiments. I would also like to thank Sharon Milgram, who recruited me to UNC through the Interdisciplinary Biomedical Sciences program. Sharon was really invested in helping me succeed as a person and a scientist. I particularly want to thank her for support in regards to my participation in the Cell and Molecular Biology training grant program. I would not be the person or the scientist I am today without my best friend and my love, David Hubert. I want to thank David for his unconditional love and understanding. Finally, I would like to thank my family. First, I want to thank my big brother, Derek, for giving me brotherly advice from across the country and reminding me of what it means to lead a well-balanced and successful life. My parents have given me the best parental support imaginable during graduate school; in the form of expensive house repairs, cars and every nonperishable good they could stock my home with. My parents have always taught me to be confident and determined. But most important to my survival in graduate school, they taught me the value of hard work. vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................... x LIST OF FIGURES ................................................................................................................ xi LIST OF ABBREVIATIONS.................................................................................................. xiii Chapter 1 Introduction........................................................................................................ 20 A two-tiered model of the plant innate immune system. ............................................ 21 The phytohormone ethylene plays multiple roles in the plant defense response. ..... 26 Successful pathogens suppress PTI /ETI activation and signaling. .......................... 28 The type III secretion system and its effectors are the main virulence determinants of Pseudomonas syringae. .................................................................. 28 References ................................................................................................................ 39 Chapter 2 Bacterial virulence factors modulate eukaryotic host cell signaling systems via deamidation.................................................................................................... 50 Preface ...................................................................................................................... 50 Abstract ..................................................................................................................... 50 Introduction................................................................................................................ 51 Cytotoxic Necrotizing Factor Toxins .......................................................................... 53 Burkholderia Lethal Factor 1 inhibits activity of translation factor eIF4A. .................. 56 Vibrio parahaemolyticus type III effector VopC deamidates small GTPases. ........... 57 Pasteurella multocida toxin ....................................................................................... 58 Cycle Inhibiting Factors ............................................................................................. 61 OspI inhibits host immune responses by deamidating an E2conjugating enzyme. ................................................................................................. 67 Figures ...................................................................................................................... 72 viii References ................................................................................................................ 81 Chapter 3 P. syringae type III effector HopAF1 suppresses plant immunity by targeting the methionine recycling pathway .................................................................... 90 Preface ...................................................................................................................... 90 Abstract ..................................................................................................................... 91 Introduction................................................................................................................ 92 Results ...................................................................................................................... 95 Discussion ............................................................................................................... 105 Materials and Methods ............................................................................................ 109 Figures .................................................................................................................... 118 References .............................................................................................................. 135 Chapter 4 Conclusions and Future Directions ............................................................... 142 Using a shotgun approach and mass spectrometry to determine if HopAF1 modulates MTN function via deamidation. .............................................................. 143 Determine whether a functional deamidation site variant of MTN1 is impervious to HopAF1 activity in planta.................................................................................... 145 Generate a HopAF1/MTN1 co-crystal to determine the molecular mechanism behind HopAF1 inhibition of MTN activity. .............................................................. 147 Figures .................................................................................................................... 150 References .............................................................................................................. 155 ix LIST OF TABLES Table 1.1 P. syringae type III effectors: defense suppression, localization, activities and targets ............................................................................................................. 38 Table 2.1 Bacterial virulence factors that use deamidation to modify host proteins. ........... 80 x LIST OF FIGURES Figure 1.1 P. syringae type III effectors target multiple host cellular components. .............. 37 Figure 2.1 A schematic representation of enzymatic deamidation in proteins. .................... 72 Figure 2.2 The catalytic domains of diverse deamidases. ................................................... 73 Figure 2.3 Crystal structure of the C-terminal region of Pasteurella multocida toxin. .......... 74 Figure 2.4 PMT deamidates and activates heterotrimeric Gα proteins. ............................... 75 Figure 2.5 Model of Cif inactivation of CRL activity and ubiquitination................................. 76 Figure 2.6 Crystal structures of cell cycle-inhibiting factors. ................................................ 77 Figure 2.7 Crystal structure of the CifYp deamidase/NEDD8 complex. ................................. 78 Figure 2.8 Crystal structures of unbound OspI and the OspI/Ubc13 complex. .................... 79 Figure 3.1 The predicted structure of HopAF1 exhibits structural homology to CheD, a bacterial deamidase. ........................................................................................................... 118 Figure 3.2 Alignment of HopAF1 sequences from P. syringae pathovars and other plant pathogens. .......................................................................................................................... 119 Figure 3.3 HopAF1 transgenic lines are susceptible to weak pathogens. .......................... 120 Figure 3.4 Arabidopsis transgenic lines expressing wild-type HopAF1 inhibit flg22-mediated protection against P. syringae pv. tomato DC3000. ................................... 121 Figure 3.5 HR triggered by AvrXv3 is dependent on putative catalytic residues................ 122 Figure 3.6 Establishing a system to study localization of transiently expressed HopAF1 in N. benthamiana. .............................................................................. 123 Figure 3.7 HopAF1 is targeted to the plasma membrane via acylation.............................. 124 Figure 3.8 The acylation-minus variant of HopAF1 co-localizes with free YFP, a soluble protein. ................................................................................................................. 125 Figure 3.9 The Yang cycle contributes to ethylene biosynthesis, which is suppressed by Pto DC3000. ............................................................................................... 126 Figure 3.10 HopAF1 associates with Arabidopsis MTN proteins at the plasma membrane .............................................................................................................. 127 Figure 3.11 Establishing P. fluorescens as a tool to determine the effect of HopAF1 on PAMP-induced ethylene accumulation. .............................................................................. 128 xi Figure 3.12 HopAF1 inhibits PAMP-induced ethylene biosynthesis. ................................. 129 Figure 3.13 Pfo-1 is unable to induce high levels of ethylene biosynthesis in Yang cycle mutants, mtn1 mtn2 and mtk. ....................................................................... 130 Figure 3.14 HopAF1 domain architecture and circular dichroism spectra of HopAF1, HopAF1H186A, and MTN1. .................................................................................................... 131 Figure 3.15 HopAF1 inhibits MTN1 activity in vitro in a manner dependent on catalytic activity. ................................................................................................................................ 132 Figure 3.16 The Yang cycle is required for PTI in Arabidopsis. ......................................... 133 Figure 3.17 Proposed model of HopAF1 suppression of plant innate immunity. ............... 134 Figure 4.1 Alignment and structural localization of MTN1 conserved putative deamidase target residues. ................................................................................... 150 Figure 4.2 MTN1 and MTN2 variants display loss-of-function phenotypes when residues N113 and N194 are ‘deamidated’. .............................................................. 151 Figure 4.3 Circular dichroism spectra of MTN1 ‘deamidated’ variants does not vary from wild-type MTN1. .................................................................................................. 152 Figure 4.4 Purification of wild-type MTN1. ......................................................................... 153 Figure 4.5 Purification of ∆130HopAF1. ............................................................................. 154 xii LIST OF ABBREVIATIONS A/E attaching and effacing ABA abscisic acid ACS aminocyclopropane carboxylate synthase ADP adenosine diphosphate ADR1 activated disease resistance 1 AMP adenosine monophosphate APX ascorbate peroxidase ARF-GEF ADP-ribosylation factor guanine nucleotide exchange factor ATP adenosine triphosphate avr avirulence BAK1 bri1-associated receptor kinase 1 BCL-10 B-cell lymphoma 10 BiFC bimolecular fluorescence complementation BIK1 Botrytis-induced kinase 1 BKK1 BAK1-like kinase 1 BLF1 Burkholderia Lethal Factor 1 BRI1 brassinosteroid insensitive 1 C caryboxyl CaCo2 human colon carcinoma cell line cAMP cyclic adenosine monophosphate CARD caspase recruitment domain CATERPILLER CARD transcription enhancer, purine binding, pyrine, lots of leucine repeats CBL3 calcineurin B-like protein 3 CC coiled-coil xiii CD circular dichroism Cdc42 cell division control protein 42 CDK cyclin dependent kinase cDNA complementary DNA Cdt1 cdc10-dependent transcript 1 CeBIP chitin elicitor binding protein CERK1 chitin elicitor receptor kinase 1 cfu colony forming units CHBP Cif homolog Burkholderia pseudomallei CHPA Cif homolog Photorhabdus asymbiotica CHPL Cif homolog Photorhabdus luminescens CHYP Cif homolog Yersinia pseudotuberculosis Cif cell inhibiting factor CNF1 cytotoxic necrotizing factor 1 CNF2 cytotoxic necrotizing factor 2 CNF3 cytotoxic necrotizing factor 3 CNFY cytotoxic necrotizing factor isolated from Yersinia Col-0 Arabidopsis ecotype Col-0 COP9 constitutive photomorphogenesis CRL cullin ring ligases CSN COP9 signalosome cYFP carboxyl terminus of YFP DAG diacylglycerol DNA deoxyribonucleic acid DNT dermonecrotic toxin DTT dithiothreitol xiv EDTA ethylenediamine tetraacetic acid EFR elongation factor Tu receptor EF-Tu elongation factor Tu EHEC enterohemorrhagic E. coli eIF4A eukaryotic initiation factor 4A EIL1 ethylene insensitive 3-like 1 EIL2 ethylene insensitive 3-like 2 EIN2 ethylene insensitive 2 EIN3 ethylene insensitive 3 elf18 18 amino acid peptide derived from elongation factor Tu EPEC enteropathogenic E. coli ERF ethylene response factor ETI effector-triggered immunity ETS effector-triggered susceptibility Fla 216 Florida 216 Fla 7060 Florida 7060 flg22 22 amino acid peptide derived from flagellin FLS2 flagellin sensing 2 GDP guanosine diphosphate GmHID1 2-hydroxyisoflavanone dehydratase from Glycine Max GMP guanosine monophosphate GRP7 glycine-rich RNA binding protein 7 GRP8 glycine-rich RNA binding protein 8 GR-RBP glycine-rich RNA binding protein GTP guanosine triphosphate HA hemaglutinin xv HeLa cell line derived from Henrietta Lacks Hop Hrp-dependent outer protein HR hypersensitivity response hrc HR and conserved genes hrcC HR and conserved protein C; structural type III secretion system protein hrp HR and pathogenicity genes HrpL HR and pathogenicity protein L; alternative sigma factor Hsp70 heat shock protein 70 Hsp90 heat shock protein 90 IPTG isopropyl β-D-1-thiogalactopyranoside JA jasmonic acid JAK/STAT Janus kinase/ signal transducer and activator of transcription kb kilobase LEE locus of enterocyte effacement LPS lipopolysaccharide LRR leucine-rich repeat LysM lysin motif MALTI mucosa-associated-lymphoid-tissue-lymphoma-translocation gene 1 MAMP microbe-associate molecular pattern MAPK mitogen-activated kinase MBP maltose binding protein MES 2-(4-morpholino)ethanesulfonic acid MTA methylthioadenosine MTK methylthioribose kinase MTN1 methylthioadenosine nucleosidase 1 MTN2 methylthioadenosine nucleosidase 2 xvi N amino NADPH nicotinamide adenine dinucleotide phosphate NB nucleotide-binding NEDD8 neural precursor cell expressed, developmentally down-regulated 8 NLR nucleotide-binding domain and leucine-rich repeat-containing NOD nucleotide-binding oligomerization domain nYFP amino terminus of YFP OD optical density ORF open reading frame PBL PBS1-like protein PBS1 AvrPphB susceptible 1 PDB protein databank PEPR1 Pep1 receptor 1 Pfo-1 Pseudomonas fluorescens PI3K phosphoinositide-3-kinase PLC2 phospholipase C 2 PLCβ phospholipase C β PMT Pasteurella multocida toxin PR pathogenesis-related PRR pattern recognition receptor PsbQ oxygen-evolving enhancer protein 3 PTI PAMP-triggered immunity Pto DC3000 Pseudomonas syringae pv. tomato DC3000 pv pathovar Rac Ras-related C3 botulinum toxin substrate 1 RAR1 required for Mla-12 resistance xvii RbohD respiratory burst oxidase homolog D Rbx RING box protein 1 RhoA Ras homology A RIN4 RPM1-interacting protein 4 RING really interesting new gene 1 RIPK RPM1-induced protein kinase 1 RLK receptor-like kinase RLP receptor-like protein rmsd root-mean-square deviation RNA ribonucleic acid ROS reactive oxygen species rpm revolutions per minute RPM1 resistance to Pseudomonas syringae pv. maculicola 1 RPS2 resistance to Pseudomonas syringae pv. syringae 2 RPS5 resistance to Pseudomonas syringae pv. syringae 5 SA salicylic acid SID2 salicylic acid induction deficient 2 STAND signal transduction ATPases with numerous domains T3E type III effector T3SS type III secretion system TAB1 TAK1-binding protein 1 TAB2 TAK1-binding protein 2 TAK1 TGFβ-activated kinase 1 TEV tobacco etch virus TIR Toll, interleukin-1 receptor TRAF6 tumor-necrosis factor receptor-associated factor 6 xviii TTSS1 type III secretion system 1 TTSS2 type III secretion system 2 UBC13 ubiquitin-conjugating enzyme 13 UBL ubiquitin-like protein UEV1A ubiquitin-conjugating enzyme variant 1a WB western blot Xcv Xanthomonas campestris pv. vesicatoria YFP yellow fluorescent protein ZED1 HopZ-ETI deficient xix Chapter 1 Introduction Plants constantly encounter pathogens such as insects, oomycetes, viruses, fungi, and bacteria in their natural habitats. However, many of these plant-microbe interactions do not result in disease. Plant species may be resistant to microbes that are successful pathogens on other plant species. This is called non-host resistance, and can be thought of as reflecting situations where the microbe in question is not evolutionarily adapted to life on that plant species (Senthil-Kumar and Mysore, 2013). Many mechanisms of non-host resistance involve physical barriers that prevent the pathogen from accessing nutrients in the plant apoplastic fluid. The waxy cuticle layer, the epidermis, and the plant cell wall are structural barriers common in many plants. Bacterial pathogens may access the apoplastic fluid through the stomatal openings in the plant leaf surface (Senthil-Kumar and Mysore, 2013). However, another mechanism of non-host resistance is the closure of stomata in the presence of microbes (Melotto et al., 2006). Moreover, the presence of non-host microbes also induces reinforcements in the cell wall in the form of callose, lignin, or suberin near sites of infection (Senthil-Kumar and Mysore, 2013). Once microbes reach the apoplastic fluid, antimicrobials prevent further colonization. Non-host Arabidopsis apoplastic fluid extracts can restrict the growth of P. syringae pathovars (Senthil-Kumar and Mysore, 2013). However, P. syringae pathovars that are capable of causing disease in Arabidopsis are not inhibited by the apoplastic extracts (Fan et al., 2011). Some antimicrobials are constitutively produced, while others are induced by non-host microbes. A two-tiered model of the plant innate immune system. Adapted microbes successful at surpassing the passive non-host immunity detailed above must overcome an active two-tiered plant immune system (Jones and Dangl, 2006). The first stage is PTI or PAMP-triggered immunity. As a key virulence strategy, pathogens have evolved effector molecules, such as type III effector (T3E) proteins in P. syringae and RXLR effectors in oomycetes, that target PTI, resulting in disease and effector-triggered susceptibility (ETS) (Jones and Dangl, 2006) (Table 1.1). Plants evolved the second tier of the immunity termed ETI for effector-triggered immunity. In ETI, plants use NLR (nucleotidebinding domain and leucine-rich repeat-containing) proteins to detect effectors, often triggering a rapid and localized cell death response called HR. PAMP-triggered immunity The first line of active defense begins with transmembrane pattern recognition receptors (PRRs) recognizing conserved molecular patterns from pathogens called pathogen-associated molecular patterns or PAMPs (Boller and Felix, 2009). Since these microbial signatures are not limited to pathogenic microbes, they are perhaps more aptly referred to as microbial-associated molecular patterns or MAMPs. For the purposes of this introduction, we will use the terms PAMPs and PTI. PAMPs are often highly conserved across a wide range of microbes, fulfill a function essential to the pathogen’s lifecycle, and yet are not present in the host (Boller and Felix, 2009). Common bacterial PAMPs are lipopolysaccharide (LPS), peptidoglycan, elongation factor Tu (EF-Tu), and flagellin (Boller and Felix, 2009). Common fungal PAMPs include chitin and ergosterol, both derived from cell walls of fungi (Boller and Felix, 2009). A 22 amino acid peptide derived from a conserved region of the amino terminus of bacterial flagellin (flg22) and the first 18 amino acids of EF-Tu (elf18) are derivatives of flagellin and EF-Tu that are sufficient for recognition by host cell surface receptors (Felix et al., 1999; Kunze et al., 2004). 21 PAMP recognition occurs via pattern recognition receptors (PRRs) that are analogous to Toll-like receptors in animals. PRRs are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs). RLKs contain an N-terminal extracellular domain, a single transmembrane domain, and a C-terminal kinase domain. RLPs contain an N-terminal extracellular domain, a single transmembrane domain, and a C-terminal short cytoplasmic tail. Some well-studied PRRs include FLS2, EFR, and CERK1 (Monaghan and Zipfel, 2012). FLS2 was the first PRR to be discovered and as a result, much of what we know about PAMP recognition stems from FLS2 studies (Gomez-Gomez and Boller, 2000). FLS2 is the RLK that recognizes flg22 (Gomez-Gomez and Boller, 2000). The C-terminal kinase domain of FLS2 is a Ser/Thr kinase, except that its catalytic loop contains a CD sequence instead of RD. For these reason, FLS2 is a non-RD kinase, which appears to be a hallmark of the kinase domain of immune system related PRRs (Dardick and Ronald, 2006). EFR is a Brassicaceae-specific RLK PRR that recognizes elf18 (Zipfel et al., 2006). CERK1, a RLK that recognizes fungal chitin and bacterial peptidoglycan in Arabidopsis, has three extracellular LysM domains for carbohydrate binding, along with the transmembrane domain and the C-terminal kinase domain (Miya et al., 2007; Wan et al., 2008). In rice, chitin recognition requires an additional RLP PRR called CeBiP (Kaku et al., 2006). CeBiP interacts with rice CERK1 and this interaction is enhanced in the presence of chitin (Shimizu et al., 2010). PAMP recognition and downstream signal transduction involve oligomerization of PRRs. BAK1 (BRI1-associated receptor kinase 1), a positive regulator of PTI signaling forms ligand-induced complexes with FLS2 and EFR (Chinchilla et al., 2007; Heese et al., 2007; Roux et al., 2011). This leads to the recruitment of several other RLKs, such as BIK1 and BKK1, to form heterocomplexes with EFR and FLS2 (Roux et al., 2011). BAK1 has been implicated in complex formation with multiple PRRs, such as PEPR1, an RLK that recognizes endogenous peptides, after induction with their cognate MAMPs (Schulze et al., 22 2010; Yamaguchi et al., 2006) Heterocomplex formation between the BAK1 and PRRs leads to trans-phosphorylation and signal transduction (Schulze et al., 2010). Schulze et al., demonstrated that triggering downstream flg22-mediated responses requires BAK1 with a functional kinase domain (Schulze et al., 2010). BAK1 is critical to PTI-related signaling because bak1 mutants have severely reduced responses to flg22 and are completely insensitive to elf18 (Chinchilla et al., 2007). BAK1 is also required for responses triggered by other PAMPs such as LPS and peptidoglycan (Shan et al., 2000). However, not all PTI signaling requires BAK1. For example, instead of interacting with BAK1, CERK1 undergoes ligand-induced homo-dimerization leading to activation of downstream immune responses (Liu et al., 2012). Perception of PAMPs triggers PTI (Jones and Dangl, 2006). PTI is a critical component of the two-tiered immune system because plants unable to trigger this response are more susceptible to infection. For example, Arabidopsis and N. benthamiana plants encoding a non-functional fls2 are more susceptible to bacterial infection (de Torres et al., 2006; Hann and Rathjen, 2007; Li et al., 2005; Zipfel et al., 2004). Conversely, ectopic expression of EFR in non-Brassicaceae plants Nicotiana benthamiana and tomato increases their resistance to a range of bacterial pathogens (Lacombe et al., 2010). The timing of PTI responses after PAMP recognition ranges from seconds to hours. Early PTI responses include an influx in ions such as H+, K+, Cl- and Ca2+. Ca2+ activates calcium-dependent protein kinases and RbohD (Ma and Berkowitz, 2007). RbohD is a NADPH oxidase that contributes to reactive oxygen species (ROS) burst in Arabidopsis (Ogasawara et al., 2008). This ROS burst