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Identification and characterization of two enhancers of the suppressor of npr1, constitutive 1 (snc1) Woloshen, Virginia Ashley 2012

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Identification and characterization of two enhancers  of the suppressor of npr1, constitutiv e 1 (snc1)     by    Virginia Ashley Woloshen   B.Sc., University of British Columbia, 2010     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   The Faculty of Graduate Studies  (Botany)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   December 2012     © Virginia Ashley Woloshen, 2012  ! ""! Abstract  Out of necessity, plants have evolved robust, multi-layered defence responses to combat pathogen attack. Resistance (R) protein-mediated defence is one such layer that provides resistance to pathogens that have successfully overcome plant cell surface defences. R proteins, such as SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1), recognize pathogenic molecules within the plant cell and initiate down-stream defence responses. A gain-of-function mutation in SNC1, snc1, results in constitutive defence responses, dwarf stature and dark green, deformed leaves. Using the snc1 mutant, many MODIFIER OF SNC1 (MOS) genes were found through a snc1 suppressor screen, which identified mutants based on increased size and decreased resistance responses. The wild-type-like phenotype of mos snc1 mutants, allowed another screen to identify enhancers of snc1 termed MUTANT, SNC1 ENHANCING (MUSE) genes. Here I describe the discovery of snc1-4d, an allele of snc1, and muse8, a partial loss-of-function mutant of AtCDC48A . snc1-4d is a unique allele of snc1 in that it possesses a mutation in the Nucleotide-Binding (NB) region of this R-like protein. The enhanced snc1 phenotype observed in this mutant is thought to be due to an increase in SNC1 activity as western blot analysis revealed no increase in snc1 protein level. This novel allele of SNC1 provides a unique opportunity to investigate the nature of the NB region of R proteins and how it may contribute to R protein activation. The partial loss-of-function allele of AtCDC48A, muse8, is also distinctive, as unlike previously characterized alleles, it remains viable when homozygous. This novel allele will be instrumental in the study of AtCDC48A’s many functions and provides the first evidence of AtCDC48’s involvement in plant innate immunity.! """! List of publications #"$%"&"’!()*)+,-&.!/,0’"!10’&%!’&2!3"&!4"!567889!:;<=>"&2"&%!?$)@-"&+!"&!?*’&@! "AA0&"@BC!!"#$%&’(")(*&+,"-.%/!<$@"D*-!EFG!6HIJKHC!! <!?)$@")&!)L!@,"+!’$@"D*-!,’+!>--&!"&D)$?)$’@-2!"&@)!M,’?@-$!8!)L!@,"+!@,-+"+C! "N! Table of contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  i i  Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  i i i  Table of contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  iv List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  vi List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  vii  List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  viii  Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xi Dedication .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  xii  1 Introduction to plant innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1    1.1 Summary .............................................................................................................................. 1    1.2 Plant innate immunity .......................................................................................................... 1    1.3 SNC1, a Resistance-like protein .......................................................................................... 6    1.4 Modifiers of the suppressor of npr1, constitutive 1 (MOS) ................................................. 9     1.5 Concluding remarks and thesis objective .......................................................................... 12 2 Mutant snc1-enhancing (MUSE) screen revealed a novel dominant allele of the suppress of npr1, constitutiv e 1  (snc1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14    2.1 Summary ............................................................................................................................ 14    2.2 Introduction ........................................................................................................................ 14    2.3 Materials and methods ....................................................................................................... 18 2.3.1 Plant growth, mutagenesis, primary and secondary screens .................................. 18 2.3.2 Genetic mapping .................................................................................................... 18 2.3.3 Oomycete infection assay ...................................................................................... 18 2.3.4 Protein extraction and western blot analysis .......................................................... 19 2.3.5 Phylogenetic analysis ............................................................................................. 20    2.4 Results ................................................................................................................................ 21 2.4.1 Mutant 119 mos2 snc1 npr1 carries an intragenic mutation in snc1 ...................... 21 2.4.2 snc1-4d protein stability ......................................................................................... 22 2.4.3 Conservation of the NB domain in R proteins ....................................................... 22    2.5 Discussion .......................................................................................................................... 24 3 A tCDC48A  negatively regulates plant innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  25    3.1 Summary ............................................................................................................................ 25 ! N!    3.2 Introduction ........................................................................................................................ 25    3.3 Materials and methods ....................................................................................................... 29 3.3.1 Plant growth and mutant isolation ......................................................................... 29 3.3.2 muse8 mos2 snc1 npr1 characterization ................................................................ 29 3.3.3 Genetic mapping .................................................................................................... 30 3.3.4 Molecular cloning .................................................................................................. 30 3.3.5 Phylogenetic analyses ............................................................................................ 31 3.3.6 Microarray gene expression analysis ..................................................................... 31    3.4 Results ................................................................................................................................ 33 3.4.1 muse8 mos2 snc1 npr1 characterization ................................................................ 33 3.4.2 muse8 contains a mutation in AtCDC48A  ............................................................. 33 3.4.3 Phylogenetic analysis of AtCDC48A protein ........................................................ 36 3.4.4 Expression analysis of the AtCDC48A family ...................................................... 36    3.5 Discussion .......................................................................................................................... 46 4 Future directions and concluding statements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  52    4.1 snc1-4d ............................................................................................................................... 52    4.2 AtCDC48A ........................................................................................................................ 52    4.3 Concluding statements ....................................................................................................... 54 Bibliography .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  55! N"! List of tables  Table 3.1 Next generation sequence analysis: list of potential candidate mutations in the mapped muse8 region ............................................................................................................................... 39 ! N""! List of figures Figure 2.1 Analysis of mutant snc1-4d ....................................................................................... 23 Figure 3.1 Characterization of muse8 mos2 snc1 npr1 ............................................................... 38 Figure 3.2 Cloning of AtCDC48A .............................................................................................. 40 Figure 3.3 Crosses of T-DNA insertion lines of candidate genes with snc1 .............................. 41 Figure 3.4 Phylogenetic analysis of CDC48A across kingdoms ................................................ 42 Figure 3.5 Phylogenetic analysis of AtCDC48 Arabidopsis orthologs ...................................... 43 Figure 3.6 Microarray expression analysis of AtCDC48 orthologs post-infection .................... 44 Figure 3.7 Microarray expression analysis of AtCDC48 orthologs in tissues and in response to chemical treatment ...................................................................................................................... 45 Figure 3.8 Proposed model of AtCDC48A in protein degradation: regulation of plant innate immunity ..................................................................................................................................... 51! N"""! List of abbreviations  ATP – Adenosine Tri-Phosphate Avr – Avirulence BAK1 – BRASSINOSTEROID ASSOCIATED KINASE 1 BAP1 – BON1 ASSOCIATED PROTEIN 1 BIR1 – BAK1 INTERACTING RECEPTOR-LIKE KINASE 1 BON1 – BONZAI 1 CC – Coiled Coil CDC – CELL DIVISION CYCLE Cf – CLADOSPORIUM FULVUM RESISTANT CIP29 – CYTOKINE INDUCIBLE PROTEIN 29 Col-0 – Columbia-0  CPR1 – CONSTITUTIVE EXPRESSER OF PR GENES 1 E – Glutamic Acid EDS1 – ENHANCED DISEASE SUSCEPTIBILITY 1 EFR – EF-TU RECEPTOR EMS – Ethyl Methanesulfonate ERAD – Endoplasmic-Reticulum-Associated Degradation ETI – Effector-Triggered Immunity ETS – Effector-Triggered Susceptibility flg22 – flagellin conserved domain 22 FLS2 – FLAGELLIN SENSITIVE 2 GPKOW/T54 – human MOS2 homolog  GUS – beta-Glucuronidase H.a. Noco2 – Hyaloperonospora arabidopsidis Noco2 HR – Hypersensitive Response K - Lysine Ler – Landsberg erecta MAC – MOS4 ASSOCIATED COMPLEX MAPK – MAP-KINASE MKP1 – MAP KINASE PHOSPHATASE 1 MLA10 – MILDEW A 10 ! "O! MOS – MODIFIER OF SNC1 MUSE – MUTANT SNC1 ENHANCING N - NICOTIANA N terminal – amino terminal NAHG – NAPHTHALENE/SALICYLATE HYDROXYLASE G NB-LRR – Nucleotide-Binding Leucine Rich Repeat NDR1 – NON-RACE SPECIFIC DISEASE RESISTANCE 1 NIM1 – NONINDUCIBLE IMMUNITY 1 NOD – Nucleotide-binding Oligomerization Domain Npl4 – NUCLEAR PROTEIN LOCALIZATION 4 NPR1 – NONEXPRESOR OF PATHOGENESIS RELATED 1 Nup – NUCLEAR PORE PROTEIN PAD4 – PHYTOALEXIN DEFICIENT 4 PAMP – Pathogen Associated Molecular Pattern PKA – PROTEIN KINASE A PR – PATHOGENESIS RELATED Prf – PSEUDOMONAS RESISTANCE AND FENTHION SENSITIVITY PRR – Pathogen Recognition Receptors PRL1 – PLEIOTROPIC REGULATORY LOCUS 1 P.s.m. – Pseudomonas syringae pv. maculicola PTI – PAMP-TRIGGERED IMMUNITY R proteins – Resistance proteins RAD23 – RADIATION SENSITIVE 23 RIN4 – RPM1 INTERACTING PROTEIN 4 RLK – Receptor-Like Kinase RPM1 – RESISTANCE TO PSEUDOMONAS SY RINGAE PV. MACULICOLA RPS4 – RESISTANCE TO PSEUDOMONAS SY RINGAE SA – Salicylic Acid SAG101 – SENESCENCE ASSOCIATED GENE 101 SAI – SA INSENSITIVE SAR – Systemic Acquired Resistance SCF – SKP1-CULLIN-F-BOX SGT1 – SUPPRESSOR OF THE G2 ALLELE OF SKP1 ! O! SNC1 – SUPPRESSOR OF NPR1, CONSTITUTIVE 1 SRFR1 – SUPPRESSOR OF rps4-RLD) STAND – Signal Transduction ATPases with Numerous Domains T3SS – Type 3 Secretion System T4SS – Type 4 Secretion System TIR – Toll/Interleukin-1 Receptor Ub – Ubiquitin UBA – Ubiquitin Associating UFD – UBIQUITIN FUSION DEGRADATION VCP – VALOSIN CONTAINING PROTEIN ! O"! Acknowledgements  First and foremost I would like to thank Dr. Xin Li, without whom this project would not have been possible. Her guidance, patience and expertise taught me how to successfully execute and analyze scientific procedures specifically in the field of molecular genetics. She took a chance on a new student with limited experience, guided me and supported me financially through research assistantships for which I am truly grateful. I will miss our philosophical chats and her little pearls of wisdom. I would also like to thank all of the members of the Li Lab. Their assistance and friendship has been indispensable. I would like to especially thank two visiting professors Dr. Jin Zhao and Dr. Shitou Xia who taught me many aspects of molecular biology. Special thanks also goes to Kaeli Johnson whose friendship has helped guide me through both the fun and challenging times of graduate school. To my committee members Dr. Ljerka Kunst, Dr. Carl Douglas and my academic advisor Dr. Quentin Cronk, I would like to extend the sincerest of thanks for their advice in and out of committee meetings. You’ve provided me with both academic and emotional support.  