occurs approximately 2 minutes after PAMP recognition. ROS burst contributes to PTI by activating defense gene expression, strengthening the cell wall, and initiating cell death (Torres et al., 2006). PTI responses also include the activation of mitogen-activated kinases (MAPKs), which in turns leads to activation of the defense-related WRKY transcription factors (Asai et al., 2002). This is only 23 a fraction of the massive reprogramming of gene expression that occurs after PAMP recognition (Zipfel et al., 2006; Zipfel et al., 2004). Later PTI responses include the induction of ethylene biosynthesis and callose deposition. Callose deposition, an easily measured output and classic PTI response, consists of an accumulation of β-1,3-glucan polymers in the plant cell wall near the sight of infection to form physical barriers against further pathogen attack. We will address the role of ethylene further in the next section of this introduction. Effector-triggered immunity The second line of defense is the gene-for-gene recognition of pathogen effectors by plant disease resistance gene products. This line of defense evolved due to the ability of virulent pathogens to inhibit PTI and thus render the plant susceptible to infection. This condition is termed effector-triggered susceptibility or ETS (Jones and Dangl, 2006). Plants evolved disease resistance (R) proteins to combat ETS. Disease resistance proteins are often referred to as NB-LRR, or NLR, proteins due to their domain architecture, which consists of a central nucleotide-binding (NB) domain and highly polymorphic C-terminal LRR region. The N-terminal domains of NLRs commonly consist of either a TIR (Toll, interleukin1 receptor) domain or a CC (coiled-coil) domain. There are approximately 150 NLRs in the Arabidopsis Col-0 genome (Meyers et al., 2003). NLRs can detect effectors from a wide variety of plant pathogens including fungi, oomycetes, viruses, and bacteria. NLR proteins may detect effectors directly or indirectly. For example, AvrPita, an effector protein from Magnaporthe grisea, interacts directly with the NLR Pi-ta from rice in yeast and in vitro (Jia et al., 2000). The indirect detection of an effector by a NLR is more common. The ‘guard hypothesis’ describes the ability of a NLR to detect a biochemical modification of a host protein by a pathogen effector (Dangl and Jones, 2001; Van der Biezen and Jones, 1998). In doing so, the host takes advantage of the pathogen’s virulence strategy to initiate a successful immune response. The guard 24 hypothesis implies that multiple effectors modify the same host target in different ways and that the host target can guarded by multiple NLRs (Jones and Dangl, 2006). This is because ETI is a driving force of the host-pathogen arms race and is consistent with the finding that pathogen effectors converge on a subset of host proteins (Mukhtar et al., 2011). A classic example of the guard hypothesis that fulfills these tenets is the targeting of the Arabidopsis protein RIN4 (RPM1-Interacting Protein 4) by multiple P. syringae T3Es. AvrRpt2-mediated cleavage of RIN4 and phosphorylation of RIN4 in the presence of AvrRpm1 and AvrB, trigger the activation of NLRs RPS2 and RPM1, respectively (Axtell and Staskawicz, 2003; Mackey et al., 2003; Mackey et al., 2002). The molecular mechanisms that govern NLR activation and signaling are not fully understood and there are few generalizable themes describing NLR activation (Bonardi et al., 2012; Bonardi and Dangl, 2012; Eitas and Dangl, 2010). This is likely due to their rapid evolution in the arms race and the ability to detect virulence effectors from a diverse range of plant pathogens. Plant NLRs are similar to CATERPILLER/NOD/LRR proteins and STAND ATPases (Leipe et al., 2004; Ting and Davis, 2005). STAND ATPases are molecular switches that cycle between an ADP-bound off state and an ATP-bound on state. Similarly, NLRs contain a critical catalytic sequence, called the P loop, in their nucleotidebinding domain. The P loop domain is not required for the function of all NLRs. For example, NLR activation, basal resistance to virulent pathogens, and salicylic acid accumulation mediated by the ADR1 family of NLRs does not require P loop function (Bonardi et al., 2011). Proper activation of NLRs requires a conformational change to release the intramolecular interactions. Additionally, homotypic or heterotypic interactions or the formation of large complexes may be required for NLR activation (Bonardi and Dangl, 2012). ETI is a more rapid and higher amplitude form of PTI. The PTI outputs described above such as ion influx, ROS burst, MAPK cascade signaling, and reprogramming of gene expression also occur during ETI. However, a rapid and localized cell death at the site of 25 infection called the hypersensitivity response or HR often occurs once ETI is triggered (Jones and Dangl, 2006). Although the exact mechanism by which ETI restricts pathogen growth is unknown, mutation of R genes or in genes encoding chaperones required for NLR stability results in an increase in susceptibility to pathogens. The phytohormone ethylene plays multiple roles in the plant defense response. There are three main hormones in plants that regulate the plant defense response against abiotic and biotic stresses: salicylic acid (SA), jasmonic acid (JA), and ethylene. The simplest model of how these hormones function states that ethylene and JA mediate resistance to necrotrophs and herbivorous insects, while SA mediates resistance to biotrophs (Robert-Seilaniantz et al., 2007). Although there are a few exceptions, the ethylene and JA pathways are synergistic to each other and antagonistic to SA (Ecker and Davis, 1987; Penninckx et al., 1998; Schenk et al., 2000). There is cross-talk between these pathways to allow the plant to fine tune its defense response against the multiple invaders it may encounter. In recent years, there have been numerous examples of plant pathogens that hijack specific hormone-regulated signaling pathways, supporting the hypothesis that phytohormones are important components of the plant defense response. A well-studied example is coronatine, a toxin from P. syringae pv. tomato DC3000 (hereafter Pto DC3000). Coronatine triggers a hyper-induction of ethylene- and JA-mediated responses. This results in the suppression of SA-dependent responses that are responsible for resistance against biotrophs like P. syringae, leading to enhanced bacterial growth (Cui et al., 2005; LaurieBerry et al., 2006; Schroeder and Hilbi, 2008; Zhao et al., 2003). The P. syringae T3E HopI1 suppresses SA accumulation (Jelenska et al., 2007). Additionally, AvrPtoB stimulates ABA (abscisic acid) biosynthesis and ABA responses that also antagonize SA biosynthesis and SA-mediated defenses (de Torres-Zabala et al., 2007). AvrRpt2 alters the auxin physiology to promote host susceptibility (Chen et al., 2007). XopD, a T3E from Xanthomonas 26 campestris pv. vesicatoria, (Xcv) was recently found to target a tomato ethylene response factor (ERF) called SIERF4. SIERF4 mutants display reduced ethylene levels and an increase in susceptibility to Xcv (Kim et al., 2013), supporting the hypothesis that ethylene is involved in plant defense. Ethylene is a gaseous plant hormone that is required for normal plant development processes such as seed germination, leaf senescence, and leaf and petal abscission (Kieber, 1997). As mentioned above, ethylene biosynthesis is induced during the plant defense response. This occurs after recognition of PAMPs and the induction of the MAPK signaling cascade. Phosphorylation stabilizes ACS (aminocyclopropane carboxylate synthase) proteins and thus increases the biosynthesis of ethylene (Liu and Zhang, 2004). There is a growing body of work focused on elucidating the role of ethylene in defense against pathogens. Ethylene up-regulates the expression of pathogenesis-related (PR) proteins and increases the accumulation of hydroxyproline-rich, cell-wall strengthening proteins (Ecker and Davis, 1987; Penninckx et al., 1998). Ethylene also increases SAinduced expression of PR-1. This suggests that in addition to functioning synergistically with JA, ethylene can also support SA-dependent defense responses (Lawton et al., 1994). Furthermore, ethylene directly up-regulates the expression of FLS2. FLS2 transcription is directly controlled by the binding of ERFs EIN3/EIL1/EIL2 to the FLS2 promoter and in ethylene insensitive mutants (ein2), both FLS2 expression and FLS2 protein accumulation are suppressed. As a result, ein2 plants are more susceptible to weakened strains of P. syringae (Boutrot et al., 2010; Mersmann et al., 2010). However, there is also data supporting the idea that pathogens up-regulate ethylene as a part of their virulence strategy. For example, P. syringae T3Es AvrPto and AvrPtoB promote increases in the biosynthesis of ethylene to promote disease symptoms in tomato (Cohn and Martin, 2005). ET-responsive transcription factors EIN3 and EIL1 repress PAMPinduced genes, including the SA biosynthesis gene SID2 and the suppression of SA 27 promotes susceptibility to P. syringae (Chen et al., 2009). Furthermore, ethylene perception is also necessary for manifestation of disease symptoms, following Pto DC3000 infection in Arabidopsis (Bent et al., 1992). In conclusion, the role of ethylene in plant defense signaling pathways is complex and needs more attention. Further experimentation is required for complete understanding of how all phytohormones regulate defense pathways and how pathogens promote disease by disrupting hormone homeostasis. Successful pathogens suppress PTI /ETI activation and signaling. Well-adapted pathogens have evolved methods to breach the multiple lines of plant defense described above. Pathogens employ effectors to block components of PTI, ETI, or both. Fungi and oomycetes encode hundreds of effectors as part of their virulence strategies. However, little is known about their function and mechanisms by which they thwart plant immune systems. For that reason, in this section, we will mainly focus on T3Es from bacterial phytopathogens, mostly from Pseudomonas syringae. Bacterial pathogen strains can each inject approximately 20 to 30 virulence effectors into the host cell during infections, and strains across one widespread species, Pseudomonas syringae deploy approximately 60 protein families, many of which have highly variable members (Baltrus et al., 2011) (Figure 1.1; Table 1.1). The type III secretion system and its effectors are the main virulence determinants of Pseudomonas syringae. Before detailing how T3Es target various defense machinery in the plant cell to suppress immune system input signaling and outputs responses, it is important to digress to first introduce the type III secretion system. This will be followed by a detailed discussion of T3E functions, largely in the context of the plant pathogen P. syringae. The type III secretion system In order to cause disease, pathogenic bacteria interact with their host in a variety of ways. These include using various secretion systems, toxins, and adhesions to exploit or 28 disrupt normal host processes and defense responses. One widespread and critical bacterial virulence mechanism is the type III secretion system (T3SS), which consists of a multi-ring base located in the bacterial inner and outer membranes and a protruding needlelike appendage. This needle-like pilus is called the Hrp pilus in phytopathogenic bacteria. The Hrp pilus is used to deliver T3Es into the host cell to cause disease. The T3SS is conserved across a broad range of Gram-negative pathogens including animal pathogens such as Salmonella, Yersinia pestis, and Escherichia coli and phytopathogens Pseudomonas syringae, Ralstonia solanacearum and various Xanthomonas species (Chatterjee et al., 2013; Deslandes and Rivas, 2012; Raymond et al., 2013). Arabidopsis-P. syringae model system P. syringae causes disease in agronomically important crops such as tomatoes, tobacco, and beans. P. syringae also infects Arabidopsis thaliana, a genetically tractable model plant system (Whalen et al., 1991). Since P. syringae was also considered to be genetically tractable, this established a new plant-pathogen system for studying molecular mechanisms utilized by pathogens to causes disease progression and the subsequent host defense responses. P. syringae can survive in a surprisingly large number of environmental reservoirs such as rivers, snow, and clouds (Morris et al., 2007; Morris et al., 2010; Morris et al., 2008). Additionally, P. syringae can infect a wide range of plant hosts. The life cycle of P. syringae begins with epiphytic growth on the surfaces of the aerial parts of plants, such as leaves and flowers. Some strains of P. syringae, such as P. syringae pv. syringae B728A, can multiply to high titers as an epiphyte, while others, such as Pto DC3000, must enter the apoplast to multiply significantly (Feil et al., 2005). Given the proper environmental conditions, such as during high humidity or heavy rain, P. syringae can enter the apoplastic region inside of leaves via stomatal openings. In the apoplast, transcription of the T3SS genes, which are encoded by the tightly linked hrp (hypersensitivity response and pathogenicity) and hrc 29 (hypersensitivity response and conserved) genes, is up-regulated. Although the specific in planta signal that is responsible for up-regulating the hrp and hrc genes is unknown, Huynh et al., demonstrated that fructose, sucrose, and mannitol upregulate AvrB transcription in defined media (Huynh et al., 1989). Although P. syringae remains extracellular, the T3SS translocates T3Es into the host cell to suppress components of PTI and ETI and contribute to pathogenicity (Grant et al., 2006). P. syringae type III secretion system mutants are nonpathogenic; therefore the type III secretion system and its effectors are required for the pathogenicity of P. syringae (Lindgren et al., 1986). Furthermore, Lindgren et al., suggested that these same effectors are responsible for HR triggered in non-host plants (Lindgren et al., 1986). Bacterial genomics, next generation sequencing, and definition of T3E families The effort to identify T3E families in all strains of P. syringae is ongoing and has involved many approaches over the years. Scoring robust effector loss-of-function phenotypes using transposon mutagenesis screens is difficult because, due to functional redundancy of sequence un-related T3Es, there is often only a weak, or even undetectable, contribution of any single effector to virulence. Therefore, identification of T3Es has relied heavily on bioinformatics-based methods. Research on the molecular mechanisms of P. syringae T3Es has focused on the three ‘gold standard’ P. syringae strains that were genome sequenced first; Pto DC3000, P. syringae pv. syringae B728A, and P. syringae pv. phaseolicola 1448A (Buell et al., 2003; Feil et al., 2005; Joardar et al., 2005). Type III effectors have several conserved features that allow for pattern-based genome searches. Studies were performed that searched the promoters of type III effectors for the hrp-box, a cis-acting element transcriptionally up-regulated by the alternative sigma factor HrpL. Chang et al., published the results of a differential fluorescence induction screen that identified type III effectors in Pto DC3000 and P. syringae pv. phaseolicola 30 1448A (Chang et al., 2005). This was followed by validation of the translocation of the candidate effector proteins (Chang et al., 2005). Next generation DNA sequencing has made it possible to quickly generate data on multiple, diverse P. syringae genomes. Using a combination of Illumina and 454 platforms, Baltrus et al., generated draft genome sequences for 14 previously unsequenced, phylogenetically diverse strains of P. syringae (Baltrus et al., 2011). The search for type III effectors among these genomes led to the identification of eight new type III effectors families, resulting in a total of approximately 60 T3E families in this diverse collection of P. syringae strains that expands the five distinct phylogroups (Baltrus et al., 2011). Effectors that target PTI Multiple effectors target RLKs at the plasma membrane and downstream components of PTI signaling pathways. AvrPto and AvrPtoB share no primary or structural homology, but they are redundant P. syringae T3Es that suppress the function of RLKs. AvrPtoB has an N-terminal kinase domain that is sufficient to suppress flg22-induced PTI (Gohre et al., 2008). The C-terminus of AvrPtoB is an E3 ubiquitin ligase domain that can ubiquitinate and degrade RLKs FLS2 and CERK1 (Gimenez-Ibanez et al., 2009; Gohre et al., 2008). AvrPto can bind and inhibit the activity of FLS2 and EFR (Xiang et al., 2008). Although, it has been reported that AvrPto and AvrPtoB can inhibit BAK1 function, there is conflicting evidence for this interaction (Shan et al., 2008; Xiang et al., 2011). AvrPphB cleaves kinases, such as BIK1, that function downstream of PAMP recognition and integrate signals from multiple PRRs to block PTI responses (Zhang et al., 2010). Interestingly, AvrPphB cleavage of PBS1 triggers RPS5-mediated ETI in Arabidopsis inbreds that carry the functional RPS5 NLR receptor gene (Ade et al., 2007; Shao et al., 2003). AvrAC, a T3E from Xanthomonas campestris pv. campestris also inhibits PTI by targeting BIK1 and RIPK (Feng et al., 2012). HopF2 and HopAI1 suppress PTI by inhibiting MAPK cascades. HopF2 ADP-ribosylates MKK5 and inhibits its kinase activity (Wang et al., 2010). HopAI1 uses 31 phosphothreonine lysase activity to inhibit the function of MPK3 and MPK6 (Zhang et al., 2007). Similar to HopF2, disruption of PTI by targeting MAPK cascades triggers activation of a NLR and ETI (Zhang et al., 2012). Using effectors as probes of plant cell biology and plant defense has led to the identification of components of the plant immune system that extend beyond PRRs and NLRs. HopM1 targets vesicle trafficking by inhibiting the function of an Arabidopsis ARFGEF protein MIN7. HopM1 degradation of MIN7 is proteasome-dependent (Nomura et al., 2006). Interestingly, when ETI is triggered by AvrRpt2, AvrPphB, and HopA1, HopM1mediated destabilization of MIN7 is inhibited (Nomura et al., 2011). This demonstrates a novel situation in which ETI contributes to plant defense by preventing bacterial suppression of a key component of PTI (Nomura et al., 2011). HopU1 is a P. syringae type III effector mono-ADP-ribosyltransferase that targets Arabidopsis glycine-rich RNA binding proteins (GR-RBPs) GRP7 and GRP8 (Fu et al., 2007). HopU1 negatively affects the ability of GRP7 to bind RNA (Jeong et al., 2011). Specifically, HopU1 blocks the interaction between GRP7 and FLS2 and EFR transcripts (Nicaise et al., 2013), leading to a reduction in FLS2 accumulation after infection with a virulent P. syringae strain expressing HopU1 (Nicaise et al., 2013). This is consistent with HopU1 inhibiting PAMP responses triggered by flg22, such as callose deposition (Fu et al., 2007). T3Es HopZ1a and HopZ1b have also been implicated in PTI suppression. HopZ1a and HopZ1b degrade the soybean protein GmHID1 (2-hydroxyisoflavanone dehydratase), a protein involved in the biosynthesis of antimicrobial phytoalexins (Zhou et al., 2011). Soybean plants with GmHID1 silenced are more susceptible to infection (Zhou et al., 2011). HopZ1a also interacts with and acetylates tubulin to inhibit formation of stable cytoskeletons (Lee et al., 2012). HopZ1a expression blocks callose formation (Lee et al., 2012). Additionally, HopI1 localizes to the chloroplast where it inhibits Hsp70 function, which leads to a decrease in SA and suppression of PTI (Jelenska et al., 2010; Jelenska et al., 2007). Overexpression of HopN1 leads to a decrease 32 in PAMP-induced ROS burst and callose deposition (Rodriguez-Herva et al., 2012). Finally, HopG1 also inhibits callose and ROS burst, although the molecular mechanism is currently unknown (Block et al., 2010). Effectors that block ETI The first evidence of type III effectors blocking HR came from studies in the 1990s in which Xanthomonas campestris pv. campestris hrp mutants induced a programmed cell death in the vascular system of crucifers (Jackson et al., 1999). This suggested that there are type III effectors in the hrp regulon that are blocking this HR-like response. More recent studies have identified P. syringae type III effectors that are involved in suppressing ETI. For example, Jackson et al. demonstrated that VirPphA, AvrPphC, and AvrPphF from P. syringae pv. phaseolicola were able to inhibit NLR activation (Jackson et al., 1999). AvrPtoB suppresses ETI-associated programmed cell death in tomato plants via ubiquitin ligase activity in its C-terminus (Abramovitch et al., 2003). HopF2 targets RIN4 and blocks ETI (Wilton et al., 2010). AvrAC can also block ETI (Feng et al., 2012). Furthermore, Jamir et al., showed that effectors could inhibit cell death triggered in yeast, suggesting those effectors target programmed cell death pathways conserved in eukaryotes (Jamir et al., 2004). Subcellular localizations and enzymatic functions of P. syringae type III effectors As described above, T3Es can inhibit PTI and ETI and thus contribute to virulence. Given the diversity of targets noted above for particular T3Es, it is not surprising to learn that they function in various subcellular locations. Thus, determining the role of any T3E in bacterial virulence requires multiple approaches and gathering pertinent forms of data, such as the subcellular localization of the effector, the enzymatic function, and a potential host target. Determining the subcellular localization of a T3E in a plant cell can help determine which host pathways or host defense mechanisms are targeted by the effectors. AvrRpm1 and AvrB were the first T3Es found to be targeted to the plasma membrane via the 33 eukaryotic-specific posttranslational modification of fatty acid acylation at the N-terminus (Nimchuk et al., 2000). At the plasma membrane that AvrRpm1 and AvrB trigger the phosphorylation of RIN4, leading to the elicitation of RPM1-mediated ETI. Myristoylation sites of AvrRpm1 and AvrB are required to trigger ETI (Nimchuk et al., 2000). Since then multiple type III effectors such have been shown to localize to the plasma membrane via acylation and their functions are dependent on their proper targeting (Dowen et al., 2009; Lewis et al., 2008; Robert-Seilaniantz et al., 2006; Shan et al., 2000). The chloroplasts, mitochondria, and nucleus are all organelles targeted by T3Es. HopI1 is targeted to the chloroplasts via a noncanonical targeting sequence (Jelenska et al., 2007). HopI1 alters thylakoid structure, suppresses SA accumulation and SA-related defenses and interacts with Hsp70 (Jelenska et al., 2010; Jelenska et al., 2007). HopN1 also is targeted to the chloroplast, where it degrades a photosystem II protein, PsbQ (Rodriguez-Herva et al., 2012). HopG1 is targeted to the mitochondria and although its specific protein target it currently unknown, HopG1 causes a reduction in PAMP-induced callose deposition and ROS burst (Block et al., 2010). Although there are no published examples of P. syringae T3Es being targeted directly to the nucleus, the AvrBs3 family of T3Es from Xanthomonas spp. are targeted to the nucleus, where they act as transcriptional activators to directly regulate host gene expression (Boch and Bonas, 2010). Along with specific localizations, many T3Es have specific enzymatic activities used to disrupt host processes and suppress PTI/ETI. Unfortunately, these diverse biochemical functions have only recently started to be deciphered. Several type III effectors have been shown to possess cysteine protease activity. AvrRpt2 cleaves RIN4, which activates the RIN4-associated NB-LRR receptor, RPS2 (Kim et al., 2005). AvrPphB is a cysteine protease that cleaves PBS1, which activates RPS5 (Shao et al., 2003). HopN1 is also a cysteine protease that cleaves PsbQ (Lopez-Solanilla et al., 2004). There are other identified enzymatic functions for T3Es. HopAO1 is a protein tyrosine phosphatase (Espinosa et al., 34 2003). HopU1 is an ADP ribosyltransferase (Fu et al., 2007). AvrAC is a uridylyl transferase from Xanthomonas campestris pv. campestris (Feng et al., 2012). HopX1 is predicted to be a transglutaminase (Nimchuk et al., 2007). While the transglutaminase function has not been shown, the putative catalytic residues are required for HopX1-triggered ETI in beans (Nimchuk et al., 2007). Assigning enzymatic functions to T3Es is challenging. T3Es have relatively low conservation at the primary amino acid sequence level to proteins of known biochemical function. This suggests that convergent evolution into conserved folds to modify host signaling pathways. Solving crystal structures of T3Es is way to help determine a conserved structure associated with a function and/or specific residues required for function (Boutemy et al., 2011; Jeong et al., 2011; Lee et al., 2004; Singer et al., 2004). In lieu of an actual crystal structure, generating tertiary structure predictions can also provide some information about the function of a T3E. AvrRpm1 is predicted to have a fold similar to the catalytic domain of poly-ADP-ribosyl polymerase and the central domains of HopQ1 is predicted to have homology to nucleoside hydrolase enzymes (Cherkis et al., 2012; Li et al., 2013). Additionally, SseI from Salmonella is predicted to have deamidase function based on homology to the bacterial deamidase PMT. However, despite many efforts, SseI has not been shown to have deamidation activity (Bhaskaran and Stebbins, 2012). Deamidation is the irreversible conversion of glutamine and asparagines to glutamic acid and aspartic acid, respectively. Since deamidation is emerging as a posttranslational modification utilized by multiple bacterial toxins to modify host signaling pathways, we will explore bacterial deamidases further in Chapter 2 (Washington et al., 2013). Although the enzymatic activities of multiple T3E proteins have been discovered, the link between enzymatic activity, the function of each effector’s host cellular target protein, and the mechanism of suppression of both ETI and PTI often remain unclear. Unraveling how T3Es use enzymatic activity to disrupt the function of host proteins will reveal details of 35 the molecular mechanisms by which these host proteins function normally. For some highly conserved host proteins this may have implications beyond pathology that expand into the realms of host cell signaling pathways and cell biology. 36 Figure 1.1. P. syringae type III effectors target multiple host cellular components. Adapted from Xin and He, 2013. Bacterial PAMPs, such as flagellin, trigger PTI. P. syringae injects type III effector proteins into the plant cell, via the type III secretion system, to block components of PTI and ETI. AvrPto and AvrPtoB inhibit the activity of PRRs at the plasma membrane through inhibition of PRR kinase activity or degradation, respectively. AvrPphB cleaves BIK1, PBS1, and PBS1-like proteins (PBLs). HopF2 ADP-ribosylates MKK5 and inhibits its activity. HopF2 may also inhibit PTI at the site of the plasma membrane by blocking BIK1 phosphorylation. HopAI1 also targets the MAPK signaling cascade by using phosphothreonine lyase activity to inhibit the function of MPK3 and MPK6. HopU1 is a mono-ADP-ribosyltransferase that targets glycine-rich RNA-binding proteins (GR-RBPs). HopU1 prevents GRP7 from interacting with FLS2 and EFR transcripts, leading to a reduction in FLS2 transcript and protein accumulation. HopM1 targets vesicle trafficking by inhibiting the function of the ARF-GEF protein MIN7. HopZ1a has been reported to have multiple roles. HopZ1a degrades HID1, a protein involved in biosynthesis of antimicrobial phytoalexins in soybean. HopZ1a also acetylates tubulin. Recently, HopZ1a has been reported to target a pseudokinase, ZED1, which triggers ZAR1-mediated recognition in Arabidopsis. Multiple effectors target RIN4. AvrRpt2 cleaves RIN4, triggering RPS2mediated ETI. HopF2 blocks ETI triggered by AvrRpt2 by interacting with RIN4. Phosphorylation of RIN4 in the presence of AvrRpm1 and AvrB is mediated by RIPK. AvrPto and AvrPtoB can degrade RIN4. Finally, AvrB targets RIN4 in complex with MPK4/RAR1/Hsp90 to alter jasmonate signaling. HopI1is targeted to the chloroplast, where it alters thylakoid structure, suppresses SA acculumation, and SA-related defenses. HopN1 is also targeted to the chloroplast. HopN1 degrades photosystem II protein, PsbQ. Although HopG1 localizes to the mitochondria, its direct target and function remain unknown. 37 Table 1.1. P. syringae type III effectors: defense suppression, localization, activities and targets Effector AvrB Host target(s) Biochemical Activity Subcellular RIN4/RAR1/MPK4 ? Plasma membrane PBS1/BIK1/PBLs Cysteine protease Plasma membrane Pto/Fen/EFR/FLS2/ BAK1? ? Plasma membrane Pto/Fen/BAK1/FLS2/RLKs/BAK1? E3 ubiquitin ligase ? AvrRpm1 AvrRpt2 RIN4 Plasma membrane RIN4 Poly-ADPib lt f ? Cysteine protease ? HopAI1 HopAO1 HopI1 MPK3/MPK6 ? Phosphothreonine lyase Tyrosine phosphatase ? ? Hsp70 J domain Chloroplast AvrPphB AvrPto AvrPtoB 38 HopF2 HopG1 HopM1 HopN1 HopU1 HopX1 HopZ1a/b HopZ2 HopZ3 RIN4/MKK5 ? AtMIN7 PsbQ GRP7, GRP8 ? GmHID1; ZED1 ? ? Mono-ADPribosyltransferase ? ? Cysteine protease Mono-ADPib lt f Transglutaminase? Acetyltransferase?/cysteine protease? Acetyltransferase?/cysteine protease? Acetyltransferase?/cysteine protease? Plasma membrane Mitochondria Vesicles Chloroplast Nucleo-cytoplasmic ? Plasma membrane Microtubules (a) Plasma membrane ? 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Zipfel, C., Robatzek, S., Navarro, L., Oakeley, E.J., Jones, J.D., Felix, G., and Boller, T. (2004). Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, 764-767. 49 Chapter 2 Bacterial virulence factors modulate eukaryotic host cell signaling systems via deamidation Preface The following chapter was published in September 2013 in Microbiology and Molecular Biology Reviews under the title “What a difference a Dalton makes: Bacterial virulence factors modulate eukaryotic host cell signaling systems via deamidation” (2013, 77, 527-539). I wrote this manuscript while planning and completing experiments for the work described in Chapter 3. I received direction and edits from our collaborator, Mark Banfield, from the John Innes Centre in Norwich, UK and my thesis advisor, Jeff Dangl. Abstract Pathogenic bacteria commonly deploy enzymes to promote virulence. These enzymes can modulate the function of host cell targets. While the actions of some enzymes can be very obvious (e.g. digesting plant cell walls), others have more subtle activities. Depending on the lifestyle of the bacteria these subtle modifications can be crucially important for pathogenesis. In particular, if bacteria rely on a living host, subtle mechanisms to alter host cellular function are likely to dominate. Several bacterial virulence factors have evolved to use enzymatic deamidation as a subtle post-translational mechanism to modify the function of host protein targets. Deamidation is the irreversible conversion of amino acids glutamine and asparagine to glutamic acid and aspartic acid, respectively. Interestingly, all currently characterized bacterial deamidases affect the function of the target protein by modifying a single glutamine residue in the sequence. Deamidation of target host proteins can disrupt host signaling and downstream processes by either activating or inactivating the target. Despite the subtlety of this modification, it has been shown to cause dramatic, context-dependent, effects on host cells. Several crystal structures of bacterial deamidases have been solved. All are members of the papain-like superfamily and display a cysteine-based catalytic triad. However, these proteins form distinct structural subfamilies and feature combinations of modular domains of various functions. Based on the diversity of pathogens that use deamidation as a mechanism to promote virulence, and the recent identification of multiple deamidases, it is clear that this enzymatic activity is emerging as an important and widespread feature in bacterial pathogenesis. Introduction Many bacterial pathogens use diverse suites of virulence factors to contribute to pathogenicity. These virulence factors include toxins and type III effectors, proteins injected into host cells via specialized type III secretion systems. Effectors often modify eukaryotic host target proteins with posttranslational modifications that alter normal cellular function. Commonly described posttranslational modifications utilized by effectors include ubiquitination, acetylation, and AMPylation (Cui and Shao, 2011; Ribet and Cossart, 2010; Yarbrough and Orth, 2009). Recently, enzymatic deamidation has emerged as a common posttranslational modification utilized by a broad range of bacterial pathogens of both plants and animals to alter the function of host proteins. Deamidation is the substitution of an amide group with a carboxylate group (Figure 2.1). Therefore, it converts glutamines and asparagines to glutamic acid and aspartic acid, respectively. This irreversible amino acid conversion results in an increase of approximately 1 Dalton in the mass of the target protein, an increase in the negative charge of the target protein, and the release of ammonia. Nonspecific deamidation can occur spontaneously as proteins age and are degraded (Robinson and Rudd, 1974). In contrast, specific enzymatic deamidation can regulate normal cellular functions, such as chemotaxis and protein turnover in prokaryotes, or to 51 disrupt eukaryotic host cell function during infection (Chao et al., 2006; Striebel et al., 2009). Here, we focus on deamidases that contribute to bacterial virulence. The topic of this review is the six currently known families of bacterial virulence factors that use deamidation to modulate host functions during infection (Table 2.1). Cytotoxic Necrotizing Factors, CNFs, are a family of deamidases from E. coli (CNF1, 2, and 3) and Yersinia pseudotuberculosis (CNFY). The CNFs target a glutamine residue (either Gln63 or Gln61) in the switch II domain of GTPase proteins that is critical for function (Flatau et al., 1997; Schmidt et al., 1997). Deamidation of this glutamine leads to constitutive activation of the target GTPases, resulting in cytoskeletal rearrangements. Reorganization of the actin cytoskeleton is one mechanism used by invasive bacteria to promote entry into host cells (Doye et al., 2002; Galan and Zhou, 2000). BLF1 is a toxin from Burkholderia pseudomallei that is lethal to mice and tissue culture cells (Cruz-Migoni et al., 2011). BLF1 inhibits host protein synthesis via deamidation of eIF4A (Cruz-Migoni et al., 2011). VopC is a type III effector from Vibrio parahaemolyticus that deamidates and constitutively activates small GTPases (Zhang et al., 2012). Pasteurella multocida toxin (PMT) is the major virulence factor of Pasteurella multocida, the causal agent of a variety of mammalian and avian diseases. PMT constitutively activates various G proteins including Gαq/11, Gαi, and Gα12/13 via deamidation of a glutamine that is also in the switch II region (Orth et al., 2009). Cell cycle inhibiting factors, Cifs, are type III effectors and cyclomodulins from multiple bacterial species. Cifs inhibit ubiquitination pathways by deamidating glutamine 40 of ubiquitin and the ubiquitin-like protein, NEDD8 (Cui et al., 2010). OspI is a type III effector protein from Shigella flexneri that dampens host immune responses by deamidating UBC13 and disrupting the TRAF6-mediated signaling pathway (Sanada et al., 2012). We review the details of each of these six families, specifically with respect to their three-dimensional structures and the impact that deamidation has on the function of their host target proteins. 52 We conclude that deamidation, as a non-reversible modification, is likely an all or nothing virulence switch to alter diverse cellular functions across diverse pathosystems. Cytotoxic Necrotizing Factor Toxins Cytotoxic Necrotizing Factor toxins deamidate Rho GTPases. Cytotoxic Necrotizing Factor, CNF1, from E. coli was the first bacterial deamidase identified (Flatau et al., 1997; Schmidt et al., 1997). CNF1 is expressed in uropathogenic strains of E. coli (Caprioli A, 1983). Following the discovery of CNF1, a number of homologues were identified in different strains of E. coli. For example, CNF2 is produced by enteropathogenic E. coli strains isolated from cows and sheep (Oswald et al., 1989). The amino acid sequence of CNF2 is 85% identical to CNF1. CNF3 was isolated from necrotoxigenic E. coli that infects sheep and goats (Orden et al., 2007). CNF3 shares 70% amino acid identity with CNF1. Interestingly, CNFs are not restricted to E. coli. Yersinia pseudotuberculosis produces CNFY, which is 61% identical to CNF1 (Lockman et al., 2002). Further, dermonecrotizing Toxin, DNT, from Bordetella is a virulence factor that also shares sequence homology with CNF1. However, DNT behaves most efficiently as a transglutaminase in vitro, active in the presence of polyamines (Masuda et al., 2000; Schmidt et al., 2001). Therefore, we will not include a detailed discussion of DNT here. Schmidt et al. and Flatau et al. demonstrated that CNF1 converts Gln63 of the critical RhoA GTPase switch II region to glutamic acid via enzymatic deamidation (Flatau et al., 1997; Schmidt et al., 1997). Importantly, neither Gln29 nor Gln52, the other two glutamine residues in RhoA, were modified by CNF1 (Flatau et al., 1997; Schmidt et al., 1997). Deamidation of Gln63 triggers constitutive activation of RhoA, functionally mimicking the GTP-bound state. Furthermore, the RhoA Gln63Glu variant, that mimics the product of the deamidation reaction, phenocopies the CNF1-treated RhoA on microinjection into Vero cells (Flatau et al., 1997; Schmidt et al., 1997). 53 RhoA is not the only target of CNFs. CNF deamidases possess different GTPase substrate specificities. CNF1 can deamidate Rac and Cdc42 in vitro and Cdc42 in intact HeLa cells (Lerm et al., 1999; Schmidt et al., 1997). CNF2 can modify RhoA and Rac when co-expressed in E. coli (Sugai et al., 1999). CNF3 can deamidate RhoA, Rac, and Cdc42 in intact HeLa cells treated with recombinant toxin (Stoll et al., 2009). Although RhoA, Rac, and Cdc42 serve as substrates for CNFY deamidation in vitro, only RhoA could be deamidated by CNFY in intact cells (Hoffmann et al., 2004). Rho GTPases are molecular switches that cycle between an inactive, GDP-bound state and an active, GTP bound state (Heasman and Ridley, 2008). The active state initiates a variety of downstream responses, including reorganization of the actin cytoskeleton. Rho GTPases that are unable to hydrolyze GTP are maintained in a constitutively active state. Activation of different members of the Rho GTPases family leads to specific effects on the cytoskeleton. RhoA activation leads to actin reorganization into stress fibers, Rac activation causes membrane ruffling, and Cdc42 activation causes the formation of filopodia (Nobes and Hall, 1995; Ridley and Hall, 1992; Ridley et al., 1992). Treatment with CNF1 induces stress fiber formation, microspikes, membrane ruffles, and multinucleation in intact cells (Flatau et al., 1997; Lerm et al., 1999; Schmidt et al., 1997). CNF3 and CNFY also trigger strong formation of actin stress fibers in intact cells (Hoffmann et al., 2004; Stoll et al., 2009). Surprisingly, there is no evidence of CNF3-triggered membrane ruffling or filopodia formation. It has been demonstrated that CNF1 is required for the invasion of uropathogenic E.coli into uroepithelial cell monolayers (Doye et al., 2002). This is consistent with other bacterial pathogens, such as Salmonella, that attack the host cytoskeletal to facilitate bacterial entry (Galan and Zhou, 2000). The C-terminus of CNF1 forms a single compact catalytic domain. CNFs are modular proteins. The N-terminal domains of these toxins are required for binding and entry into host cells via receptor-mediated endocytosis (Contamin et al., 2000; 54 Lemichez et al., 1997). The structure of the catalytic C-terminus of CNF1 (residues 7201014) revealed a three-layered α/β/β sandwich, consisting of two mixed 5-stranded β-sheets that are flanked by two α-helices (Figure 2.2A) (Buetow et al., 2001). The structure of CNF1 identified a novel overall protein fold that contained an arrangement of residues in the active site reminiscent of the papain-like superfamily of enzymes that include the transglutaminase blood coagulation factor XIII and GMP synthetase (Buetow and Ghosh, 2003). More recently, it was determined that CNF1 is also structurally similar to Thermotoga maritima deamidase protein, CheD, and a bacterial protein of unknown function, YfiH (Chao et al., 2006). Papain-like superfamily enzymes typically contain a catalytic triad consisting of histidine, cysteine, and asparagine/aspartic acid. The cysteine and histidine form a thiolateimidazolium pair that is critical for activating the cysteine nucleophile, with the asparagine/aspartic acid thought to be involved in orienting the histidine. CNF deamidases contain the invariant cysteine and histidine residues (Schmidt et al., 1998). Interestingly, in CNF1, valine is the third member of the catalytic triad and this residue is involved in orienting the histidine through a main chain carbonyl hydrogen bond (Buetow et al., 2001). Structure-based mutagenesis of conserved residues in the catalytic pocket identified Asn835 and Ser864 that are also required for CNF1 activity (Figure 2.2A) (Buetow and Ghosh, 2003). Further, deletion of five individual loop regions surrounding the CNF1 catalytic pocket revealed that loops 8 and 9 are required for deamidation of RhoA (Buetow and Ghosh, 2003). Most likely Asn835 and Ser864 have indirect roles in catalysis and loops 8 and 9 are important for substrate recognition. A peptide derived from RhoA residues 59-69 could be deamidated by CNF1, thus defining the minimal region of RhoA required for CNF1 deamidation (Flatau et al., 2000). Mutations outside of these key residues did not interfere with the ability of CNF1 to deamidate RhoA (Flatau et al., 2000). This suggests that CNF1 interacts with a narrow 55 surface of RhoA, and likely other GTPase substrates, and does not require a significant binding surface for substrate recognition. Burkholderia Lethal Factor 1 inhibits activity of translation factor eIF4A. Burkholderia Lethal Factor 1, formerly known as BPSL1549, is a potent toxin from Burkholderia pseudomallei. Melioidosis is a disease caused by infection with Burkholderia pseudomallei, a pathogen endemic in areas in Southeast Asia and northern Australia (Wiersinga et al., 2006). It is associated with severe septicemia and can remain dormant in the host for decades. The molecular basis for the pathogenicity of B. pseudomallei is poorly understood. BLF1 interacts with the human translation factor eIF4A in a human cell lysates (CruzMigoni et al., 2011), suggesting that BLF1 might inhibit translation. This interaction was confirmed by co-immunoprecipitation. BLF1 reduces endogenous host cell protein synthesis and triggers increased stress granule formation, which is associated with translational blocks. In order to determine the outcome of the BLF1/elF4A interaction, FLAG-tagged eIF4A was purified from human cells expressing BLF1. Subsequent mass spectrometric analysis of eIF4A revealed deamidation of Gln339 to Glu. To determine how deamidation of this residue altered eIF4A activity, the Gln339Glu variant was generated. Although this variant was capable of binding both RNA and ATP, and maintained wild-type levels of ATPase activity, helicase activity was reduced by 47% (Cruz-Migoni et al., 2011). The crystal structure of BLF1 revealed an α/β fold comprising a sandwich of two mixed β-sheets surrounded by loops and α-helices (Figure 2.2B) (Cruz-Migoni et al., 2011). The β-sheet core of the catalytic pocket is structurally similar to that of the deamidase domain of CNF1, with an rmsd of 3.9 Angstroms over 170 residues (Cruz-Migoni et al., 2011). Interestingly, these deamidase domains share only 9% amino acid identity. BLF1 lacks the receptor binding and translocation domains of CNF1, an expected evolutionary innovation, since the intra-cellular lifestyle of B. pseudomallei renders these domains 56 unnecessary. However, the invariant catalytic residues found in CNF1, cysteine and histidine, are conserved in the catalytic pocket. The third residue of the catalytic triad in BLF1 is a threonine. BLF1 deamidation of eIF4A and efficient inhibition of protein translation is dependent on the catalytic cysteine as mutation of this residue to a serine renders BLF1 essentially non-functional (Cruz-Migoni et al., 2011). Vibrio parahaemolyticus type III effector VopC deamidates small GTPases. A recent addition to the CNF family of deamidases is VopC from Vibrio parahaemolyticus. V. parahaemolyticus is an enteric pathogen that contains two type III secretion systems, TTSS1 and TTSS2 (Broberg et al., 2011); TTSS2 is encoded within a pathogenicity island. The V. parahaemolyticus TTSS2 is necessary and sufficient for invasion of and replication in both HeLa cells and CaCo2 cells. Among the type III effectors encoded in the T3SS2 pathogenicity island is VopC, which shares 20% amino acid identity to the catalytic domain of CNF from E. coli. As VopC is a type III effector protein it does not share the same mechanism for host cell delivery as CNF1. As a result, VopC and CNF1 have different N-terminal domains. Both the catalytic cysteine and histidine residues of CNF1 are conserved in VopC (Cys220 and His235). Although a VopC structure has yet to be determined, homology modeling suggests that the carbonyl oxygen of Leu187 completes the papain-like catalytic triad. VopC deamidates and constitutively activates small GTPases, Rac and Cdc42, to facilitate host cell invasion (Zhang et al., 2012). VopC is not able to activate RhoA. However, as noted above, differences in substrate specificity are common among CNF homologs. VopC activates Rac by deamidating Gln61 on the critical switch II domain. V. parahaemolyticus invasion of nonphagocytic HeLa cells is dependent on catalytically active VopC (Zhang et al., 2012). 