Thank you to the Botany Department for their financial support through teaching assistantships. Special thanks to Dr. George Haughn, Dr. Craig Berezowski, and Dr. Hugh Brock (Genetics); Dr. Mary Berbee, Dr. Clemtent Tsui, and Jacklyn Dee, MSc. (Botany) for being inspirational teaching supervisors.  To my family Mom, Dad and Sonya, without your love, support and patience this project would not have been possible. You are inspirational but also keep me grounded. I would like to thank my friends, who have become more of an extended family. And a special thank you to my loving partner Floyd, who has weathered the storm through the completion of my work.! O""! Dedication      for My Family  ! 8! 1 Introduction to plant innate immunity1 1.1  Summary  Each cell in a plant is capable of mounting a defence in response to pathogen attack. Recognition of pathogens at the plant cell surface elicits a signalling cascade that leads to expression of defence genes. However, some pathogens have evolved mechanisms to circumvent this initial response through the injection of effector molecules that inhibit such pathways. Plants, in turn, have evolved Resistance (R) proteins that act as internal surveillance systems that recognize these effectors and again initiate a down-stream defence response. In this chapter I will discuss our current understanding of the plant innate immune system, primarily focused on R protein mediated defence. I also discuss a specific R-like protein SNC1 and its regulation in plant innate immunity. 1.2 Plant innate immunity  The robust immune system in plants has evolved out of necessity due to their sessile nature. The first barrier a pathogen must overcome is the plant surface. This is comprised of rigid cell walls, epicuticular waxes, and trichomes that serve as deterrents and prevent some pathogens from entering the cell. Some pathogens can circumvent these primary defences; however, they then must overcome plant innate immunity present in each plant cell. Conserved features of pathogens or Pathogen Associated Molecular Patterns (PAMPs) such as chitin or flagellin, of fungi and bacteria respectively, are recognized by Pathogen Recognition Receptors (PRRs), transmembrane proteins present on the plant cell surface (Jones and Dangl, 2006). Many PRRs are of the Receptor-Like Kinase (RLK) variety, they possess a transmembrane domain, a !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 8!<!?)$@")&!)L!@,"+!D,’?@-$!,’+!>--&!?0>*"+,-2C!#"$%"&"’!()*)+,-&.!/,0’"!10’&%!’&2!3"&!4"C! 567889!E&G!!"#$%&’(")(*&+,"-.%/!<$@"D*-!EFG!6HIJKHC!! 6! cytoplasmic Serine/Threonine protein kinase domain and variable amino (N) terminal domains on the cell surface that confer PAMP specificity. FLS2 (FLAGELLIN SENSITIVE 2), for example, recognizes flagellin from bacteria and can also be induced by flg22 alone, the conserved domain found in flagellin (Felix et al., 1999; Gómez-Gómez and Boller, 2000). EFR (EF-TU RECEPTOR), another PRR, recognizes the bacterial PAMP EF-Tu (Kunze et al., 2004; Zipfel et al., 2006). Subsequent to pathogen detection, map-kinase (MAPK) signalling cascades lead to defence responses, such as callose deposition at the cell walls, stomata closure, production of reactive oxygen species and other defence compounds, such as salicylic acid (SA) and transcription of PATHOGENESIS RELATED (PR) genes (Nürnberger et al., 2004). This initial response is termed PAMP-Triggered Immunity or PTI (Jones and Dangl, 2006).  Successful pathogens have evolved effector molecules, which are secreted by the pathogen into the apoplast to overcome PTI. In some cases effectors can be released directly into the plant cell. Bacterial pathogens often use the Type Three Secretion System (T3SS) or the Type Four Secretion System (T4SS) to accomplish this. The T3SS is a syringe-like structure encoded by the bacteria, which is assembled on the surface of the plant and subsequently injects effectors into the hosts cytoplasm (Jin and He, 2001). The T4SS delivers effectors through bacterial membrane-associated proteins (Backert and Meyer, 2006; Cascales and Christie, 2003). Nematodes employ the use of a stylet, a different syringe-like structure that injects effectors into the host (Grundler et al., 1998). Effectors of oomycetes and fungi, on the other hand, contain translocation signals and are recognized by receptor proteins on the plant cell surface, or directly bind the plant cell surface, and enter the plant cell through endocytosis (Whisson et al., 2007). Once inside the plant cell, these effectors serve to inhibit PTI (Jones and Dangl, 2006).  ! P! Inhibition of PTI via effectors can occur in a multitude of ways. AvrPtoB of Pseudomonas syringae (P.s.), for example, is similar to plant E3 ligases and possesses E3 ligase activity (Abramovitch et al., 2006; Janjusevic et al., 2006). As an E3 ligase it is hypothesized that AvrPtoB targets positive regulators of resistance for degradation. Another P.s. effector AvrPto has been shown to prevent the phosphorylation of FLS2 and EFR by binding their respective phosphorylation sites thus preventing downstream signalling cascades (Xiang et al., 2008). Both AvrPto and AvrPtoB target BAK1 (BRASSINOSTEROID ASSOCIATED KINASE 1), the co-receptor to various PRRs. They also inhibit chitin signalling, which is not associated with BAK1 function, suggesting multiple targets for these effectors (Shan et al., 2008).  MAPKs can also be directly targeted by effector molecules, for example, Hop1A of P.s. targets both MPK3 and MPK6 (Zhang et al., 2007). Some effectors can mimic natural plant hormones that inhibit the defence response. For example, various pathovars of P.s. produce coronatine, an analog of jasmonic acid. The plant perceives this chemical as jasmonic acid and uses the jasmonic acid wound pathway to halt the production of SA (Zhao et al., 2003; Brooks et al., 2005). Introduction of coronatine to the host plant also leads to stomata opening, facilitating the entry of the bacteria (Melotto et al., 2006).  Therefore PTI can be inhibited at various steps in the signalling cascade and ultimately lead to Effector-Triggered Susceptibility (ETS) (Jones and Dangl, 2006).  Plants have in turn developed mechanisms to combat ETS through Resistance (R) proteins that recognize pathogen effectors and initiate downstream defence responses. The most abundant R proteins belong to the NB-LRR cohort named for their nucleotide-binding (NB) and leucine-rich-repeat (LRR) motifs. They are similar to mammalian proteins used in inflammatory and immune responses termed NOD (Nucleotide-binding Oligomerization Domain) LRRs ! Q! (Inohara et al., 2005). The NB domain contains motifs reminiscent of Signal Transduction ATPases with Numerous Domains (STAND), which are often used in activation (Tameling et al., 2002). The LRR domain is involved in the regulation of intramolecular interactions and recognition specificity (Moffett et al., 2002; Dodds et al., 2001). In plants there are two main categories of NB-LRR R-proteins based on their N-terminal domain. TIR (Toll/Interleukin-1 Receptor) NB-LRR and CC (coiled-coil) NB-LRRs, which are both involved in the defence response following effector recognition; however, they have distinct downstream signalling pathways. CC-NB-LRRs use the NON-RACE SPECIFIC DISEASE RESISTANCE 1 (NDR1) pathway following effector recognition (Aarts et al., 1998). TIR-NB-LRRs, on the other hand, use the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) paired with either PHYTOALEXIN DEFICIENT 4 (PAD4) or SENESCENCE ASSOCIATED GENE 101 (SAG101) (Aarts et al., 1998; Feys et al., 2005).   Effectors that are recognized by R proteins are termed Avirulence (Avr) proteins.  NB- LRR R proteins recognize the presence of effectors in various ways. One widely accepted hypothesis is the guard hypothesis in which the R protein senses the effector indirectly by monitoring an endogenous plant protein that is targeted by the effector (Van der Biezen and Jones, 1998). For example, RPM1 (RESISTANCE TO PSEUDOMONAS SY RINGAE PV. MACULICOLA ) is a CC-NB-LRR R protein that interacts with RIN4 (RPM1 INTERACTING PROTEIN 4), a protein that localizes to the plasma membrane. Various Avr proteins target RIN4. AvrRPM1 and AvrB are examples of effectors secreted by P.s. using the T3SS. Upon release into the host plant cell, these Avr proteins are acetylated and targeted to the plasma membrane (Nimchuk et al., 2000). Effectors then interact with RIN4, which is subsequently phosphorylated (Boyes et al., 1998; Mackey et al., 2002). RPM1 also localizes to the plasma ! R! membrane and has been found to interact directly with RIN4 (Boyes et al., 1998). Upon RIN4 phosphorylation RPM1 is activated and leads to downstream defence responses. Another Avr protein that targets RIN4 is AvrRpt2. This effector molecule cleaves the C-terminal of RIN4 targeting it for protein degradation (Kim et al., 2005). The absence of RIN4 at the plasma membrane is sensed by RESISTANCE TO PSEUDOMONAS SYRINGAE 2 (RPS2), another R protein that is indirectly activated by pathogen effectors and initiates a defence response (Axtell and Staskawicz, 2003).  R protein mediated defence responses are characterized by a type of programmed cell death termed the Hypersensitive Response (HR). HR begins by a sudden burst of reactive oxygen species. These reactive oxygen species can interact with other molecules/organelles within the cell, which can induce their degradation (Berlett and Stadtman, 1997; Foyer and Noctor, 2005; Breusegem and Dat, 2006). The accumulation of SA is yet another response. This has been shown to bind catalases, which convert reactive oxygen species such as H2O2 to harmless forms like H2O and O2, and prevent their action (Wendehenne et al., 1998; Conrath et al., 1995). SA also induces certain MAPKs involved in plant innate immunity (Zhang and Klessig, 1997; Romeis et al., 1999). PR gene expression is also affected by elevated levels of SA. NPR1 (NONEXPRESSOR OF PATHOGENESIS RELATED 1), NIM1 (NONINDUCIBLE IMMUNITY  1), and SAI (SA INSENSITIVE) have been shown to require SA to induce downstream PR gene expression and mount a defence response (Cao et al., 1994; Glazebrook et al., 1996; Delaney et al., 1995; Shah et al., 1997). If successfully induced, this type of cell death can halt the invasion of pathogens. By killing infected cells, the plant prevents pathogen spread to neighbouring cells: the sacrifice of some to preserve the whole. Once HR has been ! J! established, the plant attains Systemic Acquired Resistance (SAR), which increases whole plant resistance to future pathogen attacks.   