57 Pasteurella multocida toxin PMT is a multidomain toxin, with catalytic activity located at the C-terminus. Pasteurella multocida toxin, PMT, is a monomeric, 146 kDa enzyme. P. multocida causes severe diseases in animals and humans. It is associated with atrophic rhinitis in pigs and dermatonecrosis and respiratory diseases, also known as snuffles, in cattle and rabbits (Felix et al., 1992; Mushin and Schoenbaum, 1980). In humans, P. multocida can cause dermonecrosis from bite wounds and bacteremia (Garcia, 1997). PMT is a multidomain toxin. The N-terminal region of PMT has sequence homology with the N-termini of CNFs, suggesting that this region is also used for binding to the host cell surface. In fact, the first 506 N-terminal residues of PMT are sufficient to bind to cells and to compete with full-length toxin in this assay (Pullinger et al., 2001). The C-terminal region of PMT contains the catalytic center of the enzyme and is sufficient for PMT-mediated toxicity when delivered into cells by electroporation (Pullinger et al., 2001). The crystal structure of the C-terminal region of PMT reveals a multi-domain Trojan horse-like structure with the feet, head, and body consisting of different domains (C1, C2 and C3) that possess different functions (Figure 2.3A). The C1 domain, or the feet, consists of seven α-helices. The N-terminal α-helices of the C1 domain are structurally similar to the N-terminal domain of Clostridium difficile toxin B (Kamitani et al., 2010). In toxin B, these helices target the protein to the plasma membrane. Similarly, the first four αhelices of C1 are required to target the catalytic domain of PMT to the plasma membrane (Kamitani et al., 2010; Kitadokoro et al., 2007). Deletion of the C1 domain causes a reduction, but not a complete loss, of PMT function, suggesting that localization to the plasma membrane is necessary for full PMT-induced toxicity (Kamitani et al., 2010). The function of the C2 domain, the body of the Trojan horse, is currently unknown. It is the largest section of the PMT C-terminal region and consists of two subdomains. Each subdomain contains typical α/β folds. Searches using the DALI server revealed possible 58 structural homology between the second subdomain and phosphate binding enzymes (Kitadokoro et al., 2007). Therefore, the C2 domain may be enzymatic or may be involved in binding target proteins in the host cell. The C3 domain is the catalytic deamidase domain of PMT. Residues Cys1165 and His1205 (which form the thiolate-imidazolium pair) and His1223 are required for PMT function (Orth et al., 2003; Ward et al., 1998). Comparison with the structures of other members of the papain-like superfamily reveals Asp1220 of PMT is the third residue of the catalytic triad. Mutation of any of these residues resulted in a complete loss of PMT function (Kitadokoro et al., 2007). Interestingly, the function of the catalytic cysteine requires the reduction of a disulfide bond with Cys1159 (Figure 2.3B) (Kitadokoro et al., 2007). Reduction of the disulfide bond between Cys1159 and Cys1165 causes a rotation of the Cys1165 side chain towards the catalytic triad (Figure 2.3C). Therefore, the disulfide bond has to be reduced for complete deamidase activity. Additionally, a Gln1225 in the catalytic cleft was shown to be required for deamidase activity. It is postulated that this glutamine forms an oxyanion hole to stabilize the tetrahedral intermediate, similar to the role of Gln19 in papain (Kitadokoro et al., 2007). PMT deamidation of heterotrimeric G protein families affects several downstream signaling events. Unlike bacterial toxins that have high specificity for their host targets, such as the CNFs, PMT mediates its virulence effect through activation of multiple heterotrimeric Gprotein families. This leads to a plethora of changes in host signaling that lead to dysregulation of normal host cell homeostasis. A variety of cell systems and assays have been used to establish that the pleiotropic effects of PMT in target cells are due to the ability of PMT to activate multiple α subunits of diverse heterotrimeric G proteins (Hennig et al., 2008; Lacerda et al., 1996; Orth et al., 2007; Orth et al., 2008; Orth et al., 2005; Preuss et al., 2009; Staddon et al., 1990; Takasaki et al., 2004; Thomas et al., 2001; Wilson et al., 59 1997; Zywietz et al., 2001). Heterotrimeric G proteins consist of four major families; Gαs, Gαi, Gαq/11, and Gα12/13. When PMT and Gαi proteins were co-expressed in E. coli, PMT modifies Gαi via deamidation (Orth et al., 2009). PMT converts the conserved Gln205 in the switch II region of Gαi to glutamic acid. PMT-mediated activation of Gαi proteins leads to the inhibition of adenylyl cyclase and reduction of cAMP accumulation (Figure 2.4) (Orth et al., 2008). PMT activation of Gαq/11 proteins results in the stimulation of phospholipase Cβ (PLCβ) (Figure 2.4). PLCβ catalyzes the hydrolysis of phostidylinositol-4,5-bisphosphate to inositol-1,4,5-triphosphate and diacylglycerol. It was originally postulated that PMT was capable of activating Gαq (Orth et al., 2004; Zywietz et al., 2001). However, more recent in vitro expression assays coupled with a mass spectrometry output demonstrated that PMT is also able to deamidate Gα11 (Orth et al., 2012). PMT-induced Gα11 activation of PLCβ is weaker than the Gαq activation of PLCβ (Kamitani et al., 2011). Therefore, although PMT can deamidate and activate both Gαq and Gα11 in vitro, the relative contributions of these activities in vivo may differ. Regardless, activation of the Gαq/11 sub-family via PMT results in an increased release of second messengers inositol-1,4,5-triphosphate and diacylglycerol, and a consequent increase in calcium mobilization. This leads to multiple calcium-mediated responses such as activation of protein kinase C and secretion of chloride ions (Hennig et al., 2008; Staddon et al., 1990; Wilson et al., 1997). PMT also stimulates the JAK/STAT pathway in a Gαq- dependent manner (Orth et al., 2007). PMT also activates the small GTPase RhoA, resulting in reorganization of the actin cytoskeleton and formation of actin stress fibers (Lacerda et al., 1996; Thomas et al., 2001). PMT induces RhoA indirectly through activation of multiple heterotrimeric Gα protein families (Figure 2.4). PMT-induced RhoA activation can occur through Gαq (Wilson et al., 1997; Zywietz et al., 2001) and Gα12/13 (Orth et al., 2005). A novel cyclic peptide that specifically inhibits Gαq partially blocked PMT-induced RhoA activation (Orth et al., 2005; Takasaki et 60 al., 2004). Additionally, PMT-induced RhoA activation was inhibited in Gα12/13-deficient HEK293m3 cells (Orth et al., 2005). However, it was not clear whether PMT functioned through Gα12, Gα13, or both. Recently, mass spectrometric analysis revealed that PMT can deamidate both Gα12 and Gα13 (Orth et al., 2012). Finally, because Gα activation leads to the release of Gβγ from the heterotrimeric complex, PMT also stimulates increased signaling through Gβγ-specific effectors such as phosphoinositide-3-kinase (Figure 2.4) (Preuss et al., 2009). Cycle Inhibiting Factors Members of the cycle inhibiting factor (Cif) family of deamidases are found in many bacterial pathogens. Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli are causal agents of intestinal diarrhea (Croxen and Finlay, 2010; Kaper et al., 2004). EPEC is the cause of potentially fatal diarrhea in infants in developing countries. In contrast, highly infectious EHEC outbreaks occur most often in developed countries, most notably North America, Japan, and Europe. Although the diseases caused by these two E. coli pathovars differ, they both cause the formation of attaching and effacing (A/E) lesions (Croxen and Finlay, 2010). The genes responsible for attaching and effacing lesions, the type III secretion system and type III effectors, are encoded in the 35 kb pathogenicity island called the locus of enterocyte effacement (LEE) (McDaniel et al., 1995). However, several effectors are nonLEE encoded (Nle) (Dean and Kenny, 2009). Cif was the first Nle effector identified in EPEC and EHEC (Marches et al., 2003). Cif homologs are encoded in multiple bacterial pathogen genomes and are all associated with horizontally transferred genetic elements (Jubelin et al., 2009; Marches et al., 2003). Cif homologues from Burkholderia pseudomallei, Yersinia pseudotuberculosis, Photorhabdus luminescens, and Photorhabdus asymbiotica have been named CHBP, CHYP, CHPL, and CHPA, respectively. Interestingly, these proteins have low primary amino 61 acid identity compared to Cif from E. coli: 56% for CHYP, 26% for CHBP, 23% for CHPL and 26% for CHPA (Jubelin et al., 2009; Yao et al., 2009). Deamidation of NEDD8 by Cif leads to disruption of the ubiquitin-proteasome system. CifEc was shown to interact with NEDD8 in a yeast-2-hybrid system and this interaction was verified by in vitro pull-down and co-expression assays (Cui et al., 2010; Jubelin et al., 2010; Morikawa et al., 2010). Cif proteins modify NEDD8 and ubiquitin via deamidation (Cui et al., 2010). Ubiquitin and ubiquitin-like (UBL) proteins such as NEDD8 control many cellular processes by targeting various proteins for degradation through the ubiquitin-proteasome system. Ligation of ubiquitin and UBLs, such as NEDD8, to substrate proteins requires a series of three enzymes; an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Cullin-RING ligase (CRLs) are a large superfamily of E3 ubiquitin ligases (Wei et al., 2008). The core CRL complex consists of cullin proteins, which are the scaffold proteins of CRLs and are modified by NEDD8, and a small RING protein. In eukaryotic cells, cullins are continuously neddylated and deneddylated. The conjugation and removal of NEDD8 is an important cycling mechanism that regulates CRL activity. The COP9 signalosome (CSN), a conserved multiprotein complex with protease activity, is responsible for cullin deneddylation (Wei et al., 2008). When cullins are neddylated they are maintained in an active, open conformation that allows substrate proteins to interact with the Rbx protein at the cullin C-terminus (Duda et al., 2008). This flexible protein conformation and interaction leads to the ubiquitination and subsequent degradation of target proteins, some of which include important cell cycle regulators (Figure 2.5A). Cifs target residue Gln40 in NEDD8 and ubiquitin with exquisite selectivity (neither Gln39 nor Gln41 of NEDD8 is a target for deamidation) (Cui et al., 2010; Yao et al., 2012). To date, recombinant CifEc, CifBp and CifYp have all been shown to deamidate Gln40 of NEDD8 in vitro (Cui et al., 2010; Yao et al., 2012). Ectopic expression of the Gln40Glu 62 NEDD8 variant in HeLa cells phenocopies either the ectopic expression of Cifs or the effects of Cif during bacterial infection demonstrating this activity is responsible for the Cif-mediated cytopathic phenotype. The deamidation of NEDD8 by Cif results in reduction in the rate of cullin deneddylation (Boh et al., 2011; Jubelin et al., 2010; Morikawa et al., 2010). The Gln40Glu mutation in NEDD8 is sufficient to decrease the deneddylation rate by the CSN in the absence of Cif (Boh et al., 2011). Gln40 is located close to the cullin-NEDD8 conjugation site. Deamidation of Gln40 in NEDD8 may prevent CSN-mediated deneddylation of CRLs through interference with the global NEDD8-induced conformational change of cullins (which may be necessary for CSN recognition (Figure 2.5B) (Boh et al., 2011). In contrast to previous studies, it was shown in yeast that CSN can remove deamidated NEDD8 from CRLs more efficiently than wild-type NEDD8 (Toro et al., 2013). This causes an increase in deneddylated CRLs. Additionally, it is appropriate to mention that deamidation of NEDD8 and subsequent effects on CRL activity have not been reconstituted in vitro. Therefore, the exact effect of Cif-mediated deamidation of NEDD8 remains unclear. Cif causes cytopathic phenotypes such as rearrangement of the host cytoskeleton and the formation of actin stress fibers. However, the hallmark of Cif infection is the inhibition of cell cycle progression. These multiple cytopathic phenotypes can be directly attributed to disruption of the ubiquitin-protease system. Depending on the stage of the cells during infection, Cifs can arrest the cell cycle at either the G1/S transition or the G2/M transition (Marches et al., 2003; Taieb et al., 2006). G1/S and G2/M transitions require complexes that consist of a cyclin and a cyclin-dependent kinase (CDK). Inhibiting CRL activity prevents ubiquitination and degradation of cell cycle regulators such as p21 and p27, resulting in inactivation of cyclin-dependent kinases and cell cycle arrest (Marches et al., 2003; Samba-Louaka et al., 2008; Taieb et al., 2006). This triggers an increase in the inactive and phosphorylated CDKs, leading to cell cycle arrest (Marches et al., 2003). 63 Cifs also inhibit the degradation of a variety of other CRL substrates such as pIκB, Cdt1, β-catenin, and RhoA (Marches et al., 2003). Therefore, inhibition of CRL activity can account for the multiple phenotypes observed during infection with bacterial species expressing Cifs. Multiple structures reveal that Cif proteins are members of the papain-like superfamily. The crystal structures of CifEc, CHBP, CHYP, and CHPL have been solved (Figure 2.6). Although the primary amino acid similarity among the homologs is low, the structures are highly conserved (Crow et al., 2009; Hsu et al., 2008). The disordered N-termini provide type III secretion signals and are either not included in expressed proteins or are not observed in the crystal structures. To date, the only crystal structure of CifEc includes truncation of the first 100 amino acids (Hsu et al., 2008). The overall fold of Cifs comprises a head-and-tail domain organization (Figure 2.6A). The tail region, also known as the helical extension domain, is encoded in the N-terminus of the protein. The head domain, also known as the globular core, is formed by the C-terminus of the protein. The head domain consists of an anti-parallel β-sheet surrounded by α-helices. This globular domain displays overall structural homology to the papain-like superfamily. Similar to the other deamidases covered in this review, the active sites of all Cifs contain an invariant Cys-His thiolateimidazolium pair. The third residue of the catalytic triad in Cifs is a glutamine (Figure 2.6B). Each of these catalytic residues are required for Cif-induced cytopathic effects, deamidation of NEDD8 and ubiquitin, and the resulting increase in stability of cell cycle regulators, p21 and p27 (Cui et al., 2010; Hsu et al., 2008; Jubelin et al., 2009; Yao et al., 2009). An interesting feature observed in the structures of Cifs is the so-called occluding loop. This loop was first identified in CifEc and is conserved across the Cif family (Crow et al., 2009; Hsu et al., 2008). The occluding loop partially blocks access to the catalytic site (substrate binding cleft of papain) and was hypothesized to be important in defining specificity for Cif substrates (Figure 2.6B; see below). 64 Cif deamidases were the first to be co-crystallized with their substrates. The crystal structures of CHYP, CHPL, and CHBP have been determined in complex with NEDD8 (Crow et al., 2012; Yao et al., 2012). Additionally, the co-crystal structure of CHBP and ubiquitin has also been determined (Yao et al., 2012). The structure of these complexes reveals an extensive binding interface between Cif proteins and their ubiquitin/NEDD8 substrates (Figure 2.7A), which involves both the head and the tail domains of Cifs. Multiple alanine mutations in CHBP in the regions that interface with either the N-terminal β-hairpin or the residues adjacent to Gln40 in ubiquitin or NEDD8 were sufficient to abolish deamidation and Cif-induced cell cycle arrest (Yao et al., 2012). Furthermore, alanine mutations in the corresponding residues in ubiquitin and NEDD8 disrupted both the interaction and deamidation (Yao et al., 2012). Interestingly, all individual alanine mutations in CHYP (that were tested) were not sufficient to disrupt NEDD8 complex formation. Only mutations that introduced steric clashes (based on the complex structure) prevented binding (Yao et al., 2012). There is very little change in the structure of Cifs when bound to their substrates, compared to the uncomplexed states (Crow et al., 2012; Crow et al., 2009; Yao et al., 2012). However, a significant re-orientation of the flexible C-terminal tail of NEDD8 and ubiquitin is observed on binding by Cifs. Interestingly, it is the occluding loop in Cifs that appears responsible for forcing this re-orientation during substrate binding. Displacement of the flexible C-terminal tail is likely important for substrate recognition and orienting Gln40 in the Cif catalytic pocket (Crow et al., 2012; Yao et al., 2012). Consistent with this, deletion of the C-terminal domain of either ubiquitin or NEDD8 diminished deamidation by Cifs (Crow et al., 2009). Therefore, multiple areas within the extensive interface between Cif deamidases and their substrates are important for activity. CHBP has a preference for NEDD8 as a substrate (Cui et al., 2010), and CHYP deamidation of NEDD8 is ten-fold more efficient than deamidation of ubiquitin (Crow et al., 65 2012). Molecular-dynamics studies revealed that this efficiency difference may be due to decreased flexibility of NEDD8 in complex with Cif, compared to ubiquitin (Yao et al., 2012). Residue Glu31 of NEDD8 interacts electrostatically with several residues in CHBP. The residue at the same position in ubiquitin is glutamine, which is not capable of electrostatic interactions. To determine whether this residue contributes to the difference in movement and deamidation efficiency, NEDD8 Glu31Gln and the ubiquitin Gln31Glu variants were generated. NEDD8 Glu31Gln exhibited increased molecular dynamic motion in simulations and deamidation efficiency in in vitro assays was comparable to that observed for wild-type ubiquitin. Conversely, ubiquitin Gln31Glu exhibited decreased molecular-dynamic motion, bound more tightly to CHBP, and was deamidated at a level similar to wild-type NEDD8 (Yao et al., 2012). Thus, the Gln31 substrate residue may determine the difference in substrate deamidation efficiency by Cifs. The data from the Cif/NEDD8 complexed structures also suggests a molecular mechanism of enzymatic deamidation by Cifs (Figure 2.7B,C). All solved structures of currently known enzymatic deamidases contain conserved papain-like catalytic residues and α/β folds similar to those in the papain-like superfamily. As a result, the molecular mechanism of deamidation is likely to be similar to the protease and transglutaminase reactions catalyzed by other superfamily members. During these reactions, the catalytic histidine residue deprotonates the cysteine sulfhydryl group to increase its ability to act as a nucleophile. Then, this nucleophile attacks the amide substrate, resulting in an acyl-enzyme intermediate that is resolved by hydrolysis. The catalytic histidine’s imidazole ring is often coordinated by a hydrogen bond with a third residue and this is ensures optimal orientation of the thiolate-imidazolium pair (Figure 2.7B,C) (Crow et al., 2012). 66 OspI inhibits host immune responses by deamidating an E2-conjugating enzyme. Deamidation of Ubc13 by OspI inhibits host inflammatory responses. Shigella species are routinely found in patients in developing countries that suffer from severe diarrhea (Schroeder and Hilbi, 2008). The severe symptoms associated with Shigella infections are due to the invasion and destruction of the colonic and rectal epithelium and a severe inflammatory reaction (Schroeder and Hilbi, 2008). S. flexneri infections trigger changes to the host cell membrane that initiate the DAG (diacylglycerol)-CBM complex-NF-κB signaling pathway. S. flexneri invades the host cell via macropinocytosis. It is then able to escape from the phagosome and into the cytoplasm through an unknown mechanism (Dupont et al., 2009). The escape leads to the accumulation of host membrane components near the site of infection and subsequent host signaling events that lead to inflammation. This likely occurs through the activation of phospholipases that hydrolyze plasma membrane phospholipids, such as DAG, which then activate protein kinases (Rawlings et al., 2006). Subsequently, protein kinases activate the complex of caspase-recruitment domain (CARD), B-cell lymphoma 10 (BCL-10), and mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1(MALT1). This complex is also referred to as the CBM complex. The CBM complex interacts with and activates tumor-necrosis factor receptor-associated factor 6 (TRAF6). TRAF6 is an E3 ubiquitin ligase that interacts with the Ubc13, an E2 ubiquitin-conjugating enzyme, to form lysine 63 (Lys63)linked polyubiquitin chains (Takeuchi and Akira, 2010). The presence of unconjugated Lys63 polyubiquitin chains activates the TGF-β-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and TAK1-binding protein 2 (TAB2) complex. This leads to IκBα phosphorylation and degradation. This allows NF-κB to translocate into the nucleus, where it activates the expression of proinflammatory cytokine genes (Takeuchi and Akira, 2010). S. flexneri encodes a type III effector, OspI, which modulates this host response by deamidating Ubc13. Incubation of OspI with TRAF6, ubiquitin conjugating E2 enzymes 67 (UBC13 and UEV1A), and ubiquitin revealed that OspI causes a shift in the mobility of Ubc13 in SDS-PAGE gels. Mass spectrometry analysis revealed that Ubc13 is deamidated at residue Gln100 in the presence of OspI (Sanada et al., 2012). The deamidation of Ubc13 Gln100 blocks its ubiquitin-conjugating activity, thereby inhibiting the TRAF6-DAG-CBM complex and subsequent NF-κB signaling. This is made evident by the increase in phosphorylation of the NF-κB inhibitor, IκBα, and consequent NF-κB translocation to the nucleus in HeLa cells infected with S. flexneri ∆ospI (Sanada et al., 2012). Consistent with this result, NF-κB translocation to the nucleus is inhibited in the presence of OspI (Sanada et al., 2012). OspI forms a papain-like catalytic pocket that rearranges upon binding Ubc13. The crystal structure of OspI reveals an α/β fold with 4 β-strands, seven α-helices, and one 310 helix (Figure 2.8A) (Sanada et al., 2012). OspI also shares structural homology with the papain-like superfamily, as noted above for PMT and the Cif family of deamidases. It is most closely related to the Pseudomonas syringae cysteine protease effector AvrPphB (Shao et al., 2002; Zhu et al., 2004). Superimposition of the catalytic domain of AvrPphB with the OspI crystal structure revealed that the catalytic triad in OspI comprises Cys62, His145, and Asp160 (Sanada et al., 2012). OspI inhibition of the DAG-CBM-NF-κB pathway is dependent on the catalytic residues as mutation of any of these residues leads to the loss of OspI activity (Sanada et al., 2012). Recently, the crystal structure of the OspI(Cys62Ala)/Ubc13 complex has been solved (Figure 2.8B,C) (Fu et al., 2013). This structure revealed that an extensive binding surface between OspI and Ubc13 that covers 11% of the exposed surface of OspI (Fu et al., 2013). A negatively-charged region and hydrophobic pocket of OspI bind α-helix 1 and the L1 and L2 loops of Ubc13, respectively. Analysis of point mutations in these regions revealed that both interfaces are required for deamidation of Ubc13 (Fu et al., 2013). In 68 contrast to Cifs, significant structural rearrangements occur in the catalytic pocket of OspI upon substrate binding (Fu et al., 2013). In uncomplexed OspI, the catalytic pocket is blocked by Asn61 (Figure 2.8A). When OspI binds Ubc13, Asn61 rotates approximately 180 degrees. This leads to formation of a hydrogen bond with Asn54 and repositioning of Ala62. The movement of these residues opens the pocket allowing insertion of Gln100 from Ubc13 into the catalytic site. Superposition of the catalytic pockets of AvrPphB and Ubc13-bound OspI reveals an overlap of OspI Ala62 (Cys62 in the wild-type protein) and the catalytic cysteine of AvrPphB (Fu et al., 2013). This suggests that the remodeled OspI catalytic pocket (when bound to Ubc13) contains the proper orientation for catalytic activity. Additionally, Phe95 and His96 of OspI re-orient to form a hydrophobic pocket that interacts with Ubc13 loops L1 and L2 (Fu et al., 2013). The OspI/Ubc13 complexed structure confirms that the molecular mechanism of OspI is similar to that of papain-like enzymes and that suggested for Cif/NEDD8 deamidation (Figure 2.7C) (Fu et al., 2013; Storer and Menard, 1994; Zhu et al., 2004). It is interesting to note that the catalytic cysteine is capable of forming disulfide bonds with Cys65 (Figure 2.8C) (Sanada et al., 2012). Although, it has not been demonstrated that this disulfide bond affects the function and positioning of the catalytic cysteine, it is possible that it maintains the deamidase in a non-catalytic state in non-reducing conditions, as has been demonstrated for PMT (Figure 2.3B,C) (Kitadokoro et al., 2007). Conclusions Recently, it has been discovered that deamidation is a common mode of action used by several bacterial virulence factors to modify the function of host proteins. Deamidation, as an irreversible modification of host proteins, offers many advantages to invading pathogens. Unlike phosphorylation, host systems lack the ability to reverse the effects of deamidation. This makes deamidases particularly potent virulence factors. Characterized deamidases have been shown to modify specific residues on host proteins that are key to the host 69 protein’s function. Additionally, there is high specificity in the proteins that are targeted by deamidases, as can be seen in the ability of CNF1 to deamidate some by not all Rho GTPases. Although the deamidation modification is subtle and specific, the downstream impacts on host cell function are extensive. This is most evident in the case of cycle inhibiting factors, which inhibit the ubiquitin-protease system leading to both cell cycle arrest and cytoskeletal rearrangements. In fact, it is apparent that bacterial deamidase virulence factors have evolved to target key modulators of critical cell signaling systems in hosts. However, our understanding of the role of deamidases in bacterial virulence is still in its infancy. Further elucidation of the molecular mechanisms and the interactions between deamidases and their substrates is important. To date, the use of enzymatic deamidation as a generic “house-keeping” mechanism for modifying protein function has only been documented in prokaryotes. Do eukaryotes also use this activity to modulate protein activity? Or is enzymatic deamidation in eukaryotic cells an innovation of bacterial pathogen mediated via translocated proteins? Future work would be greatly aided by new methods for easier identification of deamidation. Current methods require knowledge of the specific substrate of each deamidase in order for activity to be assayed. This differs from enzymes such as cysteine proteases, for which cleavage of generic substrates can be easily measured. Additionally, the presence of nonspecific deamidation in protein samples can make the identification of enzymatic deamidation difficult. Another important future direction is to define the molecular mechanisms by which deamidation specifically impacts the function of host cell targets. At present, our knowledge is restricted to observing the phenotypic outcomes of deamidation on particular targets during diverse steps in the lifestyles of diverse pathogens. It would be valuable to understand how deamidation alters function at the level of the modified protein. For example, G proteins are commonly targets of at least some of the deamidases discussed here. But is the phenotypic outcome of these events always merely an irreversible loss of catalytic function or could the deamidated G 70 protein now act as a dominant negative on its normal cellular partners, amplifying the impact of the deamidation event to multiple output pathways, as noted above for PMT. Such understanding will be important, for example, in exploration of genome editing to target glutamine to glutamate mutations. Regardless of the challenges of these studies, it is clear that tackling the molecular mechanisms of deamidation, as has been done for other posttranslational modifications used by bacterial pathogens, is essential for our understanding of bacterial pathogenesis and, in fact, for the normal host cell biology of their targets. 