The innate immunity in plants must be highly regulated. Mis-regulation can result in a decrease in plant fitness. For example, a gain-of-function mutation in the R-like protein SUPPRESSOR OF NPR1, CONSTITUTIVE 1 (SNC1), results in heightened disease resistance; however, it also results in dwarf stature and deformed leaves (Li et al., 2001; Zhang et al., 2003). Various mechanisms have been suggested for such regulation. In the cytosol, R proteins are thought to be present in their folded, inactive form (Ade et al., 2007). A CC-NB-LRR protein of potato indicated that the CC domain interacts with the NB-LRR domains in the absence of pathogens; however, upon infection these interactions cease (Moffett et al., 2002). Conversion of R proteins from the folded, inactive form to their less folded, active form is thought to be ATP- dependant. Following this conformational change, R proteins dimerize and it is this dimerization that activates downstream defence responses (Bernoux et al., 2011; Maekawa et al., 2011). In certain cases, R proteins must be shuttled to the nucleus for successful defence responses. The nuclear localization of RPS4 of Arabidopsis, NICOTIANA (N) of tobacco and MILDEW A 10 (MLA10) of barley is required for successful resistance to their respective pathogens (Shen et al., 2007; Burch-Smith et al., 2007; Wirthmueller et al., 2007). Lack of tight regulation can have detrimental effects that affect plant fitness, as we see in mis-regulated snc1 (Zhang et al., 2003; Li et al., 2001). 1.3 SNC1, an R-like protein  SNC1 belongs to the TIR-NB-LRR class of R proteins; however, it is sometimes called an R-like proteins since its corresponding effector molecule or molecules have not yet been identified. It was isolated during a suppressor screen designed to identify mutants with enhanced ! H! disease resistance (Li et al., 2001). Arabidopsis thaliana plants, of the Columbia (Col-0) ecotype, containing a loss-of-function mutation in NPR1 were used as a background for this screen as they display enhanced disease susceptibility due to the absence of PR gene expression typically induced by functional NPR1 proteins (Cao et al., 1997). The mutation found in snc1 npr1 plants caused a gain-of-function in that double and single mutant plants had increased and constitutive resistance to pathogens, PR gene expression and SA accumulation; however, unlike the wild- type R-mediated defence response, no HR was observed (Li et al., 2001). This increase in defence responses however, also causes a dwarf, dark green, curly-leaved phenotype exemplifying the consequences of mis-regulation in R-protein mediated defence responses.  Many regulators of SNC1 have been identified (Gou and Hua, 2012; Johnson et al., 2012). BON1 (BONZAI 1) along with its functional partner BAP1 (BON1 ASSOCIATED PROTEIN 1) negatively regulates SNC1. bon1 and bap1 single mutants display a snc1-like phenotype. SNC1 transcript was found to be significantly higher in bon1 mutants and over- expression of this protein has been shown to exhibit auto-immune phenotypes (Li, et al., 2010b).  Since BON1 and BAP1 are plasma membrane localized, they are thought to accomplish such regulation indirectly (Yang et al., 2006; Yang and Hua, 2004). Typically, SNC1 activation eventually leads to the accumulation of SA, which in turn up-regulates defence gene expression, including SNC1 gene expression creating a feedback loop. When bon1 mutants contain a mutation in naphthalene/salicylate hydroxylase G (nahG) or pad4, mutants that inhibit SA synthesis or accumulation, the increase in SNC1 transcript is abolished (Yang and Hua, 2004). Therefore, negative regulation of SNC1 by BON1 is achieved indirectly through the SA feedback loop, which affects the transcription of SNC1.  BON1 interacts with both BIR1 (BAK1 INTERACTING RECEPTOR-LIKE KINASE 1) and BAK1 (Wang et al., 2011). These proteins ! I! are thought to be guarded by multiple R proteins that recognize changes in BIR1 or BAK1 and induce defence responses. Therefore, when mutated in BAK1 or BIR1, mutant plants display an autoimmune phenotype. This can be partially rescued by introducing a loss-of-function mutation in snc1, confirming BAK1 and BIR1 are also indirect, negative regulators of SNC1 (Wang et al., 2011). Another indirect negative regulator of SNC1 is MKP1 (MAP KINASE PHOSPHATASE 1). Again, mkp1 mutants display a snc1-like phenotype and can be partially rescued with the introduction of a snc1 loss of function allele, suggesting involvement in plant innate immunity. Mutant mkp1 phenotypes are dependent on MPK3 and MPK6, crucial components of PTI (Asai et al., 2002). The mkp1 phenotype was also dependent on SA accumulation; however, no change in SNC1 transcript was detectible suggesting MKP1 may negatively regulate SNC1 post- transcriptionally (Bartels et al., 2009).  CONSTITUTIVE EXPRESSOR OF PR GENES 1 (CPR1) is yet another negative regulator of SNC1. This protein is an F-box protein associated with the SCF (SKP1-CULLIN-F- BOX) protein complex, which directs target proteins to the 26S proteasome for degradation, and has been implicated in R protein regulation (Cheng et al., 2011).  Similar to bon1, bap1, bir1 and bak1, cpr1 mutants display constitutive defence responses. A lack of this protein in the SCF complex prevents degradation of certain target proteins, one of which is thought to be SNC1, since cpr1 mutants have increased SNC1 protein accumulation. Over-expression of CPR1 rescues the mutant phenotypes of the gain-of-function snc1 mutant as well as bon1, and decreases the amount of SNC1 protein (Gou et al., 2012). Altogether these results indicate CPR1 is a negative regulator of SNC1, which is accomplished through SNC1 degradation by the 26S proteasome.  ! K!  SNC1 accumulation is also regulated by SRFR1 (SUPPRESSOR OF rps4-RLD). Mutant srfr1 show an increase in SNC1 protein and therefore display the same autoimmune phenotype previously discussed. However, there is no observable change in snc1 transcript levels, therefore it is thought that SRFR1 regulates SNC1 by stabilizing the protein. Autoimmunity in srfr1 mutants can be suppressed by loss of function mutations in snc1 further confirming the role of SRFR1 in SNC1 regulation (Li, et al., 2010b). A similar role is proposed for SGT1 (SUPPRESSOR OF THE G2 ALLELE OF SKP1), which interacts with SRFR1, indicating that SRFR1 and SGT1 may act in a complex to regulate SNC1 stability (Li, et al., 2010b).  SNC1 is regulated at the DNA level by MOS1 (MODIFIER OF SNC1, 1). Mutant mos1 plants were found to repress the transcription of snc1, the gain-of-function mutant allele, which suppressed the constitutive phenotype of snc1. Upon further investigation, mos1 mutants possessed a change in DNA methylation upstream of the SNC1 gene, indicating MOS1 is crucial for transcriptional regulation of the SNC1 gene (Li et al., 2010a). 1.4 Modifiers of the suppressor of npr1, constitutive 1 (MOS)  In an attempt to identify novel components of the SNC1-mediated defence pathway, a mutant screen was conducted using snc1 gain-of-function mutants. This screen revealed fifteen possible MOS genes contributing via various ways to plant defence (Monaghan et al., 2010). MOS1, as previously discussed, was found to be involved in DNA methylation, and therefore transcription of SNC1 (Li et al., 2010a). Following transcription, RNA must be processed prior to export into the cytoplasm and translated. MOS2 and MOS4 have both been implicated in RNA processing. Mutations in MOS2 suppress the snc1 phenotype, PR gene expression is completely abolished, as is constitutive resistance to pathogens. SA accumulation; however, is only partially suppressed, suggesting that MOS2 may be involved in the SA-independent pathway. The MOS2 ! 87! protein localizes to the nucleus and contains one G-patch and two KOW domains, which function in nucleotide and protein binding. Altogether, these results suggest that MOS2 plays a role in RNA processing (Zhang et al., 2005). Recently a study was conducted using a MOS2 homolog, GPKOW/T54, from humans. This study revealed that T54 interacts in vitro with PROTEIN KINASE A (PKA), and confirmed the putative binding of RNA (Aksaas et al., 2011). This finding along with previous studies implicating PKA in RNA splicing, suggests that T54 also seems to be contributing to RNA splicing (Kvissel et al., 2007). Therefore, MOS2 could be an RNA-binding protein putatively involved in pre-mRNA splicing.  MOS4 is another nuclear protein involved in RNA processing. It was found to interact with several other components, transcription factor AtCDC5 (CELL DIVISION CYCLE 5), a WD-40 protein PRL1 (PLEIOTROPIC REGULATORY LOCUS 1), and E3 ubiquitin ligases MAC3A and MAC3b (MOS4-ASSOCIATED COMPLEX) (Palma et al., 2007; Monaghan et al., 2009). Mutations in all components display defects in plant defence. Together these proteins comprise the MOS4-Associated Complex (MAC). This complex is homologous to the Nineteen Complex found in humans and yeast (Ajuh et al., 2000; Ohi and Gould, 2002). This complex associates with the spliceosome and it is therefore thought that its Arabidopsis homolog, MAC is also involved in splicing (Hogg et al., 2010; Monaghan et al., 2009).  Subsequent to transcription and RNA splicing, mature RNA must then be transported to the cytoplasm. This is accomplished through shuttling mRNA to the nuclear envelope and exit through a nuclear pore complex. MOS3 and MOS7 both suppress the snc1 phenotype and resistance when mutated and both localize to the nuclear envelope (Zhang and Li, 2005; Cheng et al., 2009). MOS3 is a homolog of Nup96 of human and C-Nup145p of yeast, which contribute to the Nup107-160 sub-complex involved in nuclear pore stability and export (Wiermer et al., ! 88! 2007). MOS7, on the other hand, is homologous to Nup88, which has been implicated in nuclear protein retention (Roth et al., 2003). These studies suggest MOS3 and MOS7 are associated with the nuclear pore complex and therefore, protein export required for plant innate immunity. Shuttling of mature mRNA to nuclear pores must occur for export to take place. Recently, MOS11 was identified as an export protein with homology to the RNA binding protein CYTOKINE INDUCIBLE PROTEIN 29 (CIP29) of humans, which binds RNA (Lemieux and Bachand, 2009). mos11 mutants have impaired resistance to pathogens in the snc1 background and only partially suppress the snc1 phenotype. Mutants also exhibit accumulation of polyA mRNA in the nucleus, indicating that export is impaired in mos11 mutants (Germain et al., 2010). MOS11 is, therefore, implicated in mRNA shuttling to the nuclear envelope for export. Nucleo-cytoplasmic trafficking also takes place in the other direction. Transcription factors and co-factors must be imported into the nucleus for function. MOS6 encodes an !-3 importin, which functions with " importins in transporting proteins with nuclear localization signals (Goldfarb et al., 2004). The suppression of snc1 phenotypes is only partial in mos6 mutants; this is most likely due to functional redundancy as MOS6 is strikingly similar to another importin IMP!6 (Palma et al., 2005). Once exported, mRNA is translated and undergoes post-translational modifications to result in a fully functional protein. MOS5 and MOS8 are involved in such processes. MOS5 encodes a ubiquitin (Ub) activating (UBA) enzyme or E1 enzyme (Goritschnig et al., 2007). These enzymes activate ubiquitin, a ubiquitous protein typically used in marking proteins for degradation. Activated Ub is then transferred to an E2 enzyme, which brings the activated Ub to an E3 ligase that determines target specificity and, finally, transfers the Ub to the target (Chau et al., 1989; Hershkos et al., 1983; Reiss et al., 1989). Poly-ubiquitination results in the degradation ! 