71 Figures Figure 2.1. A schematic representation of enzymatic deamidation in proteins. Deamidases act on specific residues in the target protein. For all currently studied bacterial virulence factors, the targets of deamidation are glutamine side chains, which are converted to glutamic acids. However, it is also possible for the amide of the side chains of asparagines to be converted to aspartic acid. Deamidation results in an increase in the negative charge of the target protein, an increase of approximately 1 Dalton in the mass of the target protein, and the release of ammonia. Each of these outputs can be measured experimentally to characterize the activity of deamidases. 72 Figure 2.2. The catalytic domains of diverse deamidases. A) Catalytic domain of CNF1 from E. coli (PDB 1HQ0) is shown with invariant catalytic residues Cys866 and His881 shown in purple. The main chain carbonyl of Val833 (also in purple) is involved in coordinating the imidazole ring of His881. In pink are conserved residues important for CNF1 function, whilst loops 8 and 9 (likely important for substrate recognition) are shown in yellow. B) BLF1 (PDB 3TU8) is a 23 kDa toxin and deamidase that forms a compact catalytic domain structurally related to CNF1 from E. coli and other papain-like superfamily enzymes. The residues that comprise the catalytic triad (Cys94, His106, and Thr88) are shown in purple. α-helices are in blue, β-sheets in green, and loops in grey. All structure figures were prepared with PyMol. 73 Figure 2.3. Crystal structure of the C-terminal region of Pasteurella multocida toxin. A) The crystal structure of the C-terminal region of PMT has been solved (residues 5751285, PDB 2EBF). The catalytic region consists of several domains. The helical C1 domain (purple), are the feet of the Trojan horse, and contain membrane-targeting properties. The C2 “body” domain contains 2 α/β subdomains (shown in green and yellow) of unknown function. The domain responsible for the deamidase activity of PMT is the C3 “head” Cterminal domain (blue). B) The crystal structure of PMT catalytic domain reveals a disulfide bond between the catalytic cysteine, Cys1165 and Cys1159 in the binding pocket. Reduction of this disulfide bond is required for the interaction of the catalytic triad and catalytic activity. His1205 and Asp1220 form the other residues of the triad. 74 Figure 2.4. PMT deamidates and activates heterotrimeric Gα proteins. The activation of diverse G protein-couple receptor signal transduction pathways by PMT leads to several phenotypes that are disruptive to the host. PMT activates Gαi, Gαq/11, and Gα12/13, but not Gαs. PMT also triggers the release of Gβγ from the heterotrimeric complex, which leads to activation of the phosphoinositide-3-kinase (PI3K) pathway. 75 Figure 2.5. Model of Cif inactivation of CRL activity and ubiquitination. A) In uninfected cells, the COP9 signalosome (CSN) deneddylates CRLs. NEDD8 (yellow) conjugation and removal represent an important mechanism by with CRL activity is regulated. When CRLs are neddylated with wild-type NEDD8 (green), they are maintained in an open conformation. This allows substrates (blue) to bind the CRLs, become polyubiquitinated, and then degraded. B) In cells infected with bacteria that express Cif, deamidation of NEDD8 (yellow) may prevent CRL (green) from forming an open conformation. This prevents CRLs from associating with the substrate-binding module that allows downstream ubiquitination and degradation of substrate proteins (blue) to occur. This may lead to a reduction in the rate of deneddylation and a decrease in unmodified CRLs (grey). 76 Figure 2.6. Crystal structures of cell cycle-inhibiting factors. A) Crystal structures of Cif deamidases from E. coli (PDB 3EFY), B. pseudomallei (PDB 3GQM), P. luminescens (PDB 3GQJ), and Y. pseudotuberculosis (PDB 4F8C) are shown. In blue are the α-helices of the core globular domain. In teal are the α-helices of the tail helical extension domain. The antiparallel β-sheet is green and the loops are colored in grey, except for the occluding loop, which is colored in black. The conserved catalytic residues histidine, cysteine, and glutamine are in purple. B) Amino acids in the catalytic domain of CHBP are shown as sticks in purple. 77 Figure 2.7. Crystal structure of the CifYp deamidase/NEDD8 complex. A) The CHYP (αhelices in blue, β-sheets in green, and loops in grey) and NEDD8 (red) complex consists of an extensive interaction surface (PDB 4FBJ). The catalytic Cys117 position is modeled based on the CHYP(Cys117Ala)/NEDD8 complex to demonstrate the proposed molecular mechanism of deamidation. B) An up-close view of the catalytic domain of CHYP shows the deamidation target, Gln40 of NEDD8, is positioned near the Cif catalytic residues (shown in purple). C) The proposed molecular mechanism of deamidation involves deprotonation of the cysteine by the imidazolium group of histidine (step 1) followed by a nucleophilic attack by the thiol group of the catalytic cysteine on the δ-carbon of glutamine (step 2). 78 Figure 2.8. Crystal structures of unbound OspI and the OspI/Ubc13 complex. A) OspI (α-helices in blue, β-sheets in green, and loops in grey (PDB 3B21) forms a compact catalytic domain structurally similar to papain-like superfamily enzymes. The residues in the catalytic triad (Cys62, His145, and Asp160) are shown in purple. The catalytic pocket is blocked by Asn61 in free OspI. B) The OspI(Cys62Ala)/Ubc13 (red) complex includes an extensive interaction surface involving Ubc13 α-helix 1 and loops L1 and L2 (PDB 4IP3). C) An up-close view of the OspI(Cys62Ala)/Ubc13 complex showing Gln100 of Ubc13 positioned adjacent to the OspI catalytic center (purple, OspI residue 62 has been modeled as a cysteine to demonstrate the nucleophilic attack (yellow dashes) by the thiol group of Cys62 on Gln100 in Ubc13). 79 Table 2.1. Bacterial virulence factors that use deamidation to modify host proteins. Protein Species Target Effects Key References Cytotoxic Necrotizing Factors (CNFs) E. coli, Yersinia pseudotuberculos is Rho GTPases (RhoA, Rac, Cdc42) Activation of Rho GTPases Flatau et al., (1997); Schmidt et al., (1997) Burkholderia Lethal Factor (BLF1) Burkholderia pseudomallei VopC PMT Cycle inhibiting factors (Cifs) OspI Vibrio parahaemolyticus Pasteurella multocida E. coli, Burkholderia pseudomallei, Yersinia pseudotuberculos is, Photorhabdus luminescens, Photorhabdus asymbiotica Shigella flexneri eIF4A Rho GTPases (Rac, Cdc42) Heterotrimeric G proteins (Gαi, Gαq/11, and Gα12/13) NEDD8 and ubiquitin UBC13 80 Inactivation of helicase activity of translation factor Activation of Rho GTPases Activation of G protein signaling Cruz-Migoni et al., (2011) Zhang et al., (2012) Orth et al., (2009) Inactivation ubiquitinproteasome system Cui et al., (2010) Inactivation of E2 ubiquitin conjugating activity of UBC13 Sanada et al., (2012) REFERENCES Boh, B.K., Ng, M.Y., Leck, Y.C., Shaw, B., Long, J., Sun, G.W., Gan, Y.H., Searle, M.S., Layfield, R., and Hagen, T. 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Shahid Mukhtar performed the yeast two-hybrid screens and pairwise tests in which the initial interaction between HopAF1 and MTN1 was identified. I generated data for the rest of the figures, with the help of many resources, including the UNC Biology Imaging Core Facility, the UNC Macromolecular Interactions Facility, and the UNC Center for Structural Biology, along with input from members of the Dangl-Grant lab. In addition, I wrote the manuscript under the direction of my advisor, Jeff Dangl. I generated tools during the completion of this chapter that have contributed to other lab projects. I generated two Gateway-compatible vectors for estradiol-inducible expression of proteins tagged C-terminally with either citrine-HA or cerulean-HA. The former vector was published by Akimoto-Tomiyama et al., (Akimoto-Tomiyama et al., 2012) and I was included as 4th author on this paper for my contribution. pMDC7-cerulean-HA was also used in a publication (Roberts et al., 2013). Finally, a plasma membrane localization marker that I generated for confocal microscopy experiments, PLC2-YFP, contributed to work published in Gao et al (Gao et al., 2011). Abstract Many plant pathogens, including Pseudomonas syringae, encode the type III secretion system for translocating effector proteins into the host during infection. Strains of P. syringae which are not capable of delivering the type III effectors are nonpathogenic. Therefore, the functions of type III effectors are essential for disease. Although several type III effectors have been demonstrated to block components of the plant defense response, the functions of most type III effectors during disease manifestation are largely unknown. Our lab is interested in the type III effector HopAF1, a type III effector that is present in eleven of the nineteen sequenced strains of P. syringae and other plant pathogens. Although the presence of HopAF1 in multiple strains of P. syringae suggests that it plays an important role in virulence, no function has yet been associated with HopAF1. We began to determine the function of HopAF1 with structure/function analyses. Using the BioInfoBank Meta Server and HHPred structural prediction programs and homology searches, we generated a tertiary structure model for HopAF1. These predictions suggest with high confidence that HopAF1 is structurally related to bacterial deamidase proteins. Deamidation, the irreversible substitution of an amide group with a carboxylate group, is the mechanism by which several bacterial virulence factors manipulate the activity of a specific substrate. To identify a potential target of the HopAF1 putative deamidation activity, we employed a stringent yeast two-hybrid screen and identified Arabidopsis methylthioadenosine nucleosidase (MTN1) as a putative target of HopAF1. This interaction, which we extended to include Arabidopsis MTN2, was confirmed in planta using coimmunoprecipitations and bimolecular fluorescence complementation assays. MTNs are enzymes in the methionine recycling cycle, which is a cycle essential for high levels of ethylene biosynthesis. Ethylene is a key plant hormone for plant developmental processes, such as senescence and flowering. Ethylene is also induced during PTI. Therefore, we hypothesized that HopAF1 inhibits PTI by manipulating MTNs and levels of ethylene in plants. To this end, we used gas 91 chromatography to measure ethylene biosynthesis in plants treated with bacteriallydelivered HopAF1. We determined that HopAF1 inhibits ethylene biosynthesis in a manner dependent on putative catalytic residues. Additionally, Yang cycle mutants mtn1 mtn2 and mtk are more susceptible to weak pathogens. This data is consistent with the idea that HopAF1 targets a novel component of the plant immune system, the Yang cycle. Additional work on this project includes determining the enzymatic activity by which HopAF1 inhibits MTN1 and MTN2. Introduction P. syringae is a Gram negative bacterium that causes disease in agronomically important crops such as tomatoes, tobacco, and beans and the model plant, Arabidopsis thaliana. P. syringae uses the type III secretion system (T3SS) to inject type III effector (T3E) proteins into the host to cause disease in plants. P. syringae mutants lacking the type III secretion system lack the ability to cause disease in hosts, meaning that the type III secretion system is its main virulence determinant (Lindgren et al., 1986). The immune response that plants use to protect themselves against pathogens consists of multiple stages. The first line of defense begins with transmembrane pattern recognition receptors (PRRs) perceiving conserved molecular patterns from pathogens called pathogen-associated molecular patterns (PAMPs). Because these microbial signatures are not limited to pathogenic microbes, they are often referred to as microbialassociated molecular patterns or MAMPs. For the purposes of this chapter, we will use the terms PAMPs. PAMPs are often highly conserved across a wide range of microbes, fulfill a function essential to the pathogen’s lifecycle, and yet are not present in the host (Boller and Felix, 2009). Common bacterial PAMPs are lipopolysaccharide, peptidoglycan, elongation factor Tu (EF-Tu), and flagellin (Boller and Felix, 2009). A 22 amino acid peptide derived from a conserved region of the amino terminus of bacterial flagellin (flg22) is sufficient for recognition by host PRR, FLS2 (Felix et al., 1999; Gomez-Gomez and Boller, 2000). This 92 leads to a downstream signaling cascade that includes many components such as an increase in callose deposition, up-regulation of defense gene expression and an increase in ethylene biosynthesis. The second line of the plant immune system is activated by the recognition of pathogen effectors by plant disease resistance gene products. Plants use disease resistance (R) proteins, which often referred to as NB-LRR, or NLR, proteins due to their domain architecture to detect effectors from a wide variety of plant pathogens. The recognition of the presence of the effector, or more commonly the effect of an effector on a specific host protein, that leads to effector-triggered immunity or ETI. An enhanced and accelerated cell death response called the hypersensitivity response (HR) is an easily visible phenotype often associated with ETI (Jones and Dangl, 2006). Plant hormones are also key components of the plant immune response. There is significant cross-talk between the three main stress hormones in plants, salicylic acid (SA), jasmonic acid (JA), and ethylene that allow the plant to regulate its response to various classes of pathogen and herbivores. As such, recently it has become clear that the manipulation of plant hormone homeostasis is an important target of T3Es. For example, he P. syringae T3E HopI1 suppresses SA accumulation (Jelenska et al., 2007). Additionally, AvrPtoB stimulates ABA biosynthesis and ABA responses that antagonize SA biosynthesis and SA-mediated defenses (de Torres-Zabala et al., 2007). AvrRpt2 alters the auxin physiology to promote host susceptibility (Chen et al., 2007). XopD, a Xanthomonas campestris pv. vesicatoria T3E, was recently found to target a tomato ethylene response factor (ERF) called SIERF4. SIERF4 mutants display reduced ethylene levels and an increase in susceptibility to Xcv (Kim et al., 2013), supporting the hypothesis that ethylene is involved in plant defense. Ethylene is a key component of the PTI response. Ethylene accumulation upregulates the expression of pathogenesis-related (PR) proteins and increases the 93 accumulation of hydroxyproline-rich, cell-wall strengthening proteins (Ecker and Davis, 1987; Penninckx et al., 1998). Ethylene also increases SA-induced expression of PR-1 (Lawton et al., 1994). Furthermore, ethylene directly up-regulates the expression of FLS2. FLS2 transcription is directly controlled by binding of EIN3/EIL1/EIL2 to its promoter and in ethylene insensitive mutants (ein2) seedlings, FLS2 expression, and FLS2 protein accumulation are suppressed. As a result, ein2 plants are more susceptible to weakened strains of P. syringae (Boutrot et al., 2010; Mersmann et al., 2010). HopAF1 was identified as a T3E in P. syringae pv. tomato DC3000 (Pto DC3000) and P. syringae pv. phaseolicola 1448A race 6 via its HrpL-dependent transcriptional induction, a loosely defined N-terminal translocation signal sequence, and the ability to be translocated into plant cells (Chang et al., 2005). HopAF1 is confirmed to be present in 11 of 19 strains of P. syringae (Baltrus et al., 2011). There are also HopAF1 orthologs in other plant pathogens, such asAvrXv3 from Xanthomonas campestris pv. vesicatoria (AstuaMonge et al., 2000), Ralstonia solanacearum, Pseudomonas savastoni, and Aave_1373 from Acidovorax citrulli (Studholme et al., 2010). The presence of HopAF1 in multiple strains of P. syringae and other plant pathogens suggests an important role in virulence. The goal of this study was to determine the role of HopAF1 in P. syringae infections. We found that the C-terminal domain of HopAF1 is predicted to be structural homologous to bacterial deamidases, members of the papain-like superfamily. The C-terminus of HopAF1 also contains conserved putative catalytic residues in the predicted catalytic fold. HopAF1 inhibits PTI responses in a manner dependent on these putative catalytic residues. HopAF1 inhibits high levels of ethylene biosynthesis, also in a manner dependent on its putative catalytic activity. HopAF1 interacts with Arabidopsis methylthioadenosine nucleosidases (MTN) at the plant plasma membrane. These Yang cycle proteins are required for recycling methionine to supplement de novo methionine biosynthesis. We demonstrated that the Yang cycle is required during high levels of ethylene biosynthesis during pathogen attack. In 94 sum we demonstrated that HopAF1 is a key virulence factor of P. syringae and also demonstrated the role of the Yang cycle and ethylene in the PTI. Results HopAF1 is a highly conserved type III effector with a putative C-terminal catalytic domain. To determine if the structure of HopAF1 might suggest an enzymatic activity, we used the BioInfoBank metaserver and HHPred structural prediction programs to generate a structure prediction for HopAF1 from P. syringae pv. tomato DC3000 (Bujnicki et al., 2001; Soding et al., 2005). The predicted structure of the full length HopAF1 protein did not have homology to any known protein structure. However, when the C-terminus of HopAF1 (residues 130-284) was queried, the homology searches predicted with high confidence that the HopAF1 C-terminus is structurally related to CheD, a single-domain chemotaxis protein encoded by Thermotoga maritima (Figure 3.1A) (Chao et al., 2006). We validated our HopAF1 model using Verify3D (Eisenberg et al., 1997). We also generated structural models for all HopAF1 family members individually and found that each, except for the Acidovorax allele which has a C-terminal truncation, contains a predicted secondary structure with homology to CheD (Figure 3.1B). We specifically focused on the divergent allele AvrXv3 from Xanthomonas campestris, which triggers HR in tomato and pepper plants (Astua-Monge et al., 2000). Although HopAF1 and AvrXv3 are only 27% identical, structural modeling of AvrXv3 also predicts that this protein has structural homology to CheD (Figure 3.1C). CheD is a deamidase that activates methyl-accepting chemotaxis proteins (MCPs) (Chao et al., 2006). CheD is structurally related to the Cytotoxic Necrotizing Factor (CNF) family of bacterial deamidases (Flatau et al., 1997; Schmidt et al., 1997). Deamidation is the irreversible mutation of asparagine and glutamine residues to aspartic acid and glutamic acid, respectively. Deamidation is a posttranslational modification utilized by multiple bacterial virulence factors to modify the function of eukaryotic host target proteins 95 (Washington et al., 2013). The catalytic domains of all known bacterial deamidases contain homology to papain-like structures (Washington et al., 2013). Therefore, we hypothesized that the HopAF1 orthologs possess enzymatic activities associated with the papain-like superfamily of enzymes, potentially deamidation. Bacterial deamidases require a histidine and cysteine residue in their catalytic pocket for activity. We demonstrated that the C-termini of both HopAF1 and AvrXv3 are predicted to form a catalytic pocket in which the invariant catalytic residues for deamidation, cysteine and histidine, are present (Figure 3.1A,B). Interestingly, these residues are conserved among all known HopAF1 orthologs, including the divergent family members such as AvrXv3 (Figure 3.2). Therefore, we predicted that these residues are likely catalytic and critical for the enzymatic function of HopAF1. The role of the third catalytic residue of deamidases is to coordinate the imidazole ring of histidine to ensure optimal orientation of the thiolate-imidazolium pair; therefore these residues are required for function. The third residue of the CheD catalytic triad is T27 (Figure 3.1A). There is no threonine at this position in HopAF1 family members. However, adjacent to the position of T27, there is a conserved aspartic acid, D154, in HopAF1 (Figure 3.1A). We hypothesize that this residue may be involved in catalytic activity. Unfortunately, structural modeling predicts this residue is present on a flexible loop and therefore, we are not confident about the exact position of D154. Since the cysteine and histidine are invariant, we did not mutate the predicted third catalytic residue of HopAF1. We simply focused on the invariant putative catalytic residues, cysteine and histidine, in further analyses