86! by the proteasome and has been shown to be important in plant defence (Cheng and Li, 2012). Mutant mos5 snc1 plants suppress the snc1 phenotype and impair disease resistance indicating the importance of MOS5 in innate immunity (Goritschnig et al., 2007). Another post- transcriptional modification is farnesylation. This modification is implicated in protein trafficking and membrane targeting (Resh, 2006). MOS8 was found to encode the " subunit of farnesyltransferase, which when mutated, displays enhanced susceptibility and impaired R protein mediated signalling (Goritschnig et al., 2008). Although the target of MOS8 is unknown, it exemplifies the importance of protein modification in plant innate immunity. 1.5 Concluding remarks and thesis objective  Plants and their pathogens have been co-evolving for millions of years. Successful infection of plants can be devastating not only to the plant, but also humans, who rely on plants for food, shelter, revenue and oxygen. We have already witnessed the catastrophic effects of when pathogenic disease overcomes plant immunity. The Irish potato famine caused the death of an estimated 1 million people, and the emigration of countless more in the mid-19th century. It was caused by the potato blight fungus Phytophthora infestans, which remains a problem to this day (Haverkort et al., 2009). Currently, we are facing new challenges. Our monoculture farming practices make our crop species more vulnerable, as one infection may destroy the entire crop due to low genetic diversity. Furthermore, with the onset of climate change, habitats of pathogens will expand. Plants that have never been exposed to certain pathogens and therefore never evolved resistance to such pathogens, will become their new hosts (Olesen and Bindi, 2002; Roos et al., 2010). It is therefore in our best interest to investigate the nature of plant innate immunity to avoid, or better respond, to future plant pathogen outbreaks. Although plant ! 8P! innate immunity has been the focus of many studies, there are still considerable gaps in our knowledge.  Our lab has sought to utilize molecular genetic analyses in the snc1 background to dissect the plant immune systmes. The objective of my thesis was to identify novel regulators in the defence against pathogens using the snc1 background to further our understanding of the complex plant innate immune system. More specifically, I aimed to identify novel negative regulators of plant immunity using a genetic approach by identifying enhancers of snc1. ! 8Q! 2 Mutant snc1-enhancing (MUSE) screen revealed a novel dominant allele of snc1 2.1 Summary  Each plant cell possesses a multi-layered innate immune system, which serves to protect it from pathogen attack. Some pathogens have evolved mechanisms to overcome initial plant defence responses through the injection of pathogen effector molecules into the host plant cell. However, plants have evolved R proteins, which recognize these effectors, and again mount a defence response. SNC1 is one such R protein tha,t when mis-regulated, exhibits constitutive defence responses. To elucidate the SNC1 defence pathway a genetic screen was conducted, which identified genes that, when mutated, either fully, or partially, suppress the snc1 phenotype called MOS genes. MOS2 was one such gene found during this screen that fully suppresses the dominant snc1 phenotype. The mos2 mutation was then used in the snc1 background to screen for enhancers of snc1. During this screen, several alleles of snc1 were found, one of which, snc1- 4d had a mutation in the NB domain of the SNC1 protein. This allele has increased PR gene expression and heightened resistance to pathogens. Unlike other snc1 alleles that enhance snc1, snc1-4d does not increase SNC1 protein stability. Therefore, it is thought that the increased resistance observed in this allele is due to self-activation of SNC1. 2.2 Introduction  Like humans, plants have evolved complex inducible mechanisms to combat pathogen attack. Typically, plant innate immunity begins with the recognition of PAMPs, conserved features of pathogens, by PRRs located on the plant cell surface (Zipfel, 2008). This PTI may prevent pathogenesis. However, the pathogen may be capable of circumventing this defence by releasing effector molecules into the host-plant cell, which lead to ETS allowing pathogenesis to ! 8R! continue. Plants however, have evolved mechanisms to counteract ETS via R proteins, which can recognize pathogen effectors and lead to ETI (Jones and Dangl, 2006). Recognition of effectors can induce a signal transduction pathway, which leads to PR gene expression, accumulation of SA, production of reactive oxygen species and initiation of localized cell death or HR, which prevents the spread of pathogens to neighbouring cells (Nürnberger et al., 2004; Jones and Dangl, 2006).   Elucidating R-protein-mediated defence has been the focus of the research in our Lab for the past decade. One R-like protein studied extensively in our lab is SNC1 gene, which was identified in the mutant npr1 background (Zhang et al., 2003; Li et al., 2001). NPR1 is induced during pathogen attack and essential for the SA-dependent pathway of plant defence (Cao et al., 1994). Mutant npr1 plants are more susceptible to pathogen attack due to the inactive SA- dependent defence pathway upon infection. The point mutation found in snc1 suppresses the npr1 phenotype regardless of the presence of pathogens, and results in increased SA accumulation, dwarf plant stature and dark green, curly leaves. This gain-of-function mutant has become a useful resource in conducting further genetic screens due to its obvious phenotypes. snc1 also illustrates the importance of regulating defence pathways, as a lack of regulation can result in unfavourable phenotypes, and reduce plant fitness possibly due to the reallocation of resources.  Recently, a group of fifteen genes, termed MOS genes, has been identified through mutant screens in our lab using snc1 or snc1 npr1 mutant plants (Monaghan et al., 2010). These suppressor screens were conducted in an attempt to identify genes that modify the snc1 phenotype and play regulatory roles in ETI. Over ten of these MOS genes were identified by their either full, or partial, suppression of snc1 phenotypes, and found to be involved in various ! 8J! processes of the plant defence response, including epigenetic gene regulation, RNA processing, protein modification, and nucleocytoplasmic trafficking (Zhang et al., 2005; Zhang and Li, 2005; Goritschnig et al., 2007, 2008; Wiermer et al., 2007; Cheng et al., 2009; Germain et al., 2010; Palma et al., 2005).  MOS2 was identified in the snc1 npr1 screen. The mos2 mutation suppresses the dominant snc1 phenotype and completely abolishes disease resistance in mos2 snc1 npr1 mutants. Triple mutants are susceptible to both virulent pathogens, Hyaloperonospora arabidopsidis Noco2 (H.a. Noco2) and Pseudomonas syringae pv. maculicola ES4326 (P.s.m. ES4326). mos2 snc1 npr1 triple mutants also restore wild-type like plant morphology. GUS staining was used to assess the level of PR2 gene expression, which is positively associated with plant resistance against pathogens, and indicative of a functional SA-independent plant defence pathway. PR2 expression is high in snc1 plants; however, mos2 abolishes PR2 gene expression. SA accumulation was also assessed to determine if the high level of SA found in snc1 is also suppressed in mos2 snc1 npr1 triple mutants, and was indeed found to be suppressed; however, only partially. MOS2 is therefore thought to be involved in the SA-independent plant defence pathway (Zhang et al., 2005). The restoration of wild-type-like phenotypes associated with the introduction of the mos2 mutation makes mos2 snc1 npr1 plants useful in further genetic screens to elucidate snc1-mediated plant defence responses and regulation.  Due to the pronounced dwarfism of snc1, constitutive resistance to pathogens and increased PR gene expression, it is difficult to identify genes that enhance snc1. The difference in size, resistance and PR gene expression would be difficult to score between snc1 and mutant- snc1 enhancer (muse) snc1 double mutants. Therefore, maintaining the mos2 mutation, which suppresses snc1 phenotypes, in the mutant background would facilitate successful identification ! 8H! and characterization of muse mos2 snc1 npr1 mutant plants. We hypothesize that the introduction of a mutation in a muse gene will enhance the snc1 phenotype and effectively abolish mos2 wild-type-like phenotypes. Therefore, muse mos2 snc1 npr1 mutant plants should resemble snc1 mutant plants and allow the identification and characterization of negative regulators of plant innate immunity. ! 8I! 2.3 Materials and methods 2.3.1 Plant growth, mutagenesis, primary and secondary screens  Plants for most experiments were grown on soil with a 16 hour light/8 hour dark cycle with a light intensity of 80 µE m-2 sec-1. The mos2 snc1 npr1 triple mutant was previously isolated (Zhang et al., 2005). Ethyl methanesulfonate (EMS) mutagenesis was carried out using 0.2% EMS for 18 hours on 40,000 Arabidopsis thaliana seeds homozygous for the mos2, snc1, and npr1 mutations as well as the BGL2-GUS reporter gene as previously described (Bowling et al., 1994). For the first primary screen, potential mutants chosen based on their snc1-like morphology. The second primary screen identified potential mutants based on increased PR2 expression visualized by GUS-staining as previously described (Zhang et al., 2003). Mutants obtained from the primary screens were subjected to a secondary screen testing resistance to the oomycete pathogen H.a. Noco2 as previously described (Li et al., 2001). Dwarf, snc1-like plants were chosen for further analysis. 2.3.2 Genetic mapping   Selected quadruple mutant plants in the Columbia-0 background were crossed with the Landsberg erecta (Ler) ecotype. The F2 generations were grown on soil and snc1-like but smaller than snc1, plants were selected for linkage analysis. Crude mapping was conducted on 24-48 plants depending on the mutant. Mutant 119 was narrowed down to a region on chromosome 4 near snc1. 119 DNA was Sanger sequenced by Nucleic Acid Protein Service Unit (NAPS) to determine the identity of any additional mutations in snc1. 2.3.3 Oomycete infection assay   snc1-4d mos2 snc1 npr1 quadruple mutants and the controls mos2 snc1 npr1, snc1 npr1, and wild-type plants were grown on soil. Seedlings were spray-inoculated onto adaxial leafe ! 8K! surfaces with Hyaloperonospora arabidopsidis Noco2 using spray bottle containing a cell suspension of 50,000 spores/mL (Li et al., 2001). Inoculum was obtained from infected wild- type Col-0 plants. Inoculated plants were covered with a clear plastic dome and sealed. Infection was assessed through spore counting 7-14 days post-inoculation by cutting 20 randomly selected plants from each genotype and placed in 2 mL eppendorf tubes (5 plants per tube). 1 mL of ddH2O was added and the tube vortexed to dislodge oomycete spores. Spores were counted using a hemocytometer, and by scoring the average percent spore coverage per plant of twenty randomly chosen plants from each genotype. Percent spore coverage was scored using the following: 0 spores per leaf = 0, less than 5 spores per leaf = 1, more than 5 spores per leaf = 2, less than 5 spores per 2 leaves = 3, more than 5 spores per 2 leaves = 4, and more than 5 spores per more than 2 leaves = 5. 2.3.4 Protein extraction and western blot analysis   Protein extraction and western blots were conducted by Yan Huang and Xuejin Chen. Total protein was extracted from 100mg of plant tissue from 10-14 day old plate-grown plants as previously described (Cheng et al., 2011). Briefly, collected plants were placed into 2mL tubes along with two 5mm glass beads and frozen in liquid nitrogen. Tissue was ground into a fine powder and placed on ice. Tissue was suspended in extraction buffer (100mM Tris-HCl pH8, 0.1% SDS, 2% "-mercaptoethonol) and spun for 5 mins at 5000 RPM in a 4°C microcentrifuge. The supernatant was placed in a 1.5 mL tube, boiled at 100°C for 5 mins then 4 x SDS loading buffer added. Samples were separated with SDS-PAGE and electro-blotted onto a nitrocellulose membrane. SNC1 protein was detected using an anti-SNC1 antibody followed by an anti-rabbit secondary antibody conjugated to horse radish peroxidase. The membrane was then treated with ! 