of HopAF1 function. HopAF1 blocks flg22-induced PTI and this requires its putative catalytic residues. Many T3Es block PAMP-triggered immunity (PTI) and thereby contribute to bacterial virulence (Block and Alfano, 2011). To determine if HopAF1 plays a role in virulence, we obtained a Pto DC3000 derivative in which the HopAF1 gene has been disrupted (Jamir et al., 2004). We did not observe a significant growth defect of Pto DC3000 in the absence of 96 HopAF1 (data not shown), therefore we decided to see if HopAF1 could enhance the growth of a weak pathogen. To this end, we generated wild-type HopAF1 transgenic lines in the wild-type Arabidopsis Col-0 background. We also generated the putative catalytic dead version of the HopAF1 transgenic lines, by mutating both the C159 and H186 to alanine (HopAF1C159AH186A). Conditional expression of HopAF1 wild-type and HopAF1C159AH186A was evident by 6 hours after estradiol induction and was maintained for 48 hours. The removal of 28 T3Es by sequential mutagenesis from Pto DC3000 transformed this virulent strain into a weakly pathogenic derivative called Pto DC3000D28E (Cunnac et al., 2011). To determine if HopAF1 inhibits PTI, we measured the growth of Pto DC3000D28E in our transgenic lines 24 hours after induction of HopAF1 expression by estradiol treatment. We hand inoculated Pto DC3000D28E into Col-0 and the two independent HopAF1 transgenic lines for both wild-type HopAF1 and HopAF1C159AH186A. We observed enhanced growth of Pto DC3000D28E on HopAF1-expressing transgenic lines compared to Col-0, demonstrating that HopAF1 expression allowed for the enhanced growth of a weak pathogen when overexpressed in planta (Figure 3.3A). The increase in bacterial growth in the transgenic lines correlates with an increase in disease symptoms (Figure 3.3B). Importantly, the growth of Pto DC3000D28E on transgenic lines expressing the putative catalytic dead HopAF1 was similar to that on Col-0, indicating that the enhanced growth of a weak pathogen on HopAF1 transgenic lines was dependent on the putative catalytic residues of HopAF1 (Figure 3.3A,B). The expression of HopAF1 in these lines was confirmed by western blots (Figure 3.3C). The pretreatment of Arabidopsis plants with flg22 induces PTI and diminishes susceptibility to subsequent infection with virulent pathogens, such as Pto DC3000 (Zipfel et al., 2004). To further test our hypothesis that HopAF1 inhibits PTI, we examined flg22induced disease resistance in Col-0 and the HopAF1 transgenic lines. After flg22 treatment, we observed reduced growth of Pto DC3000 on Col-0 compared to untreated plants (Figure 97 3.4A). This effect was suppressed in transgenic Col-0 plants expressing HopAF1, but not in transgenic Col-0 plants expressing the HopAF1 putative catalytic dead protein (Figure 3.4A). Because Pto DC3000 is a highly virulent pathogen, HopAF1 transgenic plants are not more susceptible to Pto DC3000 than Col-0. Expression of HopAF1 in the transgenic lines in these experiments was also confirmed (Figure 3.4B). We conclude that HopAF1 suppresses flg22-induced PTI in a manner dependent on its putative catalytic residues. HR triggered by AvrXv3 in tomato is also dependent on its putative catalytic residues. AvrXv3 is recognized by, and elicits an HR-response in, tomato cultivar Florida 216 (Fla 216) but not Florida 7060 (Fla 7060) (Astua-Monge et al., 2000). In order to determine if the putative catalytic residues of AvrXv3 are required for recognition and HR, we generated clones that allowed the constitutive overexpression of wild-type AvrXv3 and the AvrXv3 putative catalytic residue mutant, AvrXv3H126A, with a C-terminal myc tag. We transiently expressed AvrXv3 wild type and mutant in both tomato cultivars via Agrobacterium mediated T-DNA transfer, we measured their expression by western blot, and scored for HR. Wildtype AvrXv3, but not AvrXv3H126A, triggered HR on Fla 216 (Figure 3.5A). As expected, we did not observe HR on Fla 7060 (data not shown). All clones were expressed (Figure 3.5B). These data suggest that the recognition of AvrXv3 in tomato requires its putative catalytic residues. HopAF1 is targeted to the plasma membrane via acylation. The N-termini of multiple HopAF1 orthologs contain putative sites for myristoylation and palmitoylation (Figure 3.2). Myristoylation is a eukaryotic-specific posttranslational modification that is used often in combination with palymitoylation to target proteins stably in the plasma membrane lipid bilayer (Resh, 2006). Multiple P. syringae type III effectors utilize these modes of acylation to localize to the plasma membrane once injected into the plant cell (Dowen et al., 2009; Lewis et al., 2008; Nimchuk et al., 2000; Robert-Seilaniantz et al., 2006; Shan et al., 2000). 98 We predicted that HopAF1 G2 and C4 are myristoylated and palmitoylated, respectively, to target HopAF1 to the plasma membrane. To test this hypothesis, we generated an acylation minus HopAF1 variant, HopAF1G2AC4S. For the HopAF1 orthologs that lack the putative acylation sequence, such as AvrXv3, we predicted that they will localize to the cytoplasm (Figure 3.2). We used confocal microscopy to detect the localization of transiently expressed estradiol-inducible HopAF1 and AvrXv3 in N. benthamiana. Both proteins were tagged on the C-terminus with a cerulean-HA tag. To avoid aberrant protein localization, we determined the lowest amount of Agrobacterium and estradiol necessary to detect HopAF1 via western blot (Figure 3.6A). We also confirmed that we were assaying the localization of HopAF1 and not free, soluble cerulean (Figure 3.6B). We used a plasma membrane marker, PLC2-YFP, as a plasma membrane localization control (Otterhag et al., 2001). HopAF1 is localized at the plasma membrane and colocalizes with PLC2 (Figure 3.7A). However, the acylation minus variant was localized in the plant cell cytoplasm, where it co-localized with free YFP, a marker for nucleocytoplasmiclocalized proteins (Figure 3.8A,B,C). Similar to the acylation minus HopAF1 variant, AvrXv3 is found in the cytoplasm (Figure 3.7A). Both of the putative catalytic dead variants, HopAF1H186A and AvrXv3H126A, remained localized to the plasma membrane, suggesting that the putative catalytic activity of HopAF1 orthologs is not required for localization (Figure 3.7A). To confirm the confocal data, we performed cell fractionation assays with N. benthamiana tissues transiently expressing the same constructs described above. The control proteins, anti-APX and anti-H+-ATPase were used as controls for the soluble and membrane fractions, respectively, and confirm that the separation of the fractions was complete. Wild-type HopAF1 is detected in the microsomal fraction, which contains the plasma membrane, whereas the acylation minus variant of HopAF1 is solely in the soluble fraction (Figure 3.7B). AvrXv3 is also in the soluble fraction. Consistent with the confocal 99 results, HopAF1H186A and AvrXv3H126A remained localized to the microsomal and soluble fractions, respectively (Figure 3.7B). We conclude that HopAF1 is targeted to the plant plasma membrane via the acylation signal sequence and mutation of the putative catalytic residues does not affect this localization. HopAF1 associates with Arabidopsis MTN proteins at the plasma membrane. We identified Arabidopsis methylthioadenosine nucleosidase (AtMTN1; At4g38800) as a protein that interacts with HopAF1 in a yeast two-hybrid screen (Mukhtar et al., 2011). This interaction was confirmed with pairwise yeast two-hybrid assays (data not shown). Methylthioadenosine nucleosidases function in the methionine recycling pathway, also known as the Yang cycle (Figure 3.9A) (Miyazaki and Yang, 1987). In Arabidopsis, two proteins, MTN1 and MTN2 (67% identical), are responsible for methylthioadenosine nucleosidase activity (Park et al., 2009). The Yang cycle generates high levels of methionine to supplement de novo synthesis of methionine. This process is essential when levels of methionine are limiting (Burstenbinder et al., 2007). This may be the case, for example, during PTI. PTI results in the induction of high levels of ethylene, and methionine is the precursor for ethylene biosynthesis. Therefore, if de novo biosynthesis of methionine does not produce sufficient levels of methionine, the Yang cycle may be required for the PTItriggered induction of ethylene biosynthesis. We hypothesized that disrupting the function of the Arabidopsis MTNs would block ethylene biosynthesis induced by PTI. Additionally, we predicted that the virulence function of HopAF1 was to inhibit MTN function and block ethylene-dependent PTI. This hypothesis is consistent with the finding that the type III secretion system deficient mutant, Pto DC3000 hrcC, triggers the accumulation of high levels of ethylene; whereas Pto DC3000 does not (Figure 3.9B). This suggests that the one or more members of the suite of T3Es from Pto DC3000 can suppress ethylene accumulation. 100 To extend the yeast two-hybrid interaction, we performed co-immunoprecipitation studies using Agrobacterium-mediated transient expression assays in N. benthamiana leaves. We co-expressed myc-tagged HopAF1 with the putative targets, MTN1-HA or MTN2-HA. The cell lysates were incubated with anti-myc conjugated beads. Bound MTN proteins were then detected with anti-HA westerns. We detected both MTN1-HA and MTN2HA interacting with HopAF1-myc (Figure 3.10A). Furthermore, HopAF1H186A-4x-myc coimmunoprecipated with MTN1-HA.The negative control demonstrates that there is very little non-specific binding of MTN1-HA to the anti-myc beads (Figure 3.10A). We conclude that HopAF1 also interacts with Arabidopsis proteins MTN1 and MTN2 when transiently overexpressed in N. benthamiana. Because HopAF1 is targeted to the plant plasma membrane, we predicted the plasma membrane to be the site of interaction with MTNs. This is consistent with literature showing that MTN1 can be found in the plant plasma membrane fraction and may interact with CBL3 in the plasma membrane (Marmagne et al., 2007; Oh et al., 2008). Therefore, MTN1 and MTN2 may be transiently tethered to the plant plasma membrane via proteinprotein interactions. We used confocal microscopy to determine if HopAF1 and MTN1 could interact at the plasma membrane. We performed bimolecular fluorescence complementation (BiFC) experiments using a Gateway BiFC vector set with ubiquitin promoters providing low but detectable levels of expression to minimize aberrant interactions due to over-expression (Grefen et al., 2010). We generated constructs with MTN1 and MTN2 fused at their Ctermini to the C-terminal half of YFP. HopAF1 variants were fused at the C-terminus to the N-terminal half of YFP. As a negative control, we assayed whether HopAF1 associated with known plasma membrane protein PLC2 (Otterhag et al., 2001). Reconstitution of YFP, resulting in a fluorescent signal emitting light at 514nm, occurred at the plasma membrane when wild-type HopAF1-nYFP was co-expressed in N. benthamiana cells with either MTN1- 101 cYFP or MTN2-cYFP (Figure 3.10B). However, there was no reconstitution of YFP when HopAF1-nYFP was co-expressed with PLC2-cYFP (Figure 3.10B). Reconstitution at the plasma membrane was not dependent on the putative catalytic activity of HopAF1 (Figure 3.10B). Consistent with the presence of Arabidopsis MTNs in the cytoplasmic fraction (Figure 3.5B, above), the acylation minus HopAF1G2AC4S-nYFP variant co-expressed with MTN1-cYFP or MTN2-cYFP reconstituted the YFP signal, but in the cytoplasm (Figure 3.10B). We conclude that wild-type HopAF1 and Arabidopsis MTNs interact specifically at the plasma membrane, consistent with the demonstrated localization of HopAF1, and the observation that MTN1 and MTN2 may transiently localize to the plasma membrane via protein-protein interactions (Marmagne et al., 2007; Oh et al., 2008). HopAF1 inhibits PAMP-induced ethylene production in a manner dependent on its putative catalytic activity and MTN proteins. Recognition of flg22 triggers activation of a MAPK cascade that leads to an increase in ethylene biosynthesis (Felix et al., 1991a; Felix et al., 1991b; Liu and Zhang, 2004). In order to determine the effect of HopAF1 on PAMP-induced ethylene biosynthesis, we established a system with which we could both induce ethylene biosynthesis with PAMPs and measure the effect of HopAF1 on subsequent ethylene biosynthesis. To induce PAMP-mediated ethylene production, we used a P. fluorescens strain (Pfo-1) lacking known T3Es but engineered to carry a functional T3SS, as a delivery vehicle for PAMPs, and, if desired, specific T3Es (Thomas et al., 2009). Pfo-1 triggers an increase in ethylene production (Figure 3.11A). We transformed HopAF1 variants expressed from the native promoter into Pfo-1. All HopAF1 alleles are expressed equally in Pfo-1 and are capable of translocation via the type III secretion system, as assayed via their ability to deliver truncated ∆79’avrRpt2 to trigger an RPS2-dependent HR (Figure 3.11B,C) (Mudgett and Staskawicz, 1999). 102 HopAF1 inhibited the PAMP-induced ethylene biosynthesis compared to Pfo-1 and empty vector controls (Figure 3.12A). Inhibition of ethylene production required the HopAF1 putative catalytic residue H186, supporting our hypothesis that HopAF1 inhibits MTN function with catalytic activity (Figure 3.12B). However, the inhibition of PAMP-induced ethylene production was not dependent on HopAF1 localization in the plasma membrane (Figure 3.12B). This is consistent with our finding (Figure 3.10B), and literature (Marmagne et al., 2007; Oh et al., 2008) showing that MTN1 and MTN2 localized to both the plasma membrane and the cytoplasm. HopAF1 phenocopies Yang cycle intermediate mutants for PAMP-induced increase in ethylene production. We predicted that HopAF1 suppression of PAMP-induced ethylene production would be phenocopied in mutants of Yang cycle intermediates. As noted above, there are two MTN paralogs in Arabidopsis, and it is likely that they function redundantly in the Yang cycle (Figure 3.9A). MTN activity is required for maximal ethylene biosynthesis in tomatoes (Kushad et al., 1985). The enzyme downstream of MTN in the Yang cycle, MTK, is also required for maximal levels of ethylene biosynthesis (Burstenbinder et al., 2007). We obtained Arabidopsis mtn1 and mtn2 mutants and generated anew the mtn1 mtn2 double mutant (Burstenbinder et al., 2010). We demonstrated that Pfo-1 induced high levels of ethylene in each mtn1 and mtn2 single mutant, consistent with these proteins functioning redundantly for Yang cycle-mediated PAMP-induced ethylene biosynthesis (Figure 3.13A). However, in both the mtk single mutant and the mtn1 mtn2 double mutant, Pfo-1 was unable to induce high levels of ethylene production, demonstrating that the Yang cycle is necessary for PAMP-induced ethylene production in this assay (Figure 3.13B). These results are consistent with our suggestion that HopAF1 can inhibit PAMP-induced ethylene biosynthesis through modulating or inhibiting the functions of Arabidopsis MTN1 and MTN2. 103 HopAF1 inhibits MTN1 activity in vitro in manner dependent on its putative catalytic residues. We used recombinant proteins purified from E. coli to determine if HopAF1 can inhibit MTN1 function directly. A protocol for purifying Arabidopsis MTN proteins from E. coli was previously established (Park et al., 2009; Park et al., 2006). To purify HopAF1, we generated N-terminal 6xHis fusion constructs for wild-type and putative catalytic dead HopAF1. Because the coding sequence of HopAF1 likely contains subdomains, we also generated 6xHisMBP-tagged truncations of HopAF1 to identify the minimal region of HopAF1 required for activity (Figure 3.14A). We used circular dichroism to confirm that MTN1, 6xHis-HopAF1, and 6xHis-HopAF1H186A are folded (Figure 3.14B). Using an established assay to measure the activity of MTN1 in vitro (Dunn et al., 1994), we demonstrated that HopAF1 inhibited the ability of MTN1 to cleave its substrate (Figure 3.15A,B). However, HopAF1H186A was not able to inhibit MTN1 activity (Figure 3.15B). Furthermore, the proposed catalytic domain of HopAF1 (residues 130-284) is sufficient to inhibit MTN1 function (Figure 3.15B). The ∆147HopAF1 truncated protein was not able to inhibit MTN1 function, suggesting that an essential part of the HopAF1 structure exists between residues 130 and 147 (Figure 3.15B). These in vitro data support our in planta findings that HopAF1 inhibits PAMP-induced ethylene biosynthesis by blocking MTN function. MTN1 and MTN2 play a role in innate immunity. To determine the role of MTNs in PTI, we assayed the susceptibility of mtn1 mtn2 and mtk to infection with Pto DC3000D28E. As a control, we used the ein2 (Ethylene Insensitive 2) null mutant (Alonso et al., 1999). Ethylene insensitive mutants allow enhanced growth of weak pathogens and are defective in flg22-mediated priming of the immune system (Boutrot et al., 2010; Mersmann et al., 2010). We show that Yang cycle mutants, similar to ein2, are more susceptible to Pto DC3000D28E (Figure 3.16). Additionally, the 104 enhanced growth of Pto DC3000D28E in mtn1 mtn2 was similar to levels of the same strain observed in transgenic plants expressing wild-type HopAF1 (Figure 3.16). Discussion Many plant bacterial pathogens use type III effectors (T3Es) as their main virulence determinants. The type III secretion system is essential to the ability of these plant pathogens to cause disease on their hosts. As such, it is critical to understand which host cellular systems T3Es are targeting and how they are disrupting those systems. Additionally, we learn how these potent virulence factors, which are common among plant and animal pathogens, function. HopAF1 is a reasonably well conserved T3E. The presence of HopAF1 in multiple strains of P. syringae and other plant pathogens suggests an important role in virulence and we sought to define its specific molecular function. Here we report that HopAF1 contributes to P. syringae virulence and we propose a model (Figure 3.17), in which HopAF1 interacts with Arabidopsis Yang cycle proteins, MTN1 and MTN2, at the plant plasma membrane. HopAF1 inhibits PAMP-induced ethylene biosynthesis and the mtn1 mtn2 phenocopies the effect of HopAF1 on PAMP-induced ethylene biosynthesis. This is consistent with HopAF1 inhibition of MTN1 activity in vitro. We have also demonstrated that MTN1 and MTN2, and, therefore the Yang cycle, are key components of the plant immune response. The role of ethylene in the plant defense response is well-documented, yet complicated because of the multiple, and sometimes conflicting, roles that ethylene plays. The contradictory results from pathology assays might result from differences in bacterial strains used and infection methods. Ethylene plays a role in defense against necrotrophs by antagonizing salicylic acid responses which are involved in defense against biotrophs (Robert-Seilaniantz et al., 2007). Ethylene is also required for symptom development during infection with virulent and avirulent pathogens (Bent et al., 1992). 105 However, more recently, it has been demonstrated in multiple studies that ethylene plays a significant role in defense against P. syringae. Ethylene directly controls FLS2 expression by inducing the activation of the EIN3/EIL family of transcription facts which directly bind the FLS2 promoter (Boutrot et al., 2010). This leads to an increase in FLS2 protein accumulation (Boutrot et al., 2010; Mersmann et al., 2010). Mutants deficient in ethylene signaling pathways, such as ein2, have reduced FLS2 transcript levels with or without flg22 treatment (Boutrot et al., 2010). This likely contributes to the susceptibility of ein2 to weakened plant pathogens such as P. syringae pv. tomato DC3000 ∆AvrPto/∆AvrPtoB (Mersmann et al., 2010). Furthermore, XopD desumoylates and represses an ethylene-responsive transcription factor and suppresses ethylene accumulation (Kim et al., 2013). Ethylene is required for the immune response against Xanthomonas campestris pv. vesicatoria (Kim et al., 2013). The role of the Yang cycle in PTI responses is thus far undocumented. This is likely due to the lack of the link between Yang cycle proteins and pathogen-induced ethylene accumulation in the literature. The Yang cycle kinase, MTK, was demonstrated to be required for accumulation of high level of ethylene by crossing mtk with an ethylene overproducing mutant, eto (Burstenbinder et al., 2007). This suggested that the Yang cycle is required for high levels of ethylene biosynthesis, such as that which occurs during PTI. However, subsequent papers have stated that the MTN proteins are not required for ethylene biosynthesis (Burstenbinder et al., 2010). Unfortunately, they did not investigate ethylene accumulation in the double mtn1 mtn2 mutant; nor did they attempt to induce high levels of ethylene accumulation (Burstenbinder et al., 2010). Therefore, the burden of proof was on us to demonstrate the requirement of MTN proteins for high levels of ethylene accumulation in Arabidopsis. This highlights the importance of the results in figure 13. We conclude that the Yang cycle is not required for steady-state levels of ethylene biosynthesis. 106 However, our results consistently show that Yang cycle proteins, MTN1 and MTN2 or MTK are required for the high levels of PAMP-induced ethylene biosynthesis. However, there are still some questions that remain unanswered. For example, are there other type III effectors from P. syringae pv. tomato DC3000 that inhibit ethylene accumulation? In figure 9, we demonstrate that the type III secretion system deficient mutant, Pto DC3000 hrcC, triggers the accumulation of high levels of ethylene; whereas Pto DC3000 does not. Assuming there is an equal presence of PAMPs associated with these pathogens, the likely conclusion is that the fully virulent Pto DC3000 is suppressing ethylene accumulation. Incorporation of the Pto DC3000∆HopAF1 mutant into this assay will help us discern whether or not other effectors are targeting ethylene accumulation. For example, if Pto DC3000∆HopAF1 accumulates ethylene similar to Pto DC3000 hrcC, HopAF1 is likely to be the only Pto DC3000 T3E that can inhibit ethylene biosynthesis. Conversely, if Pto DC3000∆HopAF1 still inhibits ethylene accumulation similar to Pto DC3000, there are still T3Es other than HopAF1 that can inhibit ethylene biosynthesis. Although we have predicted, based on structural modeling, that HopAF1 is a member of the family of bacterial toxins/effectors that are deamidases, we have not yet demonstrated that HopAF1 uses this activity to affect the function of Arabidopsis MTNs or any other proteins. We have, however, demonstrated that the ability of HopAF1 to block PTI, inhibit MTN1 function in vitro, and inhibit PAMP-induced ethylene biosynthesis is dependent on putative catalytic residues. This suggests that HopAF1 has enzymatic activity. We will attempt to determine the enzymatic function of HopAF1 using mass spectrometry to identify deamidation on MTN1 (see Chapter 4). Mass spec has been an invaluable tool in identifying posttranslational modifications on host proteins due to pathogen virulence proteins, such as type III effectors. However, identifying a 1 Dalton increase in the size of a target protein, such as would occur during deamidation, with mass spectrometry is not trivial. For this reason, we are collaborating with Mark Banfield, a biochemist at the John Innes Centre with 107 extensive experience with deamidases (Crow et al., 2012; Crow et al., 2009; Jubelin et al., 2009). Multiple P. syringae T3Es target PTI signaling pathways using diverse biochemical mechanisms. In this study, we have described a new component of the plant innate immune system, the Yang cycle. Furthermore, we will continue to determine whether a unique biochemical mechanism, namely deamidation, is being utilized by the P. syringae T3E HopAF1 to target Arabidopsis Yang cycle proteins, MTN1 and MTN2. 