67! SuperSignal West Chemiluminescent substrate (Thermo) and horse radish peroxidase substrate, then exposed to photographic film to visualize the SNC1 protein (Li, et al., 2010b).  2.3.5 Phylogenetic analysis   The SNC1 protein sequence was obtained from PubMed Protein and used to conduct a BLASTP search for homologs in Arabidopsis thaliana. Homologous sequences were chosen based on low e-values and the quality of the reference sequences. Protein alignment was achieved using Muscle (Edgar, 2004). The alignment sequence was then manually adjusted using Se-Al (Rambaut, 2002). Conserved amino acid residues were found using JalView (Waterhouse et al., 2009).  ! 68! 2.4 Results  In an attempt to find negative regulators of plant immunity, mos2 snc1 npr1 plants were mutagenized and screened. Two primary screens based on morphology and PR2 gene expression, as well as a secondary screen based on susceptibility to an oomycete pathogen, H.a. Noco2, identified over 40 putative mutants. Some have been identified and previously characterized as negative regulators of plant innate immunity. However, other putative mutants had yet to be identified and characterized such as 119 mos2 snc1 npr1.  2.4.1 Mutant 119 mos2 snc1 npr1 carries an intragenic mutation in snc1   The morphology of the quadruple mutant 119 mos2 snc1 npr1 was evaluated and the mutant was found to be significantly smaller than snc1 gain-of-function mutants (Fig. 2.1A). The quadruple mutant appears to restore the snc1-like morphology of dark green, curly leaves, which was fully suppressed by the mos2 mutation. 119 quadruple mutants are sterile. Some homozygous mutants do not survive to reproductive age; however, they are highly resistant to the oomycete pathogen H.a. Noco2 (Fig. 2.1B). The mutation in 119 fully restores plant disease resistance, comparable to that of the snc1 gain-of-function mutant, which is suppressed by mos2.   To determine the location of the mutation in mutant 119, a map-based approach was used. It revealed that the mutation in 119 was located on chromosome 4 near snc1. We therefore used Sanger sequencing to sequence the SNC1 gene in the 119 mos2 snc1 npr1 mutant. We found an additional mutation in the SNC1 gene. The original snc1 gain-of-function mutation, glutamic acid (E) 552 mutated to lysine (K), is located in the linker region of this R-like protein (Fig. 2.1C). The mutation in 119 is in to the NB region of SNC1, which is the only snc1 allele with a mutation in this region. This mutation causes an amino acid change at residue 203 from E to K. Both E and K residues often correspond to protein activation sites, or protein binding sites. ! 66! snc1 is exemplary in the constitutive activation that can occur when E is substituted by K. As an allele of snc1, 119 was renamed snc1-4d (suppressor of npr1, constitutive 1-4, dominant). Other alleles of snc1 found in these screens are also indicated in Figure 2.1C. 2.4.2 snc1-4d  protein stability  SNC1 protein stability was then assessed to determine if the snc1-4d mutation enhances SNC1 function through increased stability. A western blot analysis was carried out (by Yan Huang and Xuejin Chen) and, unlike the snc1-1d allele that enhances SNC1 stability, snc1-4d does not result in increased SNC1 protein stability (Fig. 2.1D).  2.4.3 Conservation of the NB domain in R proteins  A protein alignment was also carried out to determine if the corresponding amino acid residue mutated in snc1-4d is highly conserved among other similar R proteins. TIR-NB-LRR proteins of Arabidopsis, RESISTANCE TO PERONOSPORA PARASITICA (RPP) 4, RPP5 and RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) as well as a CC-NB-LRR of Arabidopsis, RPS2, shared high sequence homology in the NB domain. Amino acid residue 203 in SNC1, the location of the mutation was indeed highly conserved among all Arabidopsis NB- LRR proteins tested (Fig. 2.1E). However, alignments including R proteins from other plant species such as NICOTIANA (N) from tobacco and LINUM 6 (L6) from flax, do not show the same degree of conservation at this amino acid residue (Fig. 2.1F). Therefore, the residue corresponding to the snc1-4d mutation, E203K, seems only highly conserved among Arabidopsis R proteins. ! 6P! A         B       C          D     E  F           Figure 2.1 Analysis of mutant snc1-4d .  (A) Morphological analysis of snc1-4d mos2 snc1 npr1 mutants grown on soil and photographed 3 weeks after planting. Size bar represents 1 cm. (B) Growth of oomycete pathogen H.a. Noco2. (C) Schematic diagram representing SNC1 protein structure. Residue change and location of snc1 alleles are indicated with arrows. (D) Relative amount of SNC1 protein in alleles of snc1 detected by western blot analysis using anti-SNC1 antibodies. (E) Amino acid sequence alignment with NB-LRR proteins homologous to SNC1 in Arabidopsis. snc1-4d mutation indicated with a star. (F) Amino acid alignment with NB-LRR proteins including L6 (flax) and N (tobacco). snc1-4d mutation indicated with a star.! 6Q! 2.5 Discussion   R proteins are typically composed of a TIR or CC N-terminal domain. They also possess a NB and a LRR region. In their inactive form, R-proteins are folded; however, upon effector recognition by the LRR domain, the protein unfolds and dimerizes with another R-protein, which can be either another copy of itself or another R-protein altogether (Moffett et al., 2002).   Previous studies have analysed the importance of each R protein domain to determine the function and necessity of each. The LRR domain possesses ligand binding activity and recognizes effector molecules and therefore determines effector-R protein specificity (Dodds et al., 2001; Wulff et al., 2001). Effector recognition by the LRR domain leads to ATP-dependant conformational change of the NB-LRR protein resulting in activation. Despite the importance of these domains they do not appear to be essential for defence activity, merely specificity. A study conducted on RPM1, a CC-NB-LRR, mutated several components of this R gene and found that if the protein was over-produced, the LRR was dispensable whereas the CC and NB domains were crucial for the induction of downstream defence responses (Tao et al., 2000).  The variable N-terminal domains of R proteins have been implicated in protein-protein interactions and it is thought that these domains are primarily responsible for dimerization and signalling. RPM1 for example, is a CC-NB-LRR type R protein and has been found to interact with RIN4 at its CC domain to negatively regulate resistance. When inactive, RIN4 and RPM1 are bound at the plasma membrane; however, the P.s effector AvrRpm1 may phosphorylate RIN4, which is perceived by RPM1, which is then activated and leads to a signalling cascade terminating in a defence response (Mackey et al., 2002). The NB domain of NB-LRR proteins appears to be crucial for R protein activation possibly due to its ATP-binding capability. The NB domain of the flax R protein, P, is essential ! 6R! for resistance against flax-rust. Certain mutations in this region render the plant susceptible to this fungus (Dodds et al., 2001). Furthermore, the NB domains of I-2 and Mi-1, two R-proteins of tomato, were found to be capable of and necessary in ATP binding, which is required to change R-protein conformation to the active form (Tameling et al., 2002, 2006).   The quadruple mutant snc1-4d mos2 snc1 npr1 was initially identified based on its severe snc1-like morphology and its enhanced disease resistance, which abolishes the suppression imposed by mos2. Using a map-based approach I cloned the mutated gene containing a mutation resulting in an amino acid change in the NB domain of the SNC1 protein. The original gain-of- function snc1 mutation causes an increase in SNC1 protein stability, and therefore possesses constitutive resistance against pathogens. However, western blot analysis indicated that snc1-4d has comparable SNC1 protein stability to that of wild-type plants. Therefore, the increased resistance observed in snc1-4d mutants is not due to increased protein stability, unlike other snc1 alleles, and must be another mechanism that causes the enhanced snc1 phenotype. One possibility may be that the mutation in the NB domain of snc1 increases its affinity for ATP, thus increasing the rate of protein unfolding, and therefore its activation. Another possibility is that the mutation in snc1-4d prevents proper folding of SNC1 allowing it to remain in its active, less folded form. Further analyses must be conducted to determine if the mutation in snc1-4d is due to auto-activation of SNC1, or another mechanism altogether.  ! 6J! 3 AtCDC48A negatively regulates plant innate immunity  3.1 Summary  Due to their sessile nature, plants had to evolve multi-layered immune systems to defend themselves against pathogens. In addition, plants have evolved regulatory mechanisms to keep the immune response in check. Much research has focused on the production of antimicrobial compounds and proteins; however, it is becoming increasingly evident that regulation at the level of protein degradation plays a key role in plant innate immunity. Through a genetic screen we discovered a novel negative regulator of plant innate immunity, AtCDC48A. Previously this protein has been implicated in the cell division cycle and proteasome-mediated proteolysis. However, here we find that mutant atcdc48a plants have heightened PR gene expression and increased resistance to the oomycete pathogen H.a. Noco2. These results implicate AtCDC48A  as a negative regulator of plant innate immunity.  3.2 Introduction  Plants have evolved complex immune systems to protect and defend themselves against pathogens. Trans-membrane PRRs present on the plant cell surface, serve to recognize conserved PAMPs present on the surface of pathogens. This recognition leads to downstream defence responses. However, some pathogens have evolved mechanisms to inject effector molecules into the host plant cell. These effectors inhibit the defence responses of the plant and lead to successful infection of the host. In turn, plants have evolved a secondary layer of resistance through R proteins that act as internal surveillance molecules that recognize pathogen effector molecules and mount a defence response (Jones and Dangl, 2006).  R protein mediated defence responses lead to the expression of PR genes, accumulation of SA, and programmed cell death referred to as the HR. snc1 encodes an R-like protein with a ! 6H! gain-of-function mutation. It exhibits constitutive expression of PR genes, SA accumulation and has increased resistance to virulent pathogens. However, the gain-of-function mutation also results in extremely dwarf stature, and dark, deformed leaves (Li et al., 2001; Zhang et al., 2003).  Due to the complex layered nature of the plant innate immune system and the detrimental phenotypes observed when these pathways are mis-regulated, as we see in snc1 mutants, plants require stringent regulation of such processes. Much of the research in this field has primarily focused on the production of defence compounds and proteins; however, it is becoming increasingly evident that protein degradation and turnover also plays a significant role in the regulation of plant innate immunity.  