108 Materials and Methods Plant Lines and Growth Conditions We used wild-type Arabidopsis thaliana ecotype Columbia (Col-0) and all mutant Arabidopsis lines we used are in the Col-0 background. For generation of transgenic estradiol-inducible HopAF1-cerulean-HA, HopAF1C159AH186A-cerulean-HA lines, the HopAF1 coding sequences were cloned into the pMDC7-cerulean-HA Gateway-compatible vector (Akimoto-Tomiyama et al., 2012) and transformed into Agrobacterium GV3101. Arabidopsis transgenics were generated using Agrobacterium-mediated floral dip transformation (Clough and Bent, 1998). Hygromycin selection of transgenic plants was performed by growing the seedlings on plates with ½ MS media with 15 μM hygromycin in the dark for 3 days and then in light conditions for 10 days. mtn1-1 (Burstenbinder et al., 2010), mtn1-2 (Burstenbinder et al., 2010), mtn2-1 (Burstenbinder et al., 2010), mtn2-2 (Burstenbinder et al., 2010), mtn1-1 mtn2-1 (Burstenbinder et al., 2010), mtk (Sauter et al., 2004), and ein2-5 (Alonso et al., 1999) have previously been described elsewhere. Except where otherwise noted, plants were grow under controlled short day conditions (9 hours light, 21°C, 15 hours dark, 18°C). Tomato cultivars Florida 216 (Fla 216) and Florida 7060 (Fla 7060) are described elsewhere (Astua-Monge et al., 2000) and were grown in the greenhouse. Nicotiana benthamiana plants were also grown in the greenhouse. Immunoblot analysis of plant proteins Unless otherwise noted, total proteins were extracted by grinding either fresh tissue or tissue flash-frozen in liquid nitrogen in a buffer containing 50 mM Tris-HCl pH 8.0, 1% SDS, 1 mM EDTA pH 8.0, 14 mM β-mercaptoethanol, and protease inhibitor cocktail. Samples were incubated on ice for 20 minutes and then the cell debris was pelleted by centrifuging the samples for 15 minutes at 20,800 g at 4°C. The supernatant was separated and quantified using the Bradford reagent. Protein was separated on 12% SDS-PAGE gels and 109 transferred to a nitrocellulose membrane. Western blots were performed using standard methods. Anti-HA (Santa Cruz Biotechnology) antibody was used at a 1:5000 dilution, antiAPX (Agrisera) was used at a 1:5000 dilution, and anti-H+-ATPase (Agrisera) was used at a 1:1000 dilution. Anti-myc polyclonal antisera was used at a 1:5 dilution. Anti-MTN1 and antiMTN2 were used at a 1:5000 dilution (Burstenbinder et al., 2010). Homology Modeling We initially detected the homology with the C-terminal domain of HopAF1 and CheD by querying the BioInfoBank Institut’s Metaserver and the HHPred structural prediction programs (Bujnicki et al., 2001; Soding et al., 2005). The 3-D model for the HopAF1 Cterminus structure based on the Thermotoga maritima CheD (PDB 2F9Z) structure was generated with MODELLER (Sali and Blundell, 1993). The validity of the HopAF1 structural model was assessed using Verify3D (Eisenberg et al., 1997). Graphical images and structural alignments were produced with Pymol. P. syringae pv. tomato DC3000D28E Growth Curve Assay Leaves of five-week-old plants were hand inoculated with 1x105 cfu/mL of P. syringae pv. tomato DC3000D28E (Cunnac et al., 2011). Leaf bacterial populations were determined the day of inoculation and three days post-inoculation. Each data point consisted of six replicates. Plants expressing either estradiol-inducible HopAF1-cerulean-HA or estradiolinducible HopAF1C159AH186A-cerulean-HA were sprayed with 20 μM estradiol 24 hours before hand inoculations. Flg22-protection Growth Curve Assay Leaves of five-week-old plants were sprayed with 20 μM estradiol 12 hours before flg22 infiltration. Plants were infiltrated with 1 μM flg22 or water 24 hours before infiltrating with 110 1x105 cfu/mL P. syringae pv. tomato DC3000. Leaf bacterial populations were determined at the indicated times. Each data point consisted of six replicates. Transient expression of AvrXv3 in tomato The coding sequences of AvrXv3 and AvrXv3H126A were cloned in the binary Gateway vector, pGWB17, which carries a C-terminal 4x-myc tag. Triparental mating was used to transfer this construct to Agrobacterium strain C58C1. For transient expression of AvrXv3 in tomato plants, the strains expressing AvrXv3 variants were grown overnight in LB media at 28°C. The bacteria were centrifuged at 6800 g for 5 minutes and resuspended in 10 mM MgCl2 at an OD of 0.1. Fully expanded leaves were infiltrated with the bacterial strains and maintained in the greenhouse for 48 hours (immunoblot samples) or 72 hours (HR). Immunoblot analysis of protein samples was completed as previously described. Cell fractionations 200 mg of tissue was flash-frozen in liquid nitrogen and ground in sucrose buffer (20 mM Tris-HCl pH 8.0, 0.33M sucrose, 1 mM EDTA pH 8.0, 5 mM DTT, plant protease inhibitors cocktail). The homogenate was centrifuged for 10 minutes at 2000 g at 4°C. The supernatant was collected as the total fraction. A portion of the total fraction was spun again for 30 minutes at 20,000 rpm at 4°C. The supernatant from this spin was collected as the soluble fraction. The pellet, which became the microsomal fraction, was resuspended in equal volumes of sucrose buffer. Agrobacterium-mediated transient transformations in N. benthamiana 2 mL of overnight cultures were centrifuged at 10,600 g for 1 minute at room temperature. Cultures were resuspended in induction media containing 10 mM MgCl2 and 10 mM MES. Cultures were re-centrifuged and then resuspended in infiltration media plus 100 μM 111 acetosyringone and agitated at room temperature for 1 hour. Cultures were diluted to the appropriate concentrations for each experiment and hand-infiltrated into fully expanded Nicotiana benthamiana leaves. Coimmunoprecipitation For co-immunoprecipitation experiments we generated 35S::MTN1-HA, 35S::MTN2-HA, 35S::HopAF1-myc, 35S::HopAF1H186A-myc using the Gateway constructs pGWB14 (Cterminal 3x-HA tag) and pGWB17 (C-terminal 4x-myc tag). Constructs were infiltrated at concentrations of 0.2 OD each with 0.1 OD P19 (Voinnet et al., 2003). Samples were collected and flash-frozen in liquid nitrogen. Protein was ground in lysis buffer (20 mM TrisHCl pH 8.0, 1 mM EDTA pH 8.0, 150 mM NaCl, protease inhibitor cocktail), centrifuged for 15 minutes at 6000 g in 4°C. The supernatant was diluted to a concentration of 2 mg/mL. 50 μL of anti-myc beads was added to the protein sample and incubated at 4°C for 4 hours with gentle agitation. The bound protein complexes were washed in extraction buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 300 mM NaCl) and eluted in pre-warmed 6X SDSPAGE loading buffer. The input and bound proteins were analyzed by SDS-PAGE and immunoblot analysis. Subcellular localization HopAF1, HopAF1H186A, HopAF1G2AC4S, AvrXv3, and AvrXv3H126A coding sequences were cloned into the Gateway-compatible pMDC7-cer-HA vector (Akimoto-Tomiyama et al., 2012) and transferred into Agrobacterium strain C58C1 by triparental mating. Agrobacteriummediated transformations into Nicotiana benthamiana leaves were performed as described above. Agrobacterium strains expressing HopAF1, HopAF1H186A, HopAF1G2AC4S, AvrXv3, and AvrXv3H126A were inoculated at an OD of 0.01 in combination with the PLC2 plasma membrane marker at 0.1 OD. Three days post-infiltration, leaves were sprayed with 5 μM 112 estradiol for 3 hours. The subcellular localization of the proteins was observed using a Zeiss LSM 710 confocal scanning laser microscope. The plasma membrane marker, PLC2-YFP, was generated by cloning the PLC2 ORF from Col-0 cDNA into the pGWB41 Gateway vector. Bimolecular fluorescence complementation HopAF1 and MTN1 coding sequences were cloned into bimolecular fluorescence (BiFC) constructs that contain an ubiquitin promoter (Gehl et al., 2009). HopAF1-nYFP, HopAF1H186A-nYFP, HopAF1G2AC4S-nYFP were transiently co-expressed with MTN1-cYFP, MTN2-cYFP, and PLC2-cYFP in N. benthamiana leaves using Agrobacterium-mediated transient transformation. Strains expressing either HopAF1, MTN1, MTN2, or PLC2 were inoculated at 0.4 OD with 0.1 OD of P19 (Voinnet et al., 2003). 3 days after infiltration, confocal microscopy was used to image the reconstituted YFP signal. HopAF1 purification N-terminal 6xHis affinity tag constructs for HopAF1 and HopAF1H186A were generated by ligation independent cloning (Aslanidis and de Jong, 1990). 6xHisMBP-∆130HopAF1 and 6xHisMBP-∆147HopAF1 were generated by cloning a TEV protease cleavage site onto the N-terminus of the truncated HopAF1 ORFs and cloning them into the Gateway-compatible pDEST-HisMBP vector. All HopAF1 bacterial expression constructs were transformed into ArticExpress (DE3) RIL (Agilent) cells. To initiate protein production, overnight cultures were used to inoculate 5 L of LB media containing 100 μg/mL ampicillin at a 1:200 dilution. Cells were grown at 28°C until the OD reached 0.6-0.8 and then the temperature was reduced to 14°C. Protein production was induced with 1 mM IPTG and cells continued to grow overnight at 14°C. 113 Purification of 6xHis-HopAF1 and 6xHis-HopAF1H186A involved affinity purification on a HisTrap column followed additional purification with the HiPrep 16/60 Sephacryl S-200 High Resolution (GE Healthcare) size exclusion column. The HopAF1 fractions that were not in the void volume were collected for in vitro assays. Purification of 6xHisMBP-∆130HopAF1 and 6xHisMBP-∆147HopAF1 also began with a His affinity purification. This was followed by dialysis into TEV cleavage buffer (20 mM Tris pH 8.0, 50 mM NaCl, 1 mM DTT, and 0.5 mM EDTA) and TEV cleavage at room temperature for 6 hours. Reverse affinity chromatography was used to collect the HopAF1 truncations into relatively pure fractions. These fractions were dialyzed into anion exchange buffer (20 mM Tris pH 8.0, 50 mM NaCl overnight at 4°C and further purified with the anion exchange HiLoad 16/10 Q sepharose column (GE Healthcare). Size exclusion chromatography using a HiPrep 16/60 Sephacryl S-200 High Resolution column (GE Healthcare) resulted in monodispersed protein. MTN1 purification MTN1 (At4g38800) was purified as previously described (Park et al., 2009). In brief, a PreScission Protease cleavage site was cloned onto the N-terminus of MTN1, which was then cloned into the Gateway vector pDEST15, which carries an N-terminal glutathione Stransferase (GST) tag. This clone was transformed into Novagen® Rosetta E. coli BL21(DE3) cells. A 5 mL overnight culture was used to inoculate 1L of LB media containing 100 μg/mL ampicillin. Cells were grown at 37°C to an OD of 0.6-0.8. The temperature was reduced to 22°C. Protein production was induced with 1 mM IPTG and cells continued to grow at 22°C overnight. MTN1 was purified with a GST affinity purification step followed by 114 PreScission Protease cleavage and reverse chromatography to isolate “free” MTN1 in the flow through fractions. MTN activity assays MTN activity assays were performed as previously described (Dunn et al., 1994). Briefly, reactions were carried out in 50 mM imidazole pH 8.0, 0.28 units of grade III buttermilk xanthine oxidase, 1 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) and 0.12 μg recombinant MTN1. The reaction was monitored at 470 nm on a Tecan GENios microplate reader (Tecan, Ltd., Switzerland) for 15 minutes with 1 minute increments. Changes in absorbance were converted to the amount of adenine released using the molar absorption coefficient (15,400 M-1 cm-1 at pH 8.0). To generate the Michaelis-Menten plot, the experiment was completed with a range of MTA concentrations (0 μM MTA – 150 μM MTA). Specific activity assays were completed with 50 μM MTA. All enzyme reactions had a final volume at 800 μL and were performed in triplicate at 25°C. All reagents for the kinetic assay were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). Ethylene measurements Three-week-old seedlings were vacuum-infiltrated with Pfo-1 strains measured at 0D 0.04 in 10 mM MgCl2. After three hours, the fresh weight of the seedlings was measured. Seedlings were sealed into gas chromatography vials and ethylene was allowed to accumulate for 24 hours. Ethylene accumulation was measured using a Clarus 500 gas chromatograph (Perkin Elmer, USA). A pure ethylene gas control was used in each experiment. Each data point represents 3 to 4 replicates. 115 Induction of native promoter HopAF1 in minimal media Genomic clones of wild-type HopAF1, HopAF1H186A, and HopAF1G2AC4S, including the native promoter with a hrp-box, were cloned into a Gateway-compatible vector, pJC531, carrying a C-terminal HA-tag. This clone was transformed into P. fluorescens, a strain engineered to express the T3SS (Thomas et al., 2009). Strains were spread onto KB plates overnight and resuspended in minimal media (7.8 mM ammonium sulfate, 50 mM potassium phosphate, 1.7 mM sodium chloride, 1.7 mM NaCl and 10 mM mannitol) at an OD of 0.02. After growth in 28°C for 12 hours, 2 mL of each cultures was centrifuged at room temperature for 5 minutes at 8600 g. The pellet was resuspended in 100uL of 6X SDS loading buffer, boiled for 10 minutes, and 10 μL was loaded onto SDS-PAGE gel. Circular dichroism 6xHis-HopAF1, 6xHis-HopAF1H186A, and MTN1 were purified as described above and dialyzed into a buffer containing 20 mM sodium phosphate pH 7.4, 150 mM NaF. Samples of approximately 0.4 mg/mL (HopAF1 variants) or 0.5 mg/mL (MTN1) were placed in a 1.0 mm cuvette. Molar ellipticity measurements were collected at 25°C from 185 nm to 260 nm on a Chirascan Plus (Applied Photophysics, Ltd, Surrey, United Kingdom). Generating the mtn1 mtn2 double mutant In order to generate the mtn1 mtn2 double mutant, which is sterile (Burstenbinder et al., 2010), we obtained and genotyped the previously described GABI Kat lines mtn1-2 and mtn2-2 (Burstenbinder et al., 2010). We crossed mtn1-2 and mtn2-2, selfed the resulting T1s and screened the T2s for lines which were homozygous for mtn1 and segregating mtn2. We maintained several stocks of these lines, which we sowed out and PCR genotyped to identify double homozygous plants at the time of experimentation. 116 Translocation Assay from Pfo-1 and trypan blue staining Genomic HopAF1 constructs were cloned into the pJC532, a Gateway-compatible vector carrying a C-terminal tag of the C-terminal 177 residues of the type III effector protein AvrRpt2, from which the N-terminal 79 amino acids of AvrRpt2 were deleted (‘∆79AvrRpt2) (Chang et al., 2005; Mudgett and Staskawicz, 1999). HopAF1-∆79AvrRpt2, HopAF1H186A∆79AvrRpt2, HopAF1G2AC4S-∆79AvrRpt2 were transferred into Pfo-1 using triparental mating. Sixteen leaves of Arabidopsis Col-0 were hand-infiltrated per strain and the HR was scored 26 hours post inoculation. The HR was compared to leaves infiltrated with Pfo-1 alone or Pfo-1 carrying an empty vector. 117 Figures Figure 3.1. The predicted structure of HopAF1 exhibits structural homology to CheD, a bacterial deamidase. A) Homology model of HopAF1 reference allele (blue) from P. syringae pv. tomato DC3000 (Pto DC3000) residues 130-284 aligned with CheD (PDB 2F9Z) (silver). The side chains of the putative catalytic residues of HopAF1, C159, H186, and D154, are highlighted in green. The side chains of the CheD catalytic residues are in silver. B) Independent homology models for the additional HopAF1 family members: P. syringae pv. maculicola M4 (teal), P. syringae pv. aceris (grey), P. syringae pv. lachrymans str. M302278 (green), and Acidovorax citrulli Aave_1373 (pink). HopAF1 from Pto DC3000 is included in blue as a reference. C) Homology model of AvrXv3 from Xanthomonas vesicatoria (magenta) aligned with CheD (silver). Putative catalytic residues, C105 and H126, of AvrXv3 are highlighted in yellow and align with the catalytic residues of CheD (silver). . 118 Figure 3.2. Alignment of HopAF1 sequences from P. syringae pathovars and other plant pathogens. Protein sequences from HopAF1 homologs from P. syringae pv. tomato DC3000 (accession no. NP_791393), P. syringae pv. aceris strain M302273 (accession no. WP_003402453), P.syringae pv. actinadae str. M02091 (accession no. WP_003381937), P. syringae pv. lachrymans str. M302278 (Lac106) (accession no. WP_0057667510), P. syringae pv.lachrymans str. M301315 (Lac107) (WP_005746129), P. syringae pv. maculicola M4 str. ES4326 (accession no. WP_007248647), P. syringae pv. mori str. 301020 (accession no. WP_005752338), P. syringae pv. pisi (accession no. WP_003346247), P. syringae pv. phaseolicola 1448A race 6 (accession no. YP_273699), P.syringae pv. syringae B728A (accession no. YP_236881), P. syringae pv. tomato T1 (accession no. EEB59276), Pseudomonas savastoni (accession no. YP_006961546), Ralstonia solanacearum str. Po82 (accession no. YP_006028103), Xanthomonas campestris pv. muscacearum (accession no. WP_010369035), Xanthomonas vesicatoria AvrXv3 (accession no. WP_008577605), and Acidovorax citrulli AAC00-1 Aave_1373 (accession no. YP_969738) were aligned using VectorNTI AlignX. Amino acids with 80% identity are shaded in grey. Putative catalytic residues are highlighted in yellow. Sites for myristoylation and palmitoylation in denoted in red and orange, respectively. Residues highlighted in green highlight the beginning of the putative catalytic domain. 119 Figure 3.3. HopAF1 transgenic lines are susceptible to weak pathogens. A) Bacterial growth of P.syringae pv. tomato DC3000D28E (Cunnac et al., 2011) in Col-0 and two independent transgenic lines expressing either estradiol-inducible HopAF1-cerulean-HA or estradiol-inducible HopAF1C159AH186A-cerulean-HA was measured 0 and 3 days after hand inoculation with 1x105 cfu (colony-forming units)/mL bacteria. Transgene expression was induced with 20 μM estradiol 24 hours prior to bacterial inoculation. Error bars represent standard error (SEM). An analysis of variance (ANOVA) was performed among the day 3 samples, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. B) Disease symptoms in two independent lines of HopAF1-cerulean-HA and HopAF1C159AH186A-cerulean-HA were photographed 3 days after hand-inoculation with 1 x 105 cfu/mL of P. syringae pv. tomato DC3000D28E in HopAF1 transgenic lines. C) Immunoblot analysis with anti-HA shows the expression of HopAF1cerulean-HA and HopAF1C159AH186A-cerulean-HA in transgenic plants 3 days after spray application of 20 μM estradiol. 120 Figure 3.4. Arabidopsis transgenic lines expressing wild-type HopAF1 inhibit flg22mediated protection against P. syringae pv. tomato DC3000. A) Col-0, estradiolinducible HopAF1-cerulean-HA, and estradiol-inducible HopAF1C159AH186A-cerulean-HA were sprayed with 20 μM estradiol for 12 hours and then infiltrated with 1 μM flg22 or water for 24 hours prior to infiltration with 1x105 cfu/mL P. syringae pv. tomato DC3000. Bacterial growth was determined 0 and 3 days after bacterial inoculation. Error bars represent SEM. An ANOVA was performed among the day 3 samples, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. B) Immunoblot analysis with anti-HA shows the expression of HopAF1-cerulean-HA and HopAF1C159AH186Acerulean-HA in transgenic plants 3 days after bacterial infiltration, which corresponds to 108 hours (or 4.5 days) after estradiol induction. 121 Figure 3.5. HR triggered by AvrXv3 is dependent on putative catalytic residues. A) AvrXv3-4x myc and AvrXv3H126A-4x myc were transiently expressed in tomato cultivar Fla 216. Cores containing both infiltrated and uninfiltrated tissue where removed from the tomato leaves 3 dpi and trypan blue staining was used to highlight the cell death response associated with HR. B) Anti-myc immunoblot analysis demonstrates the expression of AvrXv3-4x-myc and AvrXv3H126A-4x-myc in tomato cultivar Fla 216 dpi. Ponceau staining of the membrane indicates equal loading. 122 Figure 3.6. Establishing a system to study localization of transiently expressed HopAF1 in N. benthamiana. A) Levels of HopAF1-cerulean-HA transiently expressed in N. benthamiana were titrated by varying the concentration of estradiol (μM) and OD of Agrobacterium. Immunoblots with anti-HA were used to detect the expression of HopAF1cerulean-HA. N. benthamiana leaves were sprayed with 5 μM estradiol 3 days postinfiltration with Agrobacterium and leaf samples were collected 3 hours after estradiol application for immunoblot analysis. B) Immunoblot analysis with anti-HA shows the expression of full-length HopAF1-cerulean-HA, HopAF1H186A-cerulean-HA, and HopAF1G2AC4S-cerulean-HA and the absence of free cerulean (black arrow) at approximately 27 kDa. N. benthamiana leaves were sprayed with 5 μM estradiol 3 days post-infiltration with Agrobacterium. Protein samples were collected 3 hours after estradiol application. Ponceau staining indicates equal loading. 123 Figure 3.7. HopAF1 is targeted to the plasma membrane via acylation. A,B) Estradiolinducible HopAF1-cerulean-HA, HopAF1G2AC4S-cerulean-HA, HopAF1H186A-cerulean-HA, AvrXv3-cerulean-HA, and AvrXv3H126A-cerulean HA were expressed transiently in N. benthamiana using Agrobacterium-mediated transient transformations. Transgene expression was induced with 5 μM estradiol 3 days after bacterial infiltration. A) The localization of transiently expressed proteins was determined with scanning confocal laser microscopy 3 hours after estradiol induction in N. benthamiana epidermal cells. PLC2-YFP is a marker for the plasma membrane localization. Scale bars represent 20 μm. White arrowheads indicate the plasma membrane (PM), cytoplasmic streaming (CS), and nuclei (N). B) Cellular fractionations into total (T), soluble (S), and microsomal (M) fractions were performed. Equal cell equivalents were loaded onto SDS-PAGE gels followed by anti-HA immunoblots to detect the fractions with the expressed proteins of interest. Anti-APX and anti-H+-ATPase were included as controls for the soluble (S) and microsomal (M) fractions, respectively. 124 Figure 3.8. The acylation-minus variant of HopAF1 co-localizes with free YFP, a soluble protein. A-C) Estradiol-inducible HopAF1-cerulean-HA and HopAF1G2AC4Scerulean-HA were expressed transiently in N. benthamiana using Agrobacterium-mediated transient transformations. Transgene expression was induced with 5 μM estradiol 3 days after bacterial infiltration. A) The localization of transiently expressed proteins was determined with scanning confocal laser microscopy 3 hours after estradiol induction in N. benthamiana epidermal cells. Free YFP is a soluble protein and, therefore, can be used as a marker of the plant cytoplasm. Scale bars represent 20 μm. B) Merged image of estradiolinducible HopAF1-cerulean-HA and YFP. C) Merged image of estradiol-inducible HopAF1G2AC4S-cerulean-HA and YFP. White arrowheads indicate the plasma membrane (PM), cytoplasmic streaming (CS), and nuclei (N). 125 Figure 3.9. The Yang cycle contributes to ethylene biosynthesis, which is suppressed by Pto DC3000. A) In this figure adapted from Rzewuski et al., (Rzewuski et al., 2007) the relationship between the Yang cycle (also known as the methionine recycling cycle) and the phytohormone ethylene in Arabidopsis is highlighted. Arabidopsis MTN1 and MTN2 are two Yang cycle proteins. Both can catabolize MTA by cleaving the adenine to create the product MTR. MTR is then phosphorylated by the kinase MTK. MTNs also functions in a secondary pathway to recycle methionine, in which S-adenosylhomocysteine is its substrate. Methionine is a precursor for the ethylene biosynthesis pathway. It is important to note that recycling of methionine supplements the continual de novo synthesis of methionine. B) Arabidopsis seedlings were vacuum-infiltrated with multiple concentrations of P. syringae pv. tomato DC3000 and the type III secretion system deficient mutant, P. syringae pv. tomato DC3000 hrcC. 3 hours post infiltration, the fresh weight of 4 seedlings was measured and these seedlings were sealed in a vial for 24 hours before ethylene accumulation was measured. Error bars represent standard error and an ANOVA was performed, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. 126 Figure 3.10. HopAF1 associates with Arabidopsis MTN proteins at the plasma membrane. A) Protein extracts from N. benthamiana leaves transiently expressing HopAF14x-myc, HopAF1 H186A-4x-myc, MTN1-HA, and MTN2-HA were collected 3 days after infiltration and subjected to immunoprecipitation using anti-myc-coupled magnetic beads. 400 μg of input and a 50x concentrated elution fraction were analyzed by anti-HA and antimyc immunoblots. B) HopAF1-nYFP, HopAF1H186A-nYFP, HopAF1G2AC4S-nYFP were transiently co-expressed with MTN1-cYFP, MTN2-cYFP, and PLC2-cYFP in N. benthamiana leaves using Agrobacterium-mediated transient transformation. 3 days after infiltration, confocal microscopy was used to image the reconstituted YFP signal. Scale bar represents 50 μm. 127 Figure 3.11. Establishing P. fluorescens as a tool to determine the effect of HopAF1 on PAMP-induced ethylene accumulation. A) This dose response curve demonstrates the increase in ethylene biosynthesis elicited by increasing concentrations of Pfo-1. B) Pfo-1 expressing native promoter HopAF1 variants was inoculated into Col-0 leaves and observed after 24 hours. The ability to elicit an RPS2-dependent HR indicates translocation of the fusion proteins. The number of leaves in which HR was evident compared to the number of leaves inoculated is displayed next to representative leaves for each Pfo-1 inoculation. C) Expression of native promoter HopAF1 variants in Pfo-1 was induced with minimal media for 12 hours and detected with anti-HA immunoblot. 128 Figure 3.12. HopAF1 inhibits PAMP-induced ethylene biosynthesis. A) Arabidopsis seedlings were vacuum-infiltrated with either MgCl2, Pfo-1, Pfo-1 carrying the empty vector (EV), Pfo-1 expressing HopAF1. 3 hours post infiltration, the fresh weight of 4 seedlings was measured and these seedlings were sealed in a vial for 24 hours before ethylene accumulation was measured. B) In addition to the aforementioned treatments, Arabidopsis seedlings were vacuumed-infiltrated with Pfo-1 expressing HopAF1H186A and Pfo-1 expressing HopAF1G2AC4S. 3 hours after infiltration, the fresh weight of 4 seedlings was measured and these seedlings were sealed in a vial for 24 hours before ethylene accumulation was measured. Error bars represent standard error and an ANOVA was performed, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. 129 Figure 3.13. Pfo-1 is unable to induce high levels of ethylene biosynthesis in Yang cycle mutants, mtn1 mtn2 and mtk. A) Seedlings of Col-0 and Arabidopsis mutants mtn11, mtn1-2, mtn2-1, and mtn2-2, were vacuum-infiltrated with Pfo-1 carrying the empty vector (EV) or Pfo-1 expressing wild-type, native promoter HopAF1. 3 hours post infiltration, the fresh weight of 4 seedlings was measured and these seedlings were sealed in a vial for 24 hours before ethylene accumulation was measured. B) Seedlings of Col-0 and Arabidopsis mutants mtn1-2 mtn2-2 and mtk were vacuum-infiltrated with either MgCl2, Pfo-1 carrying the empty vector (EV) or Pfo-1 expressing wild-type, native promoter HopAF1. 3 hours post infiltration, the fresh weight of 4 seedlings was measured and these seedlings were sealed in a vial for 24 hours before ethylene accumulation was measured. Error bars represent standard error and an ANOVA was performed, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. 130 Figure 3.14. HopAF1 domain architecture and circular dichroism spectra of HopAF1, HopAF1H186A, and MTN1. A) Schematic of HopAF1 predicted domains and the truncations generated for in vitro analyses. B) Circular dichroism spectra collected at 25°C at the far-UV CD spectrum (185 nm to 260 nm) demonstrates that wild-type MTN1, 6xHis-HopAF1 and 6xHis-HopAF1H186A have secondary structure. The H186A mutation in HopAF1 does not perturb the secondary structure. The buffer control (20 mM sodium phosphate pH 7.4, 150 mM NaF) was set as the baseline and subtracted from the protein-associated spectra above. 131 Figure 3.15. HopAF1 inhibits MTN1 activity in vitro in a manner dependent on catalytic activity. A) Michaelis-Menten plot of MTN1 activity measured in vitro using a xanthine oxidase-coupled spectrometric assay. Recombinant MTN1 was incubated for 18 hours in buffer at 37°C alone or with equimolar amounts of recombinant wild-type 6xHis-HopAF1 or GST. B) MTN1 enzyme specific activity measured in vitro using xanthine oxidase-coupled spectrometric assay. MTN1 was incubated at 37°C for 18 hours with equimolar amounts of 6xHis-HopAF1, 6xHis-HopAF1 H186A, ∆147-HopAF1, ∆130-HopAF1, and GST. Error bars represent standard error and an ANOVA was performed, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. 132 Figure 3.16. The Yang cycle is required for PTI in Arabidopsis. Bacterial growth of P.syringae pv. tomato DC3000D28E (Cunnac et al., 2011) in Col-0, mtn1 mtn2, mtk, and ein2 was measured 0 and 3 days after hand inoculation with 1x105 cfu (colony-forming units)/mL bacteria. Error bars represent standard error (SEM). An analysis of variance (ANOVA) was performed among the day 3 samples, followed by Tukey’s post-hoc analysis (p < 0.05). Groups that differ significantly are indicated with different letters. 133 Figure 3.17. Proposed model of HopAF1 suppression of plant innate immunity. Plant pathogens such as P. syringae and X. campestris inject multiple type III effector (T3E) proteins into plant cells, including HopAF1 and AvrXv3, respectively. Recognition of PAMPs, such as flg22, a peptide derived from bacterial flagellin, by pattern-recognition receptors (PRRs) such as FLS2 triggers a signal cascade that results in a multiple of outputs, which we collectively call PTI for PAMP-triggered immunity. Additionally, recognition of T3Es by plant Resistance proteins, often called NLRs, triggers ETI. The hypersensitive response (HR) is often associated with ETI. Through perception of flg22 and activation of a MAPK cascade, PTI leads to an increase in ethylene biosynthesis. Our data suggests that HopAF1 inhibits PTI, including flg22-induced PTI. Our model predicts that HopAF1 is targeted to the plant plasma membrane via acylation, where it inhibits the function of Yang cycle proteins, MTN1 and MTN2, which are required for the induced level of ethylene that occurs during PTI. As a result, the induced amount of ethylene that occurs during PTI may be an important output, making the Yang cycle an important component of the plant defense pathway and HopAF1 a critical T3E that prevents high ethylene accumulation during the plant defense response by targeting the Yang cycle. 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Nature 428, 764-767. 141 Chapter 4 Conclusions and Future Directions Many plant bacterial pathogens use type III effectors (T3Es) as their main virulence determinants (Deslandes and Rivas, 2012). The type III secretion system is essential to the ability of these plant pathogens to cause disease on their hosts. As such, it is critical to understand which host cellular systems T3Es are targeting and how they are disrupting those systems. Much progress has been made in this field during the course of my graduate career. Because T3Es can be used as probes of the plant immune system, the knowledge of which plant cellular systems T3Es disrupt can be used to decipher ways to make the plants more resistant to pathogen attack. Additionally, we learn how these potent virulence factors, which are common in plant and animal pathogens, function. Although recently several functions and targets for T3Es have been identified, the sheer number of plant pathogen T3Es ensures that there is still a lot of work to be done. In Chapter 3, I presented research that describes the function of a conserved type III effector, HopAF1, and I noted how it contributes to disease in Arabidopsis. HopAF1 inhibits the function of Yang cycle proteins, MTN1 and MTN2. This prevents the induction of ethylene biosynthesis that occurs during the plant immune response after the perception of PAMPs (termed PTI). The result is that HopAF1 inhibits a component of PTI, leading to enhanced growth of weakened strains of P. syringae. The research described in Chapter 3 is the beginning of our efforts to fully elucidate the function of HopAF1 and the specific effect that it has on Arabidopsis MTN proteins. Although we have predicted, based on structural modeling, that HopAF1 is a member of the family of bacterial toxins/effectors that are deamidases (Washington et al., 2013), we have not yet demonstrated that HopAF1 activity leads to deamidation of Arabidopsis MTNs or any other proteins. We have, however, demonstrated that the ability of HopAF1 to block PTI, inhibit MTN1 function in vitro, and inhibit PAMP-induced ethylene biosynthesis is dependent on putative catalytic residues. Therefore, we demonstrated that HopAF1 has enzymatic activity. In this chapter, I will discuss the future directions to significantly extend this work, which include determining the specific biochemical effect of HopAF1 on MTN1, solving the HopAF1 and MTN1 co-crystal structure, and using the Yang cycle to increase plant resistance to pathogen attack. Using a shotgun approach and mass spectrometry to determine if HopAF1 modulates MTN function via deamidation. Bacterial virulence factor deamidases typically alter a single conserved residue to modulate target protein function (Washington et al., 2013). Thus, we sought to identify conserved Arabidopsis MTN asparagine and glutamine residues that conferred loss-offunction phenotypes when mutated to the ‘deamidated’ residues, aspartic acid and glutamic acid, respectively. We discovered only three fully conserved residues across the evolutionary distance between plants and E. coli that could be targets of putative deamidases: MTN1N113, MTN1N169, and MTN1N194 (Figure 4.1). Since the crystal structures of Arabidopsis MTN1 and MTN2 are solved, we determined where the putative deamidation target residues are in relation to the MTN ligand binding site and catalytic residues. MTN1N113 is involved in ligand binding via coordination of a water molecule in the ligand binding pocket. MTN1N194 and MTN1N169 are both surface-exposed and at the end of helices α4 and α3, respectively (Figure 4.1). These conserved residues are located in approximately the same place in the MTN2 crystal structure. We generated individual deamidation mimic MTN1 variants (MTN1N113D, MTN1N169D, and MTN1N194D) and tested their function using an assay to measure MTN activity in vitro 143 (Dunn et al., 1994). For negative controls, we also cloned and purified MTN1D225N and MTN2D212N, catalytic residue loss-of-function alleles (Bretes et al., 2011). We determined that both MTN1N113D and MTN1N194D lost activity in vitro, while MTN1N169D retained wild-type levels of activity (Figure 4.2). Circular dichroism spectra confirmed that all of the MTN1 deamidation mimic variants retain elements of secondary structure, similar to wild-type MTN1 (Figure 4.3). We also determined whether the loss-of-function phenotypes we noted above are dependent on the introduction of a negative charge in the deamidation mimics. The MTN1N194A and MTN1N194V variants retained wild-type levels of activity (Figure 4.2). This suggests that a charge change at MTN1N194, as would occur during deamidation, causes a loss-of-function phenotype but that conservative replacement at MTN1N194 does not alter activity. Conversely, MTN1N113A and MTN1N113V resulted in loss-of-function (Figure 4.2), suggesting that MTN1N113 is sensitive to multiple mutations. This is consistent with the idea that MTN1N113 is involved in ligand binding. We generated the same set of variants for Arabidopsis MTN2 and determined that they functioned similar to MTN1 (Figure 4.2). Based on the results using our deamidation mimics of MTN1 and MTN2, we hypothesize that HopAF1 targets MTN1N113, MTN1N194, or both. Mass spectrometry can be a critical tool for identifying specific residues that are modified via posttranslational modifications (Witze et al., 2007). As such, mass spectrometry has been used to identify a single residue on each target protein that is deamidated by known bacterial deamidase toxins (Washington et al., 2013). Our initial attempts to identify MTN residues deamidated by HopAF1 via mass spectrometry resulted in a general identification of nonenzymatically deamidated residues (not shown). Nonenzymatic deamidation is a natural process that occurs as proteins age, leading to their eventually misfolding and degradation. We concluded that mutated residues identified in our original mass spectrometry experiments were a result of nonenzymatic deamidation because multiple MTN residues were 144 deamidated after no treatment, treatment with HopAF1, and treatment with catalytic dead HopAF1. As an alternative, we will generate peptides containing the residues described above and incubate them with HopAF1. Although, deamidation of peptides derived from a target protein has not been demonstrated using mass spectrometry, Lerm et al. demonstrated that CNF1 can deamidate a peptide derived from RhoA residues 59-78 using the glutamate dehydrogenase assay (Lerm et al., 1999). It was also demonstrated that CNF1 could deamidate a peptide derived from RhoA residues 59-69. Deamidation was evident due to increased elution times on a C18 hydrophobic column (Flatau et al., 2000). After incubating HopAF1 with the peptides derived from MTN1, we will use a hydrophobic column to purify the peptides and analyze them using mass spectrometry. We will be able to determine whether the putative target residues have been converted to aspartic acids. We will also attempt to detect deamidation of peptides generated from recombinant wild-type MTN1 protein. These peptides may allow us to detect the modification of residues not considered in our shotgun approach. Determine whether a functional deamidation site variant of MTN1 is impervious to HopAF1 activity in planta. We demonstrated above that deamidation mimics MTN1N113D and MTN1N194D lose enzymatic activity in vitro. We also showed that MTN1N194A and MTN1N194V retain activity. We are currently performing in planta complementation experiments using ubiquitin promoter driven constructs of wild-type MTN1, MTN1N194D, MTN N194V, MTN1N113D, MTN1N113V, and MTN1D225N transformed into the mtn1 mtn2 double mutant. This double mutant is viable, but fails to set seed (Burstenbinder et al., 2010). It has previously been demonstrated that a ubiquitin promoter-driven MTN1 can complement the mtn1 mtn2 infertility phenotype (Waduwara-Jayabahu et al., 2012). We will first address whether each of our constructs can complement the loss of seed set phenotype. Based on the results of our in vitro assays 145 described above, we hypothesize that MTN1 wild-type and MTN1N194V will complement the loss of seed set phenotype because these are functional variants, but that the deamidation mimic MTN1N194D will not. Similarly, we expect the MTN1N113D and MTN1D225N complementation lines to remain infertile because MTN1N113D and MTN1D225N are loss-offunction variants involved in ligand binding and catalysis, respectively. We will then repeat our experiment to measure PAMP-induced ethylene biosynthesis in transgenic seedlings expressing each MTN1 variant. If our hypothesis is true, we expect that MTN1 wild-type and MTN1N194V will support PAMP-dependent ethylene formation, but that MTN1N194D will not. We also expect that HopAF1 delivery will suppress Pfo-1-induced ethylene production in plants expressing wild-type MTN1. The key test will be whether or not MTN1N194V, which we hypothesize will still be functional based on our MTN activity determination (see Figure 4.2 above), will be unaffected by HopAF1. If so, we will conclude that MTN1N194 is the sole site of action for HopAF1. In Chapter 3, we demonstrate that the mtn1 mtn2 double mutant is more susceptible to weakened Pto DC3000D28E compared to Col-0 wild-type. We will determine the susceptibility of mtn1 mtn2 plants to Pto DC3000D28E expressing wild-type HopAF1 under a native promoter with a C-terminal HA tag. If Pto DC3000D28E(HopAF1) grows to equal levels in Col-0 as Pto DC3000D28E does in mtn1 mtn2 and there is no further increase in growth of Pto DC3000D28E(HopAF1) on mtn1 mtn2 plants, this will indicate the MTN1 and MTN2 are necessary for the HopAF1-mediated increase in the growth ofDC3000D28E that we described in Chapter 3. Furthermore, we will determine the ability of Pto DC3000D28E and Pto DC3000D28E(HopAF1) to grow in the mtn1 mtn2 complementation lines in which we express either wild-type MTN1, MTN1N194V, MTN1N194D, MTN1N113D, MTN1N113V or MTN1D225N under the ubiquitin promoter. Complementing mtn1 mtn2 with non-functional variants of MTN1, such as MTN1N194D and MTN1N113D will likely result in plants in which both Pto 146 DC3000D28E and Pto DC3000D28E(HopAF1) grow to an equally high titer. However, since MTN1N194V is functional and no longer a target of the putative deamidation activity of HopAF1, we expect MTN1N194V to be blind to HopAF1 and therefore to not be susceptible to either Pto DC3000D28E or Pto DC3000D28E(HopAF1). This result will further confirm that the ability of HopAF1 to inhibit PTI is dependent upon modification of Arabidopsis MTN1N194. Generate a HopAF1/MTN1 co-crystal to determine the molecular mechanism behind HopAF1 inhibition of MTN activity. The generation of co-crystals of bacterial deamidases and their targets addresses the critical question of how deamidases can modulate target protein activity (Crow et al., 2012; Fu et al., 2013; Yao et al., 2012). This will also provide information on how target proteins function, which is highly relevant for several known deamidase targets such as RhoA, ubiquitin, NEDD8, and Gα proteins (Cui et al., 2010; Flatau et al., 1997; Orth et al., 2009; Schmidt et al., 1997). It is equally important to understand how plant methylthioadenosine nucleosidases function, as these proteins are involved in recycling methionine, the precursor for protein translation, polyamine biosynthesis, and ethylene biosynthesis. Using published methods (Park et al., 2006), we are able to purify MTN1 from E. coli. We generated an expression construct using the Gateway-compatible pDEST15 vector (Gateway, Invitrogen). This vector includes an IPTG-inducible promoter with an N-terminal GST tag. We cloned a PreScission Protease (GE Healthcare) cleavage site between the GST tag and MTN1 to allow for specific and efficient cleavage at 4°C. We used affinity chromatography to separate GST-tagged MTN1 from the crude lysate. Then we cleaved used PreScission Protease to cleave the protein and used reverse affinity to chromatography to capture ‘free’ MTN1 in the flow-through. This protein was approximately 90% pure (Figure 4.4). To increase the purity of MTN1 for the purposes of crystallization, we 147 will run the partially purified protein over a size exclusion column. This will also determine if the protein is folded and monodispersed. After attempting many methods, we have made significant progress in optimizing the purification of HopAF1. We used a disorder prediction program, RONN, which revealed that the N-terminus of HopAF1 contains regions of disorder (Yang et al., 2005). These regions of disorder may likely make it difficult to purify large quantities of full-length HopAF1 and may also inhibit crystal formation in the future. Again using Gateway technology, we cloned a 6xHis-MBP (maltose-binding protein) on the N-terminus of HopAF1 truncations and we identified the minimal region of HopAF1 required for activity (see Figure 3.14 and Figure 3.15 in Chapter 3). 6xHisMBP-∆55HopAF1 was insoluble, while 6xHis-∆147HopAF1 was not able to inhibit MTN activity in vitro. However, purification of 6xHis-∆130HopAF1, the residues of HopAF1 that correspond to the putative catalytic domain, was successful (Figure 4.5) and ∆130HopAF1 can inhibit MTN1 activity (see Figure 3.15 in Chapter 3). Therefore, we decided to focus on purifying ∆130HopAF1 for crystallography studies. Our current protocol, similar to the purification scheme of MTN1, uses affinity chromatography was used to capture 6xHisMBP-∆130HopAF1 from crude extract (figure 5A). Then after cleavage with TEV protease, reverse affinity chromatography was used to capture ∆130HopAF1 in the flow-through (Figure 4.5B). Finally, ∆130HopAF1 was run over a size exclusion chromatography column, resulting in a monodispersed peak (Figure 4.5C). Interestingly, the ∆30HopAF1 monodispersed peak elution time suggests that the protein is forming oligomers. We will perform size exclusion chromatography multi-angle light scattering (SEC-MALS) experiments in the UNC Macromolecular Interactions Facility to determine the nature of these oligomers. To start the crystallization trials, we will first confirm that HopAF1 and MTN1 form a stable complex in solution using size exclusion chromatography. Since the crystal structure of MTN1 from Arabidopsis has been solved (Park et al., 2006), we will start with the 148 conditions used for MTN1 and begin the crystallization trials with HopAF1/MTN1 in the UNC Protein Crystallography core facility. 149 Figures Figure 4.1. Alignment and structural localization of MTN1 conserved putative deamidase target residues. A) Alignment of methylthioadenosine nucleosidase amino acid sequences from plants and E. coli. B) Ribbon representation of the MTN1 crystal structure (PDB 2H8G). Monomers are represented in sand and silver. The ligand analog (MTT) is shown in green. The ligand-binding and catalytic residues from MTN1 are represented as blue sticks. The putative conserved targets of deamidation are labeled and colored as red stick representations. Images were generated using Pymol. 150 Figure 4.2. MTN1 and MTN2 variants display loss-of-function phenotypes when residues N113 and N194 are ‘deamidated’. A) MTN1 enzyme specific activity measured in vitro using xanthine oxidase-coupled spectrometric assay. MTN1 catalytic dead mutant is MTN1D225N. B) MTN2 enzyme specific activity measured in vitro using xanthine-oxidase coupled spectrometric assay. MTN2 catalytic dead mutant is MTN2D212N. Letters represent treatments with significant difference according to the post-hoc ANOVA Tukey’s test (p < 0.05). 151 Figure 4.3. Circular dichroism spectra of MTN1 ‘deamidated’ variants does not vary from wild-type MTN1. Circular dichroism spectra were recorded for MTN1 variants in 20 mM sodium phosphate buffer pH 7.4, 150 mM NaF from 185 nm to 260 nm. The differential spectrum was obtained by subtracting the buffer spectrum before conversion to molar ellipticity. 152 Figure 4.4. Purification of wild-type MTN1. A) The construct used to purify MTN1 consists of an N-terminal glutathione S-transferase (GST) tag, followed by a PreScission Protease cleavage site (PP), and the coding sequence of Arabidopsis MTN1 (At4g38800). B) SDSPAGE analysis and Coomassie stain of affinity purification using the GST tag. Fractions 1317 represent the eluted fractions that contain full-length GST-PP-MTN1. C) SDS-PAGE analysis and Coomassie stain of MTN1 after cleavage with PreScission Protease to remove the GST tag. The cleaved protein was run over a fresh GST affinity column to capture the GST and PreScission Protease, allowing MTN1 to be collected in the flow through fractions. Black arrows denote the proteins of interest from each step of the purification. 153 Figure 4.5. Purification of ∆130 HopAF1. A) The construct used to purify ∆130-HopAF1 consists of an N-terminal 6xHis-Maltose Binding Protein (MBP) tag, followed by a TEV Protease cleavage site (TEV), and the coding sequence of P. syringae pv. tomato DC3000 HopAF1. B) SDS-PAGE analysis and Coomassie stain of affinity purification using the 6xHis tag. The eluted fractions contain full-length 6xHisMBP-TEV-∆130HopAF1. C) SDS-PAGE analysis on a 15% gel and Coomassie stain of ∆130HopAF1 after cleavage with TEV Protease to remove the 6xHisMBP tag. 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