The 26S proteasome pathway is the primary method of protein degradation in plants (Schubert et al., 2000). This complex is responsible for the turnover of many housekeeping proteins; however, it also plays a significant role in targeted protein degradation in response to internal or external cues. Degradation in this pathway is initiated by poly-ubiquitination of target proteins. Affixation of Ub, one of the most highly conserved and abundant proteins, marks the protein for degradation (Callis et al., 1995). Ubiquitination is accomplished through an E1, E2, E3 cascade in which an E1 enzyme activates Ub, which is subsequently transferred to an E2 enzyme by transesterification (Ciechanover et al., 1982; Hershkos et al., 1983). E2 then transports the activated Ub to an E3 ligase enzyme, which determines target specificity and the final transfer of Ub to the target protein typically at a lysine (K) residue (Hershkos et al., 1983; Reiss et al., 1989; Chau et al., 1989). This process is repeated until the correct length of ubiquitin chain is achieved for its degradation. Once poly-ubiquitinated, target proteins require transfer to the proteasome for degradation. Complexes are formed that specifically recognize and recruit ubiquitinated proteins, ! 6I! some of which have also been found to be associated with Cell Division Cycle 48 (CDC48) complex, a highly conserved protein complex that has been implicated in protein extraction from membranes and proteasome-mediated degradation (Richly et al., 2005; Ye et al., 2001; Baek et al., 2011). However, the exact role of CDC48 in targeted proteolysis remains unclear. Here we report the identification of a mutant snc1-enhancing 8 (muse8) gene that negatively regulates plant innate immunity as evidenced by increased PR gene expression and resistant to infection by the oomycete pathogen H.a. Noco2. Map-based cloning indicates the muse8 phenotype is due to a mutation in AtCDC48A, a CDC48 homolog in Arabidopsis thaliana. Solexa sequencing revealed that the muse8 mutation causes an amino acid change in a highly conserved residue of AtCDC48A, which forms a hexameric protein complex. This protein complex is involved in the cell division cycle and also implicated in proteasome-mediated proteolysis; however, here we observe a clear indication of its involvement in regulating plant innate immunity. ! 6K! 3.3 Materials and methods 3.3.1 Plant growth and mutant isolation   For most experiments, plants were grown on soil with a 16 hour light/8 hour dark cycle. Isolation of the mos2 snc1 npr1 triple mutant was previously described (Zhang et al., 2005). The muse8 mos2 snc1 npr1 was created by mutating 40,000 mos2 snc1 npr1 Arabidopsis thaliana seeds also carrying a BGL2-GUS construct with 0.2% EMS (ethylmethanesulfonate) for 8 hours (Bowling et al., 1994). Mutants were isolated by screening based on snc1-like morphology and PR2 gene expression, which was assessed using a GUS-reporter construct that was previously introduced (Cao et al., 1997). Mutants obtained from these primary screens were subjected to a disease resistance screen where mutants were spray-inoculated with H.a. Noco2 as described previously (Li et al., 2001).  3.3.2 muse8 mos2 snc1 npr1  characterization   muse8 mos2 snc1 npr1 morphology was characterized based on plant size, leaf shape and colour, sterility, and other deviations from wild-type or snc1 mutant phenotypes. Photographs were taken using a digital camera and size bars added using Adobe Photoshop.   Total RNA was extracted from 14 day-old plants using the Totally RNA Kit (Ambion). Total RNA was then reverse transcribed using M-MLV Rerverse Transcriptase (Takara). PR1, PR2, and Actin7 were amplified separately as previously described (Zhang et al., 2003). PCR products were then run on a 1% agarose gel containing ethidium bromide and imaged using an AlphaImager HG (AlphaInnotech).   Disease resistance was tested using muse8 mos2 snc1 npr1, mos2 snc1 npr1, snc1, and BGL2 plants grown on soil. Two-week-old plants were spray inoculated with 50,000 spores/mL of H.a. Noco2 as previously described (Li et al., 2001). Infection was assessed precisely through ! P7! spore counting on twenty randomly chosen plants from each genotype 10-14 days post- inoculation using a hemocytometer.  3.3.3 Genetic mapping  muse8 mos2 snc1 npr1 mutants in the Columbia-0 ecotype of Arabidopsis thaliana were crossed with the Landsberg erecta (Ler) ecotype. The F2 generation was grown on soil and 48 plants smaller than snc1 were selected for linkage analysis. The muse8 mutation was narrowed down to a region on chromosome 3 between markers F3L34 (1.4) MB and MGH6 (4.19 MB). The fine mapping population (F3) was obtained from three F2 lines that were heterozygous in the aforementioned region. Fine mapping further narrowed the region to between T16O11 (2.7 MB) and MGH6 (4.19 MB). Homozygous quadruple mutant plant DNA was isolated from quadruple mutant plants grown on soil and sent to the National Institute of Biological Sciences in Beijing, China for Solexa sequencing.  3.3.4 Molecular cloning  T-DNA insertion lines for At3G06660, At3G08950, and At3G09840 were obtained from Arabidopsis Biological Resource Center (ABRC). Complementation tests were done by crossing T-DNA mutant lines with muse8 snc1 double mutant lines and examining the F1 for evidence of complementation by the presence of a wild-type phenotype. The F2 generation for each cross was also grown to confirm complementation or a lack of complementation. Further cloning confirmation was obtained by crossing T-DNA lines with snc1 mutants and examining the subsequent F1 and F2 generations for snc1-enhancing phenotypes of the double mutants of snc1 and the T-DNA insertion.  ! P8! 3.3.5 Phylogenetic analyses  The AtCDC48A protein sequence was obtained from PubMed online and used to conduct a BLASTP search. Homologous sequences from each kingdom were chosen based on low e- values and high quality reference sequences. Protein alignment was achieved using Muscle (Edgar, 2004). The alignment was then manually adjusted using Se-Al (Rambaut, 2002). Conserved amino acid residues were found using Jalview (Waterhouse et al., 2009). The optimal evolutionary model was chosen using ProtTest; the output form the AICc and BIC trials were compared and the most appropriate model was found to be LG+I+G (Darriba et al., 2011; Guindon and Gascuel, 2003). Heuristic searches and bootstrapping with 5000 replicates was conducted using SeaView (Gouy et al., 2010).  For the Arabidopsis thaliana CDC48 tree, paralogs were found using a BLASTP search. Protein sequences of AtCDC48A-E were obtained from TAIR. All sequences were aligned using Muscle (Edgar, 2004). Using ProtTest, comparing the output from AICc and BIC, the optimal evolutionary model was found to be LG+I (Darriba et al., 2011; Guindon and Gascuel, 2003). Heuristic searches and bootstrapping was conducted using 5000 replicates with SeaView (Gouy et al., 2010). 3.3.6 Microarray gene expression analysis   Expression data for each AtCDC48 paralog was obtained using the AtGenExpress Visualization Tool (http://jsp.weigelworld.org/expviz/expviz.jsp). All AtGenExpress data is quantile-normalized using GeneChip Robust Multiarray Averaging (gcRMA) and absolute values provided by this database are linerarized gcRMA values. Data from various treatments was obtained by submitting the Arabidopsis gene number (ie. At3G09840), and selecting pathogen, development, or hormone from the experiment drop-down menu. Absolute values ! P6! from individual data points from the resulting expression graph in response to various treatments of pathogens, chemicals or various tissues were used in creating bar charts to determine if expression patterns of AtCDC48A  are similar to those of the other AtCDC48 paralogs. For developmental expression data, tissues were chosen based on their relative exposure to pathogens or due to the previous implication of AtCDC48A  within that tissue (Feiler et al., 1995). For hormone expression data in each gene, MG132 was chosen due to its proteasome inhibition activity and cyclohexamide was chosen due to its involvement in protein synthesis inhibition to determine the involvement of AtCDC48A  in proteasome-mediated protein degradation as previously hypothesized (Richly et al., 2005; Ghislain et al., 1996; Schuberth et al., 2004).     ! PP! 3.4 Results 3.4.1 muse8 mos2 snc1 npr1  characterization  muse8 mos2 snc1 npr1 mutant was obtained from a primary screen based on PR2 gene expression using a GUS construct that was fused to the endogenous PR2 promoter. Expression of GUS indicates the PR2 promoter is being bound and transcription is being initiated; therefore, PR2 should also be expressed. A secondary screen based on resistance to H.a. Noco2 tested the resistance of putative mutants obtained from the primary screen. The quadruple mutant displays enhanced snc1-like morphology, which is dwarf, dark green and curly leaved; however, it is slightly smaller than snc1 (Fig. 3.1A).   PR gene expression was assessed using RT-PCR. muse8 mos2 snc1 npr1 restores the PR1 and PR2 gene expression levels similar to that of snc1, which were abolished by the mos2 mutation (Fig. 3.1B). To determine if this PR gene expression confers increased disease resistance, muse8 quadruple mutants as well as controls were infected with the oomycete pathogen H.a. Noco2.  Spores were counted 10-14 days post-inoculation. Quadruple mutants indeed had increased disease resistance comparable to that of snc1 plants (Fig. 3.1C).  3.4.2 muse8 contains a mutation in A tCDC48A   The mutation responsible for the observed phenotype of muse8 mos2 snc1 npr1 mutants was crude-mapped to the top of chromosome 3, near marker MGH6 at 4.2 MB. Fine-mapping narrowed down the region to between indel markers T16O11 (2.7MB) and MGH6 (4.2 MB) (Fig. 3.1E). Further mapping did not narrow down the region any further; therefore, nuclear DNA was extracted from quadruple mutants and sent to the National Institute of Biological Sciences in Beijing for Solexa sequencing. This next generation sequencing revealed mutations in many candidate genes within the 2MB on chromosome 3 (Table 3.1). Two mutations occurred ! PQ! in a pseudogene of nodulin (At3G06433); however, as a pseudogene, it is also an unlikely candidate for the muse8 mutation. Similarly, two amino acid changes were found in MOV34 (At3G11270), which is a PAD-1 family protein. One mutation resulted in a valine to methionine subtitution, both of which are hydrophobic amino acids and serve similar functions; therefore this mutation would most likely not change the function of the protein. The second mutation in this gene caused a threonine to isoleucine substitution. At3G11402, a cysteine/histidine-rich C1 domain family protein, At3G11560 a LETM1-like protein, and At3G12800 encoding a short- chain dehydrogenase-reductase B protein also contained point mutations; however, the recombination frequency seen in the F2 and F3 population indicated the mutation should be in a gene closer to 3MB.  Three genes seemed to be the most likely candidates due to either their protein function or the observed recombination frequency in the F2 and F3 generations. At3G06660 encodes a PAPA-1-like family protein, a transcription factor located in the chloroplast. The location of the point mutation of this gene did not correspond to a functional domain, which makes it an unlikely candidate; however, due to its location in the genome it was considered further. At3G08950 encodes a phosphoglycerate mutase found in mitochondria and used in copper ion binding and cytochrome c synthesis. The location of the mutation in this gene is within the last ten amino acid residues and does not correspond to a known functional domain. Due to its location in the genome and the recombination frequency observed, it was considered a possible candidate for the location of the muse8 mutation. At3G09840 or AtCDC48A  seemed like the most likely candidate. This protein is involved in the conversion of ATP to ADP through two P- loop AAA ATPase domains. This protein forms a homohexamer creating a cylindrical structure. The location of the mutation, which causes a glycine to glutamic acid substitution at residue 274 ! PR! is in the first P-loop AAA ATPase domain (Fig. 3.2A). Glycine is the smallest amino acid and as such is often found in the centre of tight complexes; changing this amino acid may alter the structure of this complex and therefore affect its function.  Glycine is also the only amino acid that can bind phosphates from ATP and therefore may be crucial to the function of this complex.   To determine which of these three genes is responsible for the muse8 phenotype, an allelism test was conducted. T-DNA insertion lines for each gene were obtained from ABRC and crossed to muse8 snc1 double mutants obtained in the F2 generation. A T-DNA insertion mutant of At3G06660, the transcription factor in the chloroplast, complemented the muse8 snc1 double mutant phenotype, as did T-DNA insertion mutants of At3G08950, the phosphoglycerate mutase found in mitochondria (Fig. 3.2B). However, when the T-DNA mutant of At3G09840 (AtCDC48A) was crossed with the muse8 snc1 double mutant, it failed to complement in the F1 generation in that plants heterozygous for the T-DNA insertion in AtCDC48A  and heterozygous for snc1, displayed a snc1-like phenotype, indicating one non-functional copy of atcdc48a is sufficient to cause a snc1-like phenotype when heterozygous for snc1. Homozygous T-DNA insertion mutant, atcdc48a, is lethal; however, plants homozygous for muse8 are partly sterile, in that they produce very small siliques with no seeds. In the F1 generation, plants heterozygous for the T-DNA insertion and heterozygous for muse8 were also sterile, which further supports that AtCDC48A  is the gene responsible for the muse8 quadruple mutant phenotype.  To confirm that AtCDC48A  is MUSE8, T-DNA insertion lines were crossed with snc1 and allowed to self to obtain double mutants homozygous for snc1 and heterozygous for the T- DNA insertion. Since the muse8 quadruple mutant enhances the snc1 phenotype, the loss of function T-DNA insertion line corresponding to the muse8 gene should also enhance the snc1 phenotype. The PAPA-1 transcription factor mutant did not enhance snc1 (Fig. 3.3A). Similar ! PJ! results were obtained for the phosphoglycerate mutase T-DNA insertion line (Fig. 3.3B). Conversely, plants homozygous for the snc1 mutation but heterozygous for the AtCDC48 T- DNA insertion exhibit snc1-enhancing morphology (Fig. 3.3C). This again confirms that we have cloned the correct gene.  3.4.3 Phylogenetic analysis of AtCDC48A protein  To determine the level of conservation of AtCDC48A, a phylogenetic tree was constructed (Fig. 3.4A). CDC48A is highly conserved across kingdoms. The amino acid residue altered in muse8 was also highly conserved in all organisms (Fig. 3.4B). In Arabidopis thaliana there are 4 paralogs of AtCDC48A. Examining their gene structure indicates that AtCDC48A  is most closely related to AtCDC48D and AtCDC48E (Fig. 3.5A). Indeed this seems to be true based on the phylogenetic tree of the AtCDC48 paralogs (Fig. 3.5B). 3.4.4 Expression analysis of the A tCDC48 gene family  Although paralogous, little investigation into the AtCDC48 genes has taken place. Microarray analysis revealed that all AtCDC48 genes appear to be up-regulated upon infection with various strains of P.s.; however, AtCDC48A  is by far the most highly expressed (Fig. 3.6A- E). Similar results were obtained by treating plants with Phytophthora infestans, another virulent pathogen, and well-known pathogen elicitors (data not shown). A similar trend is seen among various tissues (Fig. 3.7A). AtCDC48A  expression is highest in roots and senescing leaves, relatively high in leaves, stamens, carpels and seeds and relatively low in mature pollen. Similar tissue specificity of expression is seen with AtCDC48D and AtCDC48E, which further support the observed phylogenetic relationship. Since AtCDC48A  has previously been implicated in proteasome-mediated protein degradation, AtCDC48A  expression was examined after treatment with MG132, a proteasome inhibitor, and was found to increase (Fig. 3.7B). Treatment with ! PH! MG132 had a similar effect on AtCDC48D and E expression but little to no change in AtCDC48B and C expression. Following treatment with cyclohexamide, a protein synthesis inhibitor, AtCDC48A  expression decreased significantly as did AtCDC48D and E expression to a lesser extent (Fig. 3.7B). Alternatively, AtCDC48B and C expression again showed little to no change.  ! PI! A        B                     C       D    Figure 3.1: Characterization of muse8 mos2 snc1 npr1 .  (A) Morphological analysis of muse8 mos2 snc1 npr1 in comparison to mos2 snc1 npr1, snc1, and wild-type. (B) PR1 (RT-PCR) and PR2 (GUS) gene expression of aforementioned genotypes. (C) Growth of the oomycete pathogen H.a. Noco2 on each of the aforementioned genotypes. (D) Schematic diagram of chromosome 3 indicating region containing muse8 narrowed down by a map-based approach. ! PK! Table 3.1: Next generation sequence analysis: list of potential candidate mutations in the mapped muse8 region.   Gene Predicted Protein Number of Reads Nucleotide Change Amino Acid Change At3G06433 Pseudogene of nodulin MtN3 family protein 6/8 C to T R396* At3G06433 Pseudogene of nodulin MtN3 family protein 3/4 G to A R973H At3G06437 Pseudogene, hypothetical protein 21/22 C to T None At3G06660 PAPA-1-like family protein/zinc finger 5/5 G to A G113S At3G07425 Unknown protein 3/6 C to T None At3G07670 Rubisco methyltranferase 2/3 C to T (intron) At3G08590 Phosphoglycerate mutates 2/2 G to A A329S At3G08930 LMBR1-like membrane protein 3/3 C to T None At3G09840 Cell Division Cycle 48 4/3 G to A G274E At3G10195 Putative membrane lipoprotein 2/3 C to T (intron) At3G10585 Homeodomain-like superfamily protein 2/4 G to A (intron) At3G10690 DNA GYRASE A 5/5 G to A (intron) At3G11260 WUSCHEL related homeobox 5 2/4 G to T or A (intron) At3G11270 Mov34/MPN/PAD-1 family protein 3/5 G to A V127M At3G11270 Mov34/MPN/PAD-1 family protein 2/2 C to T T142I  ! Q7!    A      B       Figure 3.2: Cloning of A tCDC48A  (A) Schematic diagram of AtCDC48A indicating location of the mutation in muse8. (B) Complementation. at3g06660 and at3g08950 T-DNA insertion lines (CS860752 and SALK_119825c) complemented muse8 snc1 mutants whereas heterozygous atcdc48a (at3g09840 het) T-DNA insertion lines (SALK_064573) failed to compliment muse8 snc1 plants. Wild-type, snc1, muse8 snc1, and T-DNA insertion line control plants are shown. ! Q8!     Figure 3.3: Crosses of T-DNA lines of candidate genes with snc1 .  F2 plants were grown on soil, genotyped and photographed 4 weeks post-planting. (A) F2 progeny of a cross between at3g06660 T-DNA insertion line (CS860752) and snc1. at3g06660 snc1 double mutants do not display an enhanced snc1-like phenotype. (B) F2 progeny of a cross between at3g08590 T-DNA insertion line (SALK_119825c) and snc1. at3g08950 snc1 double mutants do not display an enhanced snc1-like phenotype. (C) F1 progeny of at3g09840 T-DNA insertion line (SALK_064573) and snc1. Heterozygous at3g09840 snc1 double mutants display an enhanced snc1-like phenotype in comparison to snc1 and the T-DNA insertion line. ! Q6! A !"##$%&’"##$% ($%&)$%*$#$% +,),&%"-./0%12 3/0,-$%&#"/4.% 56 7"0.,&8/8., 69 78,%,-:.#"&)/#"0,’"%;/8 !"#$%&’"()*+,-#.#/"$, <== 1< (/>.*"’,&;8$0*";$#" !#?*.0/&)"@ A.*.0$%&*,))$0.% 26 B8"C.>,-%.%&;:"#."0" 22 B##.$)&*/-" 26 D,8’:$)&C.*,#,8 0&12"-,"+3" 66 <== 4#."/*$#.."-5%#..#$)%&6* E:?%*,).;8/##"&-";/0%&%$C%-F&-";/0% 5G 61 !’."51)%5%$",-&#*$’"&)+* <== (.*8,),0"%&-$%*.##" 56 7.*;?,%;/#.$)&-$8-$8/$) 11 E:?;,-:;:,8"&.0H/%;"0% 5= 1G 16 I/$8,%-,8"&*8"%%" D*:.J,%"**:"8,)?*/%&-,)C/ D"**:"8,)?*/%&*/8/4.%."/65 <== 0.1    B     *      Figure 3.4: Phylogenetic analysis of CDC48A across kingdoms. (A) Phylogenetic tree of AtCDC48A and representative homologs from other kingdoms. (B) Amino acid alignment of a portion of AtCDC48A and representative homologs from other kingdoms. ! QP! A       B !"#$#%&#’!"()*+,+* !"#$#%&-’!".)*(,/* !"#$#%&$’!"()0(.(* !"#$#%&!’!"()*1&%* !"#$#%&2’!"0)*((%* %/ +** *3.       Figure 3.5: Phylogenetic analysis of A tCDC48  A rabidopsis  orthologs. (A) Schematic diagram representing the gene structure of AtCDC48 orthologs. (B) Phylogenetic tree of AtCDC48 orthologs indicating high homology between AtCDC48A, AtCDC48D, and AtCDC48E. ! QQ!   A        B       C       D       E    Figure 3.6: Microarray expression analysis of A tCDC48  orthologs post- infection. Expression data was obtained from AtGenExpress Visualization Tool. (A) Relative expression of AtCDC48A after treatments with various strains of Pseudomonas syringae pv. tomato. (B) Relative expression of AtCDC48B after treatments with various strains of Pseudomonas syringae pv. tomato. (C) Relative expression of AtCDC48C after treatments with various strains of Pseudomonas syringae pv. tomato.(D) Relative expression of AtCDC48D after treatments with various strains of Pseudomonas syringae pv. tomato. (E) Relative expression of AtCDC48E after treatments with various strains of Pseudomonas syringae pv. tomato. ! QR!      A       B         Figure 3.7: Microarray expression analysis of A tCDC48  orthologs in tissues and in response to chemical treatment.  Expression data was obtained from AtGenExpress Visualization Tool. (A) Microarray expression of AtCDC48 orthologs in root, leaf, senescing leaf, stamen, mature pollen, carpel and seed. (B) Microarray expression of AtCDC48 orthologs in response to the proteasome inhibitor MG132 and the protein synthesis inhibitor cyclohexamide. ! QJ! 3.5 Discussion  Protein degradation is needed to mount a controlled, yet robust defence response. The 26S proteasome-mediated proteolysis is the most common type of protein degradation in plants, and requires ubiquitination of target proteins through E1, E2 and E3 enzymes (Schubert et al., 2000; Hershkos et al., 1983; Reiss et al., 1989; Chau et al., 1989). Targeted proteins are then transferred to the 26S proteasome for degradation.  Several E3 ligases have been implicated in plant defence (Cheng and Li, 2012). CPR1 of Arabidopsis thaliana is a negative regulator of plant innate immunity (Bowling et al., 1994). When mutated, cpr1 mutants have elevated levels of SNC1 protein; however, when CPR1 is over-expressed, SNC1 protein accumulation is abolished (Cheng et al., 2011; Gou et al., 2012). Therefore the E3 ligase CPR1 negatively regulates plant innate immunity by regulating SNC1 protein stability. Other examples of E3 ligases involved in plant defence are ACIF1 from tobacco, or ACRE189 of tomato. These E3s positively regulate HR mediated by various R genes such as CLADOSPORIUM FULVUM RESISTANCE 4 (CF-4), Cf-9, N, and PSEUDOMONAS RESISTANCE AND FENTHION SENSITIVITY (Prf) (Van den Burg et al., 2008). During infection, these F-box protein are up-regulated and associate with the SCF complex, which ubiquitinates target proteins to be degraded. Loss-of-function or silencing mutations in ACIF1 decrease the HR. Therefore, the role of ACIF1 in immunity is to target a negative regulator of defence for ubiquitination. Once degraded, the defence response can occur, and HR is observed.  Transfer of target proteins to the proteasome is thought to be accomplished via protein complexes that associate with both the ubiquitinated substrate, as well as the proteasome (Hartmann-Petersen et al., 2003). Some of these proteins are Ub-receptor proteins, which contain both a Ub-like (UBL) domain at the N ternimus that is recognized by the proteasome as well one ! QH! or multiple Ub-Associating (UBA) domains that function in Ub binding (Wilkinson et al., 2001). These UBL-UBA containing proteins bind target proteins via their Ub-chains and bind the proteasome, essentially delivering the target protein to the proteasome (Elsasser and Finley, 2005). These events have been shown to be crucial to plant innate immunity. A UBA-containing protein MOS5 has been previously implicated in plant defence (Goritschnig et al., 2007). The mos5 loss-of-function mutant prevents the accumulation of SNC1 protein and therefore inhibits disease resistance. Therefore, MOS5 is essential in disease resistance mediated by the R-like protein, SNC1.  Another family of Ub-associated proteins, is the UBX family, also important in proteasome-mediated degradation as well as plant innate immunity. UBX proteins contain UBA domains and UBX domains, which are similar to UBL domains in that they resemble Ub itself. In Arabidopsis, microarray analysis of wild-type controls and plants infected with the powdery mildew Golovinomyces orontii revealed an up-regulation of a number of UBX proteins implicating them in plant defence (Chandran et al., 2009).  UBX proteins are often associated with the CDC48 complex (Orme and Bogan, 2012). This complex is comprised of six monomers of the CDC48 protein and forms a ring-like structure. Each monomer contains two AAA (ATPase Associated with different cellular Activities) ATPase domains, which use the hydrolysis of ATP to ADP to perform various cellular activities through conformational changes. Each monomer contains N-terminal domains, which allow binding of substrates. Since CDC48 is a homohexameric complex, it possesses six N-terminal domains, each capable of binding a different co-factor allowing the CDC48 complex to have a multitude of functions depending on the cofactors with which it is associated.  ! QI! As its name suggests, CDC48 has been implicated in the cell division cycle and cell proliferation (Moir and Botstein, 1982; Peters et al., 1990; Park et al., 2008). However, CDC48, as well as its homologs p97 in mouse and frog, and VALOCIN CONTAINING PROTEIN (VCP) in humans, have been implicated in the proper assembly of endoplasmic reticulum and Golgi body (Kondo et al., 1997; Yuan et al., 2001; Roy et al., 2000), nuclear envelope formation (Hetzer et al., 2001), and ER-associated protein degradation (ERAD) (Alzayady et al., 2005; Schuberth and Buchberger, 2005; Romisch, 2006). In yeast, CDC48 is thought to act in the ERAD pathway through the extraction of proteins from the cytosolic side of the ER membrane via their interaction with CDC48 cofactors, UBIQUITIN FUSION DEGRADATION 1 (UFD1) and NUCLEAR PROTEIN LOCALIZATION 4 (Npl4) (Braun et al., 2002; Jarosch et al., 2002; Rabinovich et al., 2002; Ye et al., 2001, 2003). These protein substrates are subsequently poly- Ub by UFD2, an E4 ligase that has been found to bind CDC48 and utilize the E1, E2, E3 cascade to poly-ubiquitinate its target (Richly et al., 2005). This poly-ubiquitination recruits RADIATION SENSITIVE 23 (RAD23), a protein that recognizes poly-ubiquitinated substrates that binds both CDC48 and the proteasome, essentially escorting the target substrate to the proteasome for degradation (Richly et al., 2005). RAD23 binding of UFD2 and the proteasome is mutually exclusive. Recently, it has been shown that disassembly of the UFD2/RAD23 complex is facilitated by CDC48 in an ATP/UFD2 dependant manner, allowing the release of the poly-Ub subtrate/RAD23 complex and subsequent binding to the proteasome complex (Baek et al., 2011).  Despite the implication of various components involved in proteasome-mediated degradation such as various E3 ligases and UBX containing proteins in plant immunity and the association of this type of protein with CDC48, this multifunctional chaperone has yet to be implicated in plant innate immunity. Using a map-based cloning approach along with genetic ! QK! complementation, the muse8 mutation was confirmed to be in AtCDC48A, the homolog of CDC48/p97/VCP found in other eukaryotes. atcdc48a mos2 snc1 npr1 quadruple mutants display snc1-like phenotypes of dwarf stature, dark green, curly leaves as well as constitutive PR gene expression and resistance to H.a. Noco2.  Phylogenetic analysis confirmed the conserved nature of this protein. The muse8 mutation was a single nucleotide polymorphism located in the first AAA ATPase domain of AtCDC48A, which, when folded in complex, resides inside the core of the cylindrical AtCDC48A homohexamer. This mutation results in an amino acid change from glycine, the smallest of amino acids often found in the centre of tight conformations and the only amino acid capable of binding phosphates of ATP, to glutamic acid and may hinder AtCDC48A activity. This mutation does however appear to be leaky. Previous studies regarding AtCDC48A have been difficult as all previous atcdc48a mutant lines described are lethal (Park et al., 2008). The muse8 allele of atcdc48a is unique in that homozygous mutants, although sterile, do survive and may be useful in further characterization of this protein complex and its many functions. Microarray expression data further confirmed muse8 as AtCDC48. Quadruple mutant plants homozygous for muse8 are female sterile, which is reflected in the tissue specific expression analysis. AtCDC48A  is lowly expressed in mature pollen; however, it is highly expressed in carpels, as well as in developing embryos supporting the role of AtCDC48A in female fertility and embryogenesis as various atcdc48a mutants display female infertility and arrest at embryogenesis. Interestingly, AtCDC48A  is also highly expressed in tissues that may interface with pathogens such as the roots, cauline leaves, and senescing leaves. Expression data of AtCDC48A  also supports the role of AtCDC48A in proteasome-mediated proteolysis as in response to a proteasome inhibitor, MG132; without a functional proteasome, need for protein ! R7! degradation would increase, and the concentration of all positive regulators of proteasome- mediated protein degradation would be expected to increase in an attempt to compensate for this lack of degradation, which is what we observed for AtCDC48A . Similarly, a lack of protein synthesis would decrease the need for protein degradation; therefore, we would expect expression of positive regulators of protein degradation to increase, which is observed in AtCDC48A  when exposed to cyclohexamide, a protein synthesis inhibitor. AtCDC48A does appear to be involved in the regulation of plant innate immunity through proteasome-mediated protein degradation. This may be accomplished in a variety of ways; however, we propose one possible model (Fig. 3.8). Perhaps AtCDC48A regulates plant innate immunity through the degradation of positive regulators of defence. For example, it is known that SNC1 is negatively regulated by CPR1 in association with the SCF complex, which in turn poly-ubiquitinates SNC1, marking it for degradation (Cheng and Li, 2012). AtCDC48A may bind an E2 enzyme that recruits SNC1 and facilitate its association with the SCF complex. Once SNC1 is poly-ubiquitinated, AtCDC48A may disassemble the E2/SCF complex allowing the SCF complex to bring SNC1 to the proteasome for degradation, thus negatively regulating SNC1-mediated defence. Future studies must be conducted to find binding partners of this protein complex to elucidate which proteins are degraded to negatively regulate the immune response. The partial loss-of-function allele described here may prove to be an essential tool in discovering the many functions of the AtCDC48 complex. ! ! "#!                                                                                                                                               Figure 3.8: Proposed model of AtCDC48A in protein degradation: regulation of plant immunity. SNC1 is recruited by an E2 enzyme (blue), which then binds with AtCDC48A (purple). An E3 ligase/complex such as the SCF complex (green) is also recruited to AtCDC48A. The target protein, SNC1 is then transferred to the SCF complex. AtCDC48A uses energy from ATP to dissociate E2 from the SCF complex. SNC1 is poly-ubiquitinated (red) by the SCF and subsequently, poly-ubiquitinated SNC1/SCF complex dissociates from AtCDC48A. The SCF complex then brings SNC1 to the proteasome for degradation.! ! "$! 4 Future directions and concluding remarks 4.1 snc1-4d  Several alleles of the snc1 gain-of-function mutant have been found previously. In this study we have described the discovery of yet another allele of snc1, snc1-4d. Unlike other alleles of snc1, snc1-4d contains a mutation in the NB region of this R-like protein. snc1-4d mutants show enhanced disease resistance and PR gene expression. These mutants are much smaller than snc1 original mutants and are sterile. SNC1 protein level was shown to be unchanged in these mutants in comparison to wild-type Col-0 plants.  Future experiments will be conducted to determine the effects of the snc1-4d allele on the SNC1 protein. Since SNC1 protein level was not altered in this allele, the observed enhanced snc1 phenotype is not due to an increase in SNC1 stability. The mutation in the NB domain of this R-like protein may cause an increase in SNC1 activation. Since the NB domain has been implicated in ATP-binding and the conversion of ATP to ADP is required for R protein conformational change and activation, investigation into the ability of snc1-4d to bind ATP may be conducted. This allele may prove instrumental in the discovery of the activation mechanism of SNC1. 4.2 AtCDC48A  Here I have shown that a mutation in AtCDC48A  is responsible for the enhanced snc1- like phenotype observed in muse8 mos2 snc1 npr1 mutant plants. This mutation causes increased disease resistance, as well as the restoration of constitutive PR gene expression and is the first example of AtCDC48A’s involvement in plant disease resistance. Expression data analysis supports the notion that AtCDC48A  is involved in proteasome-mediated protein degradation, since it is up-regulated when the proteasome is inhibited but down-regulated when protein ! ! "%! synthesis is inhibited. Not only is AtCDC48A homologous to many other proteins in other eukaryotes, it is also homologous to other Arabidopsis proteins. The AtCDC48 family is composed of five members, A-E. AtCDC48A is by far the most highly expressed in all tissues and after pathogen attack. Previous studies on AtCDC48A revealed that homozygous atcdc48a null mutants are lethal; however, in this study we have found a unique allele of AtCDC48A. This partial loss-of-function allele is not lethal when homozygous and will therefore be an essential tool in future studies of AtCDC48A.   There are many questions about the roles of AtCDC48A. Previously this protein, which forms a homohexameric complex, has been shown to bind proteins associated with ubiquitination and proteasome degradation. Future studies will be conducted to determine if AtCDC48A binds proteins that are not only associated with ubiquitination and degradation but also plant innate immunity. CPR1, for example, seems a likely candidate as it negatively regulates the R-like protein SNC1 through association with the SCF complex, which is involved in protein degradation (Gou et al., 2012). This can be achieved by first examining the level of SNC1 protein in atcdc48a mutants. Second, direct interaction between AtCDC48A and CPR1 could be determined through a yeast-two-hybrid or Bimolecular Fluorescence Complementation (BiFC). Another possible candidate is the Arabidopsis homolog of yeast UFD2 protein, which is an E4 ligase that has been shown to bind CDC48 in yeast (Baek et al., 2011).  Since this is the first report of AtCDC48A being involved in plant defence, future studies will be conducted to determine where it acts in the plant immunity pathway. Crosses will be made with pad4/eds1 mutants, which are defective in TIR-NB-LRR signal transduction. This will determine whether AtCDC48A acts prior to TIR-NB-LRR action or after signal transduction ! ! "&! has commenced. Crosses will also be done between atcdc48a mutants and loss-of-function snc1 mutants to determine if atcdc48a relies on SNC1.  Analyses will also be conducted with the AtCDC48 homologs to determine if they share similar functions as AtCDC48A and in similar pathways. Morphology, pathogen resistance and defence responses will be assessed in loss of function T-DNA insertion lines for each AtCDC48 homolog. Subsequent experiments will be designed based on the findings from these studies. 4.3 Concluding statements  This thesis work provides tools for investigating the regulation of